THE NATIONAL ANTIMICROBIAL
RESISTANCE MONITORING SYSTEM MEETING
June 23, 2005
I N D E X
Introduction and Purpose of Meeting
by Linda Youngman, Ph.D.
NARMS Background
by David G. White, Ph.D.
Budget Issues
by Robert Walker, Ph.D.
Retail Meat Arm
by David G. White, Ph.D.
Animal Arm
by Paula Fedorka-Cray, Ph.D.
Human Arm
by Tom Chiller, M.D., Ph.D.
by Tim Barrett, Ph.D. (Continued)
Animal Isolate Sampling
by Neena Anandaraman, DVM
Retail Meat Sampling
by Terry Proescholdt
Retail Meat Reporting
by Elvira Hall-Robinson, D.
V.M., Ph.D.
Molecular Characterization
By Shaohua Zhao, D.V.M., Ph.D.
KEYNOTE: "---" Indicates inaudible in transcript.
" * " Indicates phonetic spelling.
Introduction and Purpose of Meeting
by Dr. Linda Youngman
(9:10 a.m.)
DR. YOUNGMAN: Good morning. We are just about ready to start. So, if I could ask that you take your seats, please.
(Pause.)
DR. YOUNGMAN: Good morning. I apologize for the delay in getting started this morning, but everything is here now, except for the booklets that we will be providing. The booklets will have the slides that people are presenting today. Those will be arriving shortly. I apologize that they are not here yet.
Anyway, we are very grateful that you all are here, and I want to do an introduction. My name is Linda Youngman, and on behalf of the Food and Drug Administration’s Center for Veterinary Medicine, I would like to welcome you to this NARMS Meeting.
I know that some of you have traveled quite a distance to be here, and we really appreciate you taking the time out of your busy schedules to be with us today. We look forward to your comments and advice about our NARMS program.
Rear Admiral Tollefson is not here today. I am filling in for her. She sends her regrets. She was called away by the Secretary of Health and Human Services for a bioterrorism exercise. We were looking forward to having her here, and we are a bit lost without her, but we will struggle through nevertheless.
We have asked each of you here today and tomorrow because you possess individual expertise that we believe will be critical and invaluable in helping us to prepare a review of our NARMS program. Today representatives from FDA, USDA and CDC will provide you with factual background and information on the NARMS program, and then we will ask for your individual comments once you have heard some of the background about NARMS.
For many of you this will be a repeat. You already know a lot of this. For some of you it will be completely new. But for everyone we are hoping to provide an update.
The representatives who will be speaking today can provide clarification to the questions that we are asking you to consider, but they cannot actually engage in any discussion about any particular aspect of the NARMS program. This meeting is not an advisory committee meeting, and we are asking you for your individual thoughts, rather than a consensus opinion, and there is not going to be any time allotted for you to prepare a consensus opinion. So, please. If we could ask for your individual thoughts, we would be grateful.
The meeting will be transcribed so that we can use it in drafting an internal assessment of the NARMS program, and we will draft this report to give to the Science Board, which is an external advisory board that has responsibilities to the FDA. They will advise us on the direction of our science, and the Science Board will also be doing a review of our NARMS program.
We will draft an internal assessment of the NARMS program using the transcription of today’s meeting as one piece of information that we will be providing to the FDA’s Science Board. We will wait for the Science Board review to be completed and then we are planning a follow up with either a public meeting or a publication of the Science Board’s review of the program, and possibly, also including pieces of this internal assessment. A Federal Register notice of availability with requests for comments will be put in the public docket.
Now for some housekeeping details. Most of you know Aleta Sindelar. Is she here? I guess she is trying to get the booklets for you. If you need something, Aleta can help you. I know there were some questions about getting some copies made while we are here today. She will help you with those kinds of issues.
The bathrooms are down the hall. So, if you take a left and then a right where the TV screens are, the mens and ladies rooms and telephones are back there.
You should each have an agenda now. Again, we apologize for the delay in starting, but we are going to try to stick closely to the agenda and try to make up for lost time this morning. So, we are very grateful that you are here. Thank you for coming. Especially those of you who came from a long distance. We appreciate it; flying through thunderstorms.
So, without further ado, I would like to turn this over to the first speaker, Dr. David White, who is going to be giving you an introduction to our NARMS program.
DR. VOGEL: Linda, can I ask a question?
DR. YOUNGMAN: Sure.
DR. VOGEL: You mentioned that the speakers can provide clarification, but cannot enter into discussion. Can you expand on that? What do you really mean?
DR. YOUNGMAN: We are looking for individual opinions, not a consensus; from the representatives who are here to give us their individual advice today. So we don’t want to push people toward a certain conclusion. We want to hear from each of you individually.
DR. VOGEL: But we are free to question the presenters?
DR. YOUNGMAN: Yes. Yes. Oh, I’m sorry. I also forgot one other thing. That is, parking. If you parked in the garage today, the normal cost is $8.50 a day. If you punch in this code, it will be free, and I think it is the same code tomorrow, but I will let you know tomorrow for sure. If you punch in #1470, you will be able to park for free. It is covered by your attending this conference.
DR. McEWEN: I have one question as well. Are you expecting our opinions as we go? Or are we to consider things and prepare written notes to submit after we convene?
DR. YOUNGMAN: No. We are asking for your comments today. But if you look at the agenda, we are providing questions and asking for answers after several of the presenters have presented. We scheduled in time for the meeting for questions and answers after the talks.
I am sure some questions are going to come up during the talks, however, but again, the reason for this is that we don’t want a consensus and we don’t want you to ask you to draft a consensus opinion. We are asking for your individual advice.
All right. I will turn it over to Dr. Dave White. Thank you.
NARMS Background
by Dr. David G. White
DR. WHITE: Thanks, Linda. I just want to take a quick second to look at my slides, if I can.
(Pause.)
DR. WHITE: Okay. Thank you very much. I think probably too though, Scott, in terms of your question, there is a notebook that is being created right now that will have a list of certain aspects we would like to address throughout the talk. Unfortunately, you don’t have it in front of you right now, but hopefully, most of these talks will kind of steer you in the direction of questions that will probably be forthcoming from all of you folks. And hopefully, we can answer it as best as we can.
DR. McEWEN: Is it an oral exchange of information? We won’t be preparing written answers to the questions?
DR. WHITE: I believe it is oral.
Thank you very much for coming. I appreciate it as well, and I would like to take about maybe 20, 25 minutes to go over the history of this program and try to go back in time to gleam the information from numerous documents about how this program came to be, where it has been going, and hopefully, where we are headed.
(Slide)
We keep seeing more and more articles on antimicrobial resistance. Of course, we only have to go back to 1944, when Penicillin was hailed as a miracle drug and a secret weapon of the allied effort.
Of course, my former mentor’s second edition of his Book, the Antibiotic Paradox. A good reason for those of you who are interested.
(Slide)
In terms of resistance, we all know, I think, that it is a complex phenomenon. The increasing incidence of resistance has serious implications for the future treatment and prevention of infectious diseases in both animals and humans. Although much scientific information is available on this subject, many ecological aspects of the development and dissemination of antimicrobial resistance remain uncertain.
(Slide)
What is known, however, is that the emergence and dissemination of bacterial antimicrobial resistance is the result of numerous complex interactions among antimicrobials, microorganisms and the surrounding environments. A clearer understanding of these interactions is necessary if a science-based assessment of the relative risks concerning the use of antimicrobials in the animal husbandry environment is to be made.
(Slide)
In terms of our strategy for CVM, it is aimed at assessing relationships between antimicrobial use in agriculture and potential human health consequences. We have adopted a multi-pronged approach that includes a revised safety assessment process. For those of you not familiar with Guidance 152, it is a new process by which antimicrobial drugs are looked at.
We have increased our education outreach activities. In cooperation with AVMA we published a series of prudent use guidelines on antimicrobial use in different food production environments.
Participation in international activities, like Codex and OIE. Expanded research activities and enhanced surveillance activities.
(Slide)
And, of course, the one that is relevant to us today is NARMS, in terms of surveillance activities. This is what I tried to gleam from the history of NARMS. It actually goes back to 1994, 11 years ago, when FDA held a joint Veterinary Medicine and an Anti-Infective Drugs Advisory Committee to address the specific issue of approval of fluoroquinolones for use in poultry.
The committee was united in advising the agency that if the products were to be approved, several restrictions should be placed on the use of these drugs in order to attempt to minimize the public health risks related to the development of resistant bacteria in animals.
The most relevant recommendation to us today was the establishment of a nationally representative surveillance system to monitor resistance trends among both human and animal enteric bacteria. So, that was one of the recommendations from 11 years ago.
(Slide)
So, from that the National Antimicrobial Resistance Monitoring System, Enteric Bacteria or, as we affectionately call it, NARMS, became operational in 1996. Salmonella of animal and human origin was the initial organism accepted for surveillance, and NARMS combines the activities of FDA, CDC and USDA to create a nationwide monitoring system.
It was a tripartite design, development and implementation, and the three founding fathers, so to speak, were Dr. Linda Tollefson, from FDA, Dr. Fred Angulo, from CDC and Dr. Paula Fedorka-Cray, from USDA.
The sources and types of isolates have expanded over the time of the program. Campylobacter was added in 1998 and Enterococcus and E. coli in 2000, and we added the retail arm in 2002.
Each year samples are collected from numerous origins and tested to determine if there have been changes in the susceptibility/resistance of certain enteric bacteria to selected antimicrobial drugs, and the antimicrobial drugs tested annually are selected based on their importance in both human and veterinary medicine.
(Slide)
What NARMS allows the FDA to do is to monitor changes in susceptibility/resistance to drugs used in humans and food animals. Very simply, the human samples are collected from ill people. The animal samples are gathered from healthy farm animals, animal clinical specimens, carcasses of food animals at slaughter and processing plants and ground products at processing plants. The retail meat samples are collected from grocery stores and shops that sell meat and poultry to the public.
(Slide)
In terms of the goals of the NARMS program, it is to provide descriptive data and trends on antimicrobial susceptibility/resistance patterns in zoonotic foodborne pathogens and select commensal organisms. It started out initially with the pathogens, but we have since added Enterococcus and E. coli as commensal organisms.
Also, the goal is to respond to unusual or high levels of bacterial drug resistance in humans, animals and retail meats in order to contain or mitigate resistance dissemination. Also, to design follow-up epidemiology and research studies to better understand the emergence and transfer of antimicrobial resistance and assist the FDA in decision making on approving safe and effective drugs for humans and animals, as well as promote prudent and judicious use of antimicrobial drugs.
(Slide)
There are three testing sites. The human isolates are done at CDC, at the National Center for infectious Diseases, Foodborne and Diarrheal Diseases Laboratory in Atlanta, Georgia. The animal isolates are tested at USDA, ARS in Athens, Georgia in the Bacterial Epidemiology and Antimicrobial Resistance Research Unit. The retail meat isolates are tested at the Center for Veterinary Medicine Office of Research in Laurel, Maryland.
(Slide)
From 1996 to the present, NARMS has been funded through FDA via IAGs, Interagency Agreements with both USDA and CDC, and we also have interagency agreements with APHIS and FSIS, I believe, through ARS.
(Slide)
The animal, human, retail lab testing are comparable in terms of the methodologies used. We have had methods meetings in 2002 and 2003 to standardize the methods between the three arms. In the past we have had an annual NARMS meeting to present the data, but we did not have one this year due to budget limitations. We hope to have one next year. Hopefully, teamed up, possibly, with ICEID.
In terms of the susceptibility testing methods, they are all the same at all three arms. We use the Sensititre System from Trek Diagnostics. It is a broth microdilution panel for Salmonella, E. coli, Enterococcus, and hopefully, as of next week, we will be using the Campylobacter brother microdilution panel, which is going for approval this weekend at CLSI.
So, as of this year we will have all four bugs under surveillance being performed with broth microdilution. In the past Campylobacter has been done by E-test and agar dilution.
We use the same antimicrobial plate formats. We meet generally a couple of times a year to go over what antimicrobials are on the panels. We try to come to some consensus about what should be on there.
In terms of the manufactured lots, Trek manufactures their susceptibility plates in certain lots. We distributed them between the three sites so that each site has the same lots, and we try to use the same isolate handling procedures.
Quality assurance. We have both internal and external programs to monitor quality assurance of susceptibility testing. We follow CLSI/NCCLS standards where appropriate. We do have several antimicrobials which do not have CLSI quality control or interpretive criteria, and we do note that in our reports.
In 2003 we adopted the WHO External Quality Assurance System for susceptibility testing and for Salmonella serology testing I believe.
(Slide)
In terms of the bacteria tested, we do have some similarities among the sites, but each site does have its own unique isolates under surveillance. CDC has Non-Typhi Salmonella, E. coli 0157:H7, Enterococcus, Campylobacter, Salmonella Typhi, Shigella, Listeria and Vibrio.
USDA has Non-Typhi Salmonella, E. coli 0157:H7 when available, Enterococcus and Campylobacter, and the retail meats do Non-Typhi Salmonella, E. coli, Enterococcus and Campylobacter.
(Slide)
As I mentioned before with regards to the methods. The major focus of this program is susceptibility testing, and we have designed this program so that comparable testing methods are used between all three arms. We use the broth microdilution methods using the Sensititre
Semi-Automated system, and this is a picture here.
We have all started using the new ARIS systems, which have been a little problematic at the beginning. We have started to use them more, and they have a new software that will allow us to, hopefully, dump the data right into an access program for analysis.
As I mentioned, the participating NARMS laboratories adhere to the appropriate published CLS/NCCLS performance standards. Both the veterinary and the human. The M100-S15 and the M31-A2 I believe. Or, the M31-S1, which is coming out.
(Slide)
So, in terms of the methods for Salmonella and E. coli, it is tested by broth microdilution, Sensititre. The plates have changed over time for the past several years, but there are some core antimicrobials on the plates that have not changed over the years of the surveillance program.
So, the core antimicrobials that have been tested over the length of the program include: amikacin, amoxicillin-clavulanic acid, ampicillin, ceftiofur, ceftriaxone, chloramphenicol, ciprofloxacin, gentamicin, kanamycin, naldixic acid, streptomycin, tetracycline and trimethoprim-sulfamethoxazole.
Now, we did have sulfamethoxazole from 1987 to 2003. We replaced it with sulfisoxazole in 2004, as the QC is only for sulfisoxazole with CLSI/NCCLS, not sulfamethoxazole.
Ticarcillin was on one of the first plates but is geared as an anti pseudomonal. Since we didn’t test Pseudomonas, we moved it off the plate. We had apramycin on there for several years until the manufacture was discontinued by Elanco. Apramycin is no longer being manufactured. So, we removed it from our plate.
Florfenicol, we put on there as a snapshot to see if there was any changes of susceptibility of florfenicol, and we felt that chloramphenicol was suitable as a sentinel for both drugs, because most of the isolates that are Florfenicol resistant -- actually, I think almost every isolate that is florfenicol resistant is chloramphenicol resistant as well.
Imipenim we put on there again as a snapshot, and I don’t think we saw any resistance at all. So, we took it off the plate.
Cephalothin we had on there for six years, but we didn’t feel it was giving us that much information. So we removed it as well. And we added cefoxitin in its place in 2000 to pick up primarily the blaCMY cephalosporinase that we are starting to see in our Salmonella and E. coli isolates.
So, over time the dilutions have improved to include full ranges in the past. Some of our ranges do not include the appropriate quality control or the break points. All of the dilutions on our plates today have both complete quality control ranges in there, as well as the interpretive criteria for SIR.
And we use the appropriate QC organisms for testing. In the case of our Salmonella and E. coli, we used E. coli, S. Aureus, E. Faecalis and Pseudomonas Aeruginosa, depending on the drug and the range.
Enterococcus. Again, it is much like Salmonella and E. coli. It is tested by broth microdilution. We do have several core antimicrobials that have remained the same over the plate, and these include bacitracin, chloramphenicol, ciprofloxacin, erythromycin, flavomycin, gentamicin, kanamycin, lincomycin, linezolid, nitrofurantoin, penicillin, salinomycin, streptomycin, Q/D, tetracycline, tylosin, vancomycin.
A lot of these drugs you may not be familiar with because they are considered animal production drugs. Salinomycin is a coccidiostat*, and we had that on the plate between 2000 and 2003. We removed it from the plate in 2004, and we added Daptomycin once it was approved by FDA. We felt that it was important to put it on our surveillance system.
Again, the dilutions have improved over time, much like the E. coli and Salmonella plates. We have full ranges now for all of these antimicrobials, including the appropriate QC ranges, as well as the interpretative criteria.
(Slide)
So, in terms of the ‘05 Salmonella/E. coli panel, this is probably our best panel we have put together, and like I said, we have worked hard to make sure that everything is quality controlled and we have the appropriate S/I/R in there. We have several beta lactams under surveillance: ampicillin, amoxicillin/clavulanic acid, cefoxitin, ceftiofur and ceftriaxone.
We have nalidixic acid and cipofloxacin. In terms of folic acid inhibitors we have sulfisoxazole and trimethoprim/sulfamethoxazole. In aminoglycosides; we have streptomycin, kanamycin, gentamicin and amikacin. There is no QC for streptomycin by CLSI/NCCLS standards. And then we have chloramphenicol and tetracycline on the plate. And the plate designation by Trek is CMV1AGNF.
(Slide)
This is all going to be in your packet when you get it. So, you don’t need to remember these numbers. This is just to give you the transparency of th
e ranges that we employ in the testing and the break points used.
As I mentioned in streptomycin we don’t have QC. We don’t have interpretative criteria. We do use a break point of 64 µg/mL.
(Slide)
Enterococcus. Just to give you a breakdown, we do have several production drugs, as I mentioned, that are used in veterinary medicine: Bacitracin, flavomycin, lincomycin and tylosin. We do have three aminoglycosides, and these are to measure high level aminoglycoside resistance. These are all at MICs 256 or higher to strep, kan and gentamicin.
We have vancomycin on there, as well as the two newer drugs that have been approved for gram positive infections in human medicine, daptomycin and linezolid. We also have penicillin, Q/D, ciprofloxacin, nitrofurantoin, erythromycin, chloramphenicol and tetracycline for the Enterococcus plate.
(Slide)
And again, this will be in your packet when you get it to give you an idea of the ranges used and the break points employed. Again, the red asterisks show that we do not have QC for those drugs, as they are production drugs. CLSI doesn’t do quality control for production drugs, but we hope to change that over time.
(Slide)
Campylobacter surveillance was added in 1998 at USDA and CDC, and from 1998 to 2004 the E-test was employed for antimicrobials, including azithromycin, clindamycin, erythromycin, nalidixic acid, ciprofloxacin, gentamicin, chloramphenicol and tetracycline.
When the NARMS retail arm began in 2002, it tested these isolates with E-test, as well as the agar dilution CLSI approved methodologies to ciprofloxacin, doxycycline, erythromycin, gentamicin and meropenem.
(Slide)
Different methods though between the arms and the lack of reproducibility for some drugs led to the development of a new CLSI/NCCLS approved broth microdilution method for Campy in 2004 using C. jejuni ATCC 33560 as the quality control.
The QC MIC limits of the AST of Campylobacter have been accepted by the CLSI in January of ‘05, and will be published in the upcoming CLSI documents. As I mentioned, the erythromycin was a little bit out of range and is being presented, I believe, this weekend at the VAST and AST meetings.
This broth method will be much easier to use than both agar dilution and E-tests and is amenable to semi-automation and has QC ranges for 14 different antimicrobial agents for Campylobacter. So, we are very excited about this and really can’t thank enough the group at OR that was spearheaded by Dr. Pat McDermott, Dr. Bob Walker and Sonya Bodeis for helping to champion this system through.
So, as soon as we are ready to go with Campylobacter this year; we will use the broth microdilution method. So, we are ready to jump in there and see what we get.
(Slide)
These are the antimicrobials that are going to be on the new panel, and we have several macrolides and lincosamides: azithormycin, erythromycin and clindamycin. We have nalidixic acid and ciprofloxacin. We added a new Ketolide, telithromycin, as well, and we have others on there: florfenicol, gentamicin and tetracycline. So, we will have nine antimicrobials on the Campy plate.
(Slide)
These are the ranges that are going to be employed, and in terms of the breakpoints, this is all kind of based on populations since there is no interpretative criteria for Campylobacter for CLSI or NCCLS. We have been using these types of breakpoints, and you will notice we don’t have
breakpoints for telithromycin or florfenicol at this point.
(Slide)
Other methods used by NARMS. We do have our Salmonella and Campylobacter isolates characterized by Pulsed Field Gel Electrophoresis, and to give you some background, Dr. Zhao will talk about this later on.
Genomic DNA is cut with an enzyme and resulting fragments of different sizes can indicate mutations or insertions in the genomes. Whereas PulseNet is an international program -- well, it is national and international now. It assists investigations and improves outbreak detection through rapid linking of cases by comparing bacterial DNA fingerprinting patterns. But a lot of our isolates are indeed now put into PulseNet.
(Slide)
Other capabilities, of course, were warranted. We all have molecular capabilities. All three arms have molecular capabilities to investigate emerging phenotypes and go after the appropriate genotypes. Here are just some examples of some PCRs that we have done looking at resistance genes for high level aminoglycoside resistance and then also we have several programs involved in looking at integrons among bacteria. So, all three arms are able to do this.
(Slide)
So again, just kind of summarizing everything up, this is the way NARMS is designed right now. There are three arms, the USDA, CDC and CVM sides.
(Slide)
We all have annual reports. They are all linked on the CVM home page, but USDA and CDC have their own home pages as well for NARMS, and you can access all three of them on our web link up there. These are just covers of the annual reports for the different sites.
(Slide)
So, in conclusion, we need to remember that NARMS is a public health surveillance system. It will enable informed decision making by FDA, initiated response activities if data warrants. We hope it promotes prudent and judicious use of antimicrobials and will help guide prescription practices.
We hope it encourages standardization of laboratory techniques, identify areas for more detailed investigation, promote collaboration between the three different government agencies involved, as well as academia. And ultimately, prolong the efficacy and useful life of antimicrobials.
With that, I thank you very much. Are we going to take questions now?
DR. YOUNGMAN: We can take questions.
DR. VOGEL: David, you mentioned that Campylobacter has gone to the broth microdilution. Two questions regarding that. Number one, how is the results of that comparable to the E-test? Is that going to affect your trend analysis or not?
And secondly, how does that affect the ability for your workload, your sample throughput? Will that increase your ability to do additional sampling for Campylobacter?
DR. WHITE: Thanks, Lyle. Those are good questions. I will answer the second one first. The broth dilution method is much easier than the E-test and agar dilution methods. So, if anything, it will shorten the time it takes to do susceptibility testing on those organisms.
Allowing us to do more is all dependent on the budget and everything else. You know, what we can collect for isolates. The numbers we get now through retail meats -- and I am just speaking for myself; I can’t talk for Tom or Paula. We get about now 600 to 800, and it takes a good six to eight months to do that by agar dilution.
This should probably drop us down to maybe two to three months once we are up and running. So, we are really excited about that.
The first question, in terms of the comparison to E-test, E-test is not a CLSI endorsed method. So, if anything, you need to take the E-test data and compare it to the broth, not the other way around.
We have done some studies. We have done both in the past comparing agar dilution and E-test at the same time, and that is probably because of our research laboratory and Dr. Walker’s expertise. So, we have that data to show comparability for some drugs, but that is why we went to broth, because the E-test was not giving reproducible results for several drugs, whereas the broth microdilution method is a reproducible standardized method.
D
oes that help? Okay. Dan?
DR. SAHM: Thanks for the overview, David. The question that I would like to ask has to do with the determination -- the two key resistance mechanisms to extended cephalosporins that are being seen in enterics are extended spectrum, beta lactams and AMP C mutations or AMP C acquisition.
The selection of cephalosporins you have on your gram negative panel wouldn’t be the most sensitive set of cephalosporins to help detect ESBLs emerging, and I was wondering how that data is looked at, given the cephalosporins that you use as a sentinel for potential ESBL emergence in your study organisms.
DR. WHITE: Thanks, Dan. That is a great question. Actually, we have -- that is a very good point. A lot of ours are just kind of screening. They may be adequate for 10 beta lactams and so forth, but the ESBLs -- we have actually created secondary plates. I didn’t have a chance to talk about it.
We actually have a secondary ESBL plate that has on there cefotaxime*, cefotaxime/clavulanic acid, ceftazidime, ceftazidime/clavulanic acid, cefepime* We have also added Cefquinome*, which is a fourth generation veterinary cephalosporin not approved in the United States. So, we further test anything that has a red flag for Cefoxitin. We kind of take off and put on this ESBL plate and we look for the definite, the three-fold reduction, and then further on we try to characterize from there.
We actually have also a supplementary fluoroquinolone panel as well. So, anything that is nalidixic acid resistant, we then put onto this plate that has on there marbofloxacin*, danofloxacin, enrofloxacin, levofloxacin, ciprofloxacin and so forth.
So, we try to -- you know, these are the second tier plates. So once we get a red flag on a certain phenotype we go to that. I apologize for not showing that. I will try to get that for everybody for tomorrow.
DR. SAHM: Just as a follow onto that, for instance, in the human isolates one of the more sensitive cephalosporins, and the flag for that is any MIC greater than or equal to one or two I believe. I can’t remember.
DR. WHITE: Right.
DR. SAHM: Because of a large number of TEMS that won’t show up with this as well. So, you might want to think about that flagging system.
DR. WHITE: Okay. Thank you. Good point.
DR. ALTEKRUSE: Is that being done in all three arms of NARSM?
DR. WHITE: Those plates are available to all three arms. I think right now CVM has probably done the most of that with that plate, but we had help with CDC. So, all three arms use the plates.
We don’t see much though for ESBLs. At least with the animal isolates and the retail isolates. I think we have seen one ESBL E. coli that fits the classic definition.
DR. KOTARSKI: I have two points to make. The first point is not necessarily a question, and maybe we will come back to this later. The objectives for the program that you have in your first slides don’t quite match up with what we received in our packages, and they are not necessarily the same as what is on the individual websites.
And so, as we go forward in the program, I would like to get some confirmation as to specifically what the objectives are. That might be helpful for the panel in addressing the questions that we have outlined for the afternoon. So, just a comment.
The second is a question. Quickly, what do you use to establish -- what criteria do you use to establish breakpoints for Campylobacter as you work through?
DR. WHITE: That is a great question. I will let Dr. Walker answer that question.
DR. WALKER: CLSI has a working group called, for lack of a better term, Orphan Organism, and they have been meeting -- we have been meeting for the last two years. And one of the things that we are doing in that group is to take infrequently isolated organisms and try to establish testing methods and QC and interpretative criteria with Campylobacter, because we have already gone through CLSI to establish the testing methods and the QC ranges.
What we are going to do, like Dave said, at this meeting come up this weekend and this next week, is establish interpretative criteria for Campylobacter for some drugs. So, CLSI is addressing this. They are doing it off of population distributions for the most part, and I would suspect that come Tuesday afternoon there should be some recommendations as to interpretative criteria for some drugs for Campylobacter.
DR. KOTARSKI: So then basically, it is a screening mechanism for increased MICs relative to the general population, what you would expect? It is not based on therapeutic failures or anything?
DR. WALKER: Yes. And for those who are familiar with CLSI, you know that to -- generally what they do to generate interpretive criteria is to look at population distributions, look at pharmacokinetics, pharmacodynamics and look at the results of clinical trials.
And then those interpretive criteria go into books that are referred to as standards books. For this particular bunch of organisms they are going to look at population distributions, PK/PD where they have it, but obviously the clinical trial is not going to be available.
So, this document will be called the M45, and it will be a guidance document. It will not be a standards document.
DR. WHITE: Great. Thank you very much.
DR. MILLER: One last question. You mentioned C. jejuni for the microdilution new techniques. Are you also moving in that direction for C. coli?
DR. WHITE: It is for both. All Campylobacter.
DR. ANGULO: Dave, just one clarification. CDC started Campylobacter in 1997, and thanks for highlighting the important innovation for having the broth microdilution for Campylobacter because that is really essential for us.
You will hear more, but we have changed the number of samples we are receiving. And that is because we are able to test more samples because we are more efficient at testing what manner. So, thanks for highlighting that.
DR. ALTEKRUSE: You mentioned Listeria being done I think at CDC. Is there any evidence from that that perhaps other arms should also be looking at Listeria?
DR. WHITE: No. I think every once in a while we take a snapshot of something and then we evaluate within our groups and we see. It is kind of like imipenem. We put imipenem on there for a year. We didn’t see any elevated MICs. I don’t think any of us did. And so we said, okay, let’s yank it off.
Remember, we have 96 wells. We are limited to 96 wells. Three of those are control wells. So, we are stuck with 93. So any time we tweak something, we have to take something off.
That is why we got rid of Cephalothin*, primarily because we didn’t think it was giving us information, and with those several wells that we had we were able to improve the QC ranges and cover all the ranges for all of the other drugs on the plate.
DR. ALTEKRUSE: Okay. Thanks.
DR. YOUNGMAN: Before I introduce the next speaker, I just wanted to point out that while our panelists have microphones at their desks, for the rest of you there is a microphone which is live all the time that is here in the center of the room. Please use it. That helps our transcriber in making sure we accurately reflect your questions and comments. Thank you.
If I could, I will introduce Dr. Robert Walker, who is going to be talking about the NARMS budget.
Budget Issues
by Dr. Robert Walker
DR. WALKER: Good morning. As Linda indicated,
Dr. Tollefson has been called away for other activities, and she was originally scheduled to discuss the budget. So, what you are going to hear today is the budget from my perspective, which is kind of like looking at the universe through a pinhole.
(Slide)
With that said, what I would like to do is to talk to you not so much about the specifics of the budget, but more of the mechanisms of how the NARMS budget is distributed. When the money comes into CVM, CVM generates IAGs, Interagency Agreements, with the two participating laboratories, CDC and USDA.
The monies that are allocated in these IAGs are for personnel, supplies, equipment and overhead. When we initiate get the IAGs, they may be broken down to a specific amount for personnel, a specific amount for overhead, et cetera. But once the IAGs are in place, the agencies, the participating laboratories, can redistribute that money however they need to, as long as the monies are used to generate NARMS related work.
In other words, if we gave $800,000 for personnel and they found that they had an increased need for personnel, they could take monies from equipment or some other source and add it to the personnel part of the budget. The only requirement is that however the money is spent, it is spent on NARMS-related activities.
Additional dollars that may go into the NARMS monies the labs receive is through supplies used for susceptibility testing, and Dave talked about this earlier. What we have found is, in order to increase the uniformity in the susceptibility testing work that we do, if we order a large batch of plates, we get a cost break because of the large volume that we order.
We then take that volume of plates and divide it amongst the three labs so that everybody is using the same lot numbers. We may have to do that several times throughout the year, but each time the lots are distributed throughout the three labs based on their needs.
(Slide)
So, just to give you an idea of how the mechanics of this works, if we look at the USDA budget, in the IAG they had about $800,000 for personnel. This person was a CVM person who was working at USDA to provide whatever assistance she could. This represents the central supplies, the cost of plates and other supplies relating to testing those plates. That comes out of CVM’s budget.
And then again, the different breakdown into the different types of testing that they are doing and the money that was allocated for that, for a total of a little over $2 million. And again, the specifics on Campy testing or Salmonella PFGE, that is all controlled internally by Paula and her group.
(Slide)
If we look at the CDC, it is kind of the same type of system where we have personnel monies, the CVM staff at CDC, central supplies. CDC is a little bit different than USDA in that as part of the retail meat program, in order for us to gain access to the isolates from the retail meats, CDC has contracted with 10 FoodNet sites to go out into the community -- Terry Proescholdt will talk about this later on -- and purchase meats, bring them back to their individual laboratories where they will isolate and identify the specific organisms. These isolates are then sent to CVM where we confirm the identification their identification. Once the identity is confirmed the isolates are subjected to susceptibility testing and whatever genetic analysis needs to be done.
CVM is not in a position to contract with these FoodNet sites or these state public health labs to go out and make these purchases. So, CDC has contracted with them. And the money -- approximately $500,000 goes from CVM into CDC for CDC to distribute it to the state public health labs that are participating in the retail meat study.
(Slide)
For CVM, again it is broken down basically the same way. And for those of you who are doing the math you will realize that this number down here is not correct This really should be $1,190,885. You might want to make a note on that for your handouts when you get them.
(Slide)
So , when you look at the expendable monies, and what we are talking about is the monies that are expendable from NARMS for the research or the surveillance program, this is how it breaks down for the animal arm, the human arm and the retail meat arm of NARMS. If you look at this, plus this, this would be what CDC gets. But keep in mind that not all of this money actually goes to CDC. It passes through CDC to the state labs.
This is the amount, $1,190,000 is what goes into the retail meat arm.. An additional $100,000 of NARMS monies goes to the WHO Global Salmonella Surveillance System. This value then comes out to be 5,446,000 for those of you who like to keep track of numbers.
Keep in mind that this is the mechanisms by which the NARMS money comes into CVM and then is distributed to the other arms. The specifics, in terms of the exact numbers, I think at this point in time this is not the issue. The issue is the mechanism by which the money is distributed and the amount of expendable money that is available. Are there any questions?
DR. ANGULO: In the opening slide you mentioned discretion of the agencies to move money, but we don’t -- we have to get permission. Well, CVM gives us permission to move money, but our finance office won’t allow us to move money, because we follow the interagency agreement as is given to us. So, we have to keep the money in the categories where it is coded to us when it comes to us. Unless we get an email or some written documentation from CVM for permission to move the money between.
We can’t move supply money to personnel money, et cetera. So, we have flexibility, but we have to get it in writing from CVM to make the changes and keep records of those changes.
DR. WALKER: Yes. That is an internal CDC thing, but I think you have found that if you communicate with us and ask for permission, it is given without any problem.
DR. ANGULO: That is right. We have always had permission to move, but we document the movement.
DR. WALKER: Okay. Yes, Paula.
DR. FEDORKA-CRAY: Bob, I think it is important to point out that those figures --
MS. SINDELAR: Can I make a comment here? For the purpose of transcription, for her ease, that was Dr. Fred Angulo, from CDC. And, Paula Cray. So, please identify yourself prior to your comments. Thank you.
DR. FEDORKA-CRAY: This is Dr. Paula Cray, from the U.S. Department of Agriculture. For the purpose of the interagency agreements, the interagency agreements do not reflect the numbers that have been shown on the slides, nor have these numbers been presented to at least the animal arm of NARMS in this particular fashion at any time during the past 11 years.
The inclusion of personnel and -- from CVM, Marcia Headrick was the NARMS coordinator. Our understanding is that she was not a directly -- directly placed to work exclusively with USDA. She coordinated, until very recently, all of the activities between CDC, FDA and USDA, and I think it would be inappropriate to count her salary toward a USDA figure.
In addition, the monies that have been expended on supplies, it was discussed early on in the program what the needs were with regard to the number of isolates that would be available, and it was always recognized that because of the -- particularly the slaughter and the diagnostic sources of isolates, that there would be an increased need for additional plates.
However, what the agency does not see is that in any given year we, between the three agencies, routinely trade or pass plates. We provided CDC with over 1,000 plates at one time for Salmonella. They provided us with 600 for Enterococci.
These numbers have gone back and forth over the year, in addition to supplies, and the supplies that FDA provides to us are strictly limited to Trek Diagnostic supplies, not other expendables that must go into the system, like gloves and tubes and gases and other media that are going to be required for the program. So, I think that there needs to be some additional detail that is reflected in order to accurately assess the amount of monies that each arm receives.
DR. WALKER: And I take what you say well. What I wanted to do is talk more about the mechanics. I am not involved in the budget. I am not in a position to go into the exact specifics on the budget.
You would notice that there was $67,500 listed for USDA for other supplies, and I recognize that the central supplies were Trek related supplies monies.
DR. YOUNGMAN: If I can just clarify a couple of things that you said though, Paula. The monies that were presented here, the budget figures you saw here, were for 2005 only. That is why you haven’t seen those exact numbers before. We are just talking about this year’s money.
We can never be certain of our exact budget because our FDA budget changes from year to year, and we have to work with the budget constraints we are given.
And also, to clarify about the CVM staff, the dollar figure that was put up there for a CVM staffer at USDA, we also supply a CVM staffer at CDC, and those monies do not come out of your IAG. Those monies are paid for separately out of FDA monies toward NARMS.
DR. FEDORKA-CRAY: This is Paula Cray again. I think that then that there needs to be a clarification made on those tables if the experts are looking at those figures and they don’t have an accurate assessment.
The interagency agreement that USDA received was $1.5 and 6/7 million for 2005, not $2 million that is reflected in the figures that were presented to you.
DR. WALKER: Right. And most of those figures that are on that page came directly from the IAG. The ones that would be the exception would be the $140,000 for Marcia and then the central supply monies would be different.
DR. MILLER: Marissa Miller. Bob, is there a special budgetary line item or appropriation for NARMS for FDA? And if not, how does CVM protect that money to make sure? I mean, a $5 million total is quite large part of the overall CVM budget I would think.
DR. WALKER: Well, I wish Linda was here to give you more information on that. I am not in the budget channel. My area is in the science. I’m not sure if this is a direct line.
DR. YOUNGMAN: I apologize that Linda Tollefson is not here. She might be able to answer your question. In actual fact, she is really the one who could answer questions about the NARMS budget. So, Bob and I are trying to cover as best we can for her today.
DR. WALKER: Based on the things that I hear it may be a line item or at least it is specifically discussed by Congress. But I don’t know the politics of how it channels down through the budgeting process.
DR. MILLER: Do you happen to know? Have you ever had a GAO audit and what the results were from that?
DR. WALKER: Not as far as I know. We have not had a GAO audit.
DR. McEWEN: Scott McEwen. Are there other sort of in-kind contributions that help to sustain and support NARMS that don’t appear in the budget?
DR. WALKER: Are there other sources of funding that can be fed into it?
DR. McEWEN: Well, funding or in-kind contributions from the different agencies. Salaries of people I guess that don’t show up in the budget that participate in NARMS or other stakeholder groups that helps furnish samples. That kind of thing. I am just trying to get a feel for other activities that support NARMS that may not appear in the financial statement.
DR. WALKER: I don’t know what CDC and USDA does. I am sure that they have peripheral activities that all contribute to it. I know at CVM we have a number of people that work directly, and indirectly, with the NARMS program doing things like Dave talked about, the Pulsed-Field Gel Electrophoresis that are not figured into the NARMS budget.
In other words, we don’t have anybody at OR that is directly involved with NARMS who is doing Pulsed-Field Gel Electrophoresis, but we do have an OR team that is involved with Pulsed-Field Gel Electrophoresis that deal with a lot of NARMS isolates.
DR. ALTEKRUSE: On that point, one example of -- and Dr. Kotarski brought this up. One example of an in-kind contribution would be that both APHIS and FSIS isolate Salmonella and provide them to the USDA/ARS laboratory for the characterization. So, that is a fairly substantial
in-kind contribution.
DR. WALKER: Yes, Fred.
DR. ANGULO: We all have in-kind additional support. At CDC it is on the scale of -- about matching is the word that we get from the interagency agreement. So, of all our NARMS activities, which include everything from education to core surveillance to applied epidemiology studies, et cetera, that are the total resources that are contributed to animal resistance testing and susceptibility testing, the interagency agreement provides about half of all of our resources.
DR. WALKER: Okay. Yes, Lyle.
DR. VOGEL: Mostly just a comment. I wish Linda Tollefson or Steve Sundlof were here to address the budget issues and questions, because one of our discussion questions was how do we suggest that we enhance or sustain funding for NARMS.
I think it would be important for us to know that if, for example, we went to Congress and was able to either create a line item for NARMS, if that would be acceptable to the agency. If not a line item, if we got increased funding specified for NARMS, would FDA use it for NARMS or would they divert to something else?
DR. WALKER: I think those are good points.
DR. FEDORKA-CRAY: This is Paula Cray again. I just want to go on record, like Dr. Angulo, that the Agriculture Research Service and USDA, including, like Sean Altekruse said, APHIS and FSIS, would spend well in excess of what receive for the IAG if we looked at the isolation and receipt of all of the isolates and what it would cost.
In addition, we also have directed appropriations from within our unit and a number of permanent staff who are directly associated with NARMS.
DR. WALKER: Okay.
DR. MILLER: Bob, just looking at the possibility of any redundancies, at one time CVM was talking to the sponsors about doing post-marketing surveillance, either helping to pay for NARMS or doing their own post-marketing surveillance. Particularly of the new fluoroquinolone approvals.
Was that ever set up and are there data available from any post-marketing activities?
Also, at one point CDER was looking to set up a resistance monitoring, sort of a clinical failures sort of database, and has that been done?
DR. WALKER: Dealing with the CDER question first, as far as I know, CDER has not done, and it is probably primarily due to budgets. There also are two excellent surveillance systems in human medicine that may be able to address those issues. One is Focus Technologies and the other is Sentry, which are very extensive surveillance monitoring systems that deal with hospitals throughout the country or throughout the world.
In terms of post-marketing for CVM, there is not a specific post-marketing program that has been set up. The surveillance system that they have established is NARMS and NARMS deals with the pre-marketing and the post-marketing, to the pre-approval and the post-approval. In other words,we would like to think that the data we generate is used by the pharmaceutical industry to help them in their drug approval process.
In fact, we had a VMAC Meeting this last year where one of the drug companies actually used some NARMS data to fill in some information for the Guidance 152 Document.
What we are seeing in terms of susceptibility isolates or susceptibility profiles on these organisms coming from retail meats, coming from zoonotic foodborne pathogens would represent a post-marketing approval for drugs that are being used in veterinary medicine, and we are seeing changes in susceptibility in the zoonotic foodborne pathogens.
DR. ANGULO: I think one of the important background pieces that I think we should provide is that NARMS really went through a remarkable growth spurt. I mean, we grew to this budget, our current budget approaching $2 million in this five-year window and had rapid increases, and that was because resources were provided to FDA. Actually, the lead agency is CFSAN through the President’s -- at that time it was the President’s Food Safety Initiative.
President Clinton had a five-year Food Safety Initiative which brought important new resources to the government. The first year award, for example, was $36 million and that money then got allocated to the different agencies that work in food safety, which was largely the Center for Food Safety and Applied Nutrition at FDA. But a piece of that went to CVM for NARMS, and almost all of the CVM food safety allocated money has been used to support NARMS.
A piece actually went to CDC to support surveillance activities and food safety, which was FoodNet and PulseNet, which were largely funded through the Food Safety Initiative, and each year the Food Safety Initiative during this five-year period grew and had more resources. And therefore, the CVM slice of the Food safety Initiative grew and NARMS resources grew.
That initiative is no longer active. In fact, this year we see a decline in food safety resources
government-wide and it is quite marked actually at CFSAN because CFSAN had the biggest piece of the Food Safety Initiative. It was also quite marked at CDC in our other activities related to food safety.
And also, as expected, we have a decline this year in CVM. In fact, every year that there is not an increase there is a decline because we -- I hope people noticed on the breakdown that by far our biggest part of the budget, of course, is personnel and personnel have a seven percent increase every year. So, if we don’t have an increase in the budget, in fact, it results in a decline.
So this year, the fact that we had a decline in the order of five or eight percent each, actually represents a 15 percent decline in our budget because of the increases in personnel. All that is to say it is just a different climate in atmosphere in the government in terms of resources and all programs are being reduced, and we can clearly anticipate, in fiscal year ‘06, a decline in resources in food safety and a decline, therefore, in NARMS.
I would expect that we would anticipate a
10-percent decline in our real resources, and we are wrestling with how to deal with that and I am sure other agencies are wrestling how to deal with that too. But there is not anything on the horizon that we are aware of that would replace these funds.
There is not new government initiatives in food safety, there is not a government-wide initiative on antibiotic resistance, which we have been trying to -- various groups have been trying to do. But there is not a new initiative that would bring new resources for antibiotic resistance. I think it is just the new reality that we can expect continued declines in the next five-year cycle.
DR. WALKER: I appreciate what you are saying, Fred. I think, with that in mind and we look at the 2006 and 2007 budget, while we don’t know what those budgets are going to be at this point in time, I think that we can safely assume that they will either be the same or a decrease.
And if they are the same, then just the cost of personnel is going to be a decrease to all of us. So I think what it means is those of us who are involved in this work really need to look at the work that we are doing.
And going back to Lyle’s question of how can we sustain or enhance the funding, I think it is looking at the quality of the data that we are generating and making sure that all of that data that we generate maximizes the potential for human health care, and there are a number of ways of doing it.
One of the things that you will hear in this next couple of days is the reporting system that is going to be coming forth from the three different arms. While it is important to generate data, it is also important to be able to report the data and do so in such a way so as to increase the visibility and the utility of the NARMS program to the country.
DR. ANGULO: I agree complete. And just to emphasize -- it gets exactly at Sue Kotarski’s point, which is to reassess the goals of NARMS and what are the real core functions, because the reality is we can expect these really steep cuts.
I have seen the pass through budget on the Food Safety Initiative for next year, and we are all anticipating important cuts in the food safety. You are optimistic to expect equal funding. Every scenario that we have worked on is anticipating deep cuts in food safety next year.
DR. WALKER: Yes, Dan.
DR. SAHM: Just a follow up on this topic and a point that Marissa brought up and then a question. On the human side, as part of a new drug application, CDER does want to see the sponsor’s commitment to post-marketing surveillance, and it is on their dime to see how their drug continues to perform once it is being used in the clinical environment. So, the precedent is set there on the human side.
So my question is how difficult or how much of an uphill climb would it be for CVM to have that same expectation for clients who are applying -- for sponsors applying for new drugs used in the animal industry?
DR. WALKER: Well, that is a good question and that is a very complex question. There are a couple of major differences between human medicine and veterinary medicine, in terms of the microbiology.
The first is that all of the human laboratories, or at least theoretically all of the human laboratories generating this information, do so under fairly regulated quality control testing methods, and they are tested I think twice yearly to assure that they are adhering to appropriate standard, quality control and testing methods.
One of the things that facilitates that is as a drug in human medicine moves through the approval process, one of the very first things that they do is they go to a contract laboratory and ask that laboratory to generate testing methods and quality control ranges for their drugs. So then, when they go into the clinical phase of their durug testing all of the isolates can be tested under standardized testing methods. From this the sponsor can establish a baseline data representing the pathogens susceptibility to their drug at the time of approval.
In veterinary medicine a pharmaceutical company does not have to go through quality control testing prior to the drug approval. In other words, a lot of times sponsors will present drugs packages to CVM where they have generated clinical data, but it was generated without the benefit of QC organisms and quality control testing. Without the benefit of appropriate testing methods, including the use of QC organisms and QC ranges the sponsors have no point of reference once their drug is approved.
The other thing is that a lot of times when drugs
gets approved in veterinary medicine, they do not have to go to CLSI for QC testing or interpretative criteria. The pharmaceutical company can provide values to the laboratories that they have decided for QC range should and interpretive criteria without the benefit of a appropriate testing procedures but rather based on marketing strategies.
The other thing that comes into play here is that the veterinary diagnostic laboratories, or the laboratories doing the isolation, identification and susceptibility testing for veterinary isolates, while many of them may be laboratories associated with human medicine, the pathogens they encounter from animal sources are going to be different than what they normally see. On the other hand, the majority of the laboratories processing specimens from animals are not human clinical laboratories and as such they do not use standardized testing methods including an appropriate quality control program that monitors the quality of data generated by those laboratories.
So again, we are dealing with, in human medicine, microbiological procedures that are fairly tightly controlled and regulated. In veterinary medicine we haven’t reached that stage of perfection yet. So I think it would be difficult for the pharmaceutical companies to generate the type of information that you are talking about.
I think it could be done if there was a concentrated effort to get this ball rolling. Probably within five years. But I don’t see the concentrated effort being made to do that.
DR. SAHM: Thanks. That helps understand, Bob. Just as a follow up to that, the guidance for industry from the FDA on the human side didn’t always require this either. In fact, one of the major sections for the NDA in microbiology now is how well does your drug perform according to standard methodology?
It all rolls together, as you have pointed out. So you can’t do one without first expecting that the clients or the sponsors or whatever, when they submit a drug -- do you have a good way of testing in the clinical laboratory? Maybe the analogy would be do you have a good way of diagnostic veterinary laboratories testing your drug in the real world?
It does go hand-in-hand. I understand what you are saying.
DR. WALKER: I have looked at some packages that come in where the pharmaceutical company says that they have done the testing in accordance with CLSI. And so, my question would be what QC organisms did you use, how often did you use them and what was your dilution range?
And when I get the information back, I find that they used the four standard QC organisms, but two of them had no QC range for the drug that was being tested. One of them had a QC range below the dilution range that was tested. The other one had a QC range above the dilution range that was tested.
So, in actual fact, they were using the CLSI QC organisms, and as such said they were following CLSI guidelines, but in actual fact, there was no QC there. This is a problem.
When I was in the vet diagnostic arena, that was one of the things that we tried to do; was to establish a quality control testing method among the participating diagnostic laboratories. We tried to get all of them to buy into the CLSI testing procedures.
There are two other problems that we have in relation to this. One is the veterinary pharmaceutical industry is somewhat reluctant, and perhaps rightfully so, to go public with their drug too early in the process because that gives their competitors an idea as to what is coming out.
The marketing in veterinary medicine is very important.-- I have heard that in human medicine they won’t even consider an anti-infective unless it is going to make $1 million a day. That is $365 million a year. Veterinary medicine, a good product may make -- and Sue can tell me if I am wrong. It may make $100 million a year. Considerably less than anything that is a poor drug in human medicine.
The other thing is that if a sample goes into a human diagnostic lab or a human clinical micro lab for culture and susceptibility, I would imagine it could cost upward of $75 or more. I know of some vet diagnostic labs that charge nothing because the money is just not there.
I know when I was in that arena I charged $25, and when I raised the price to $28, you could almost hear the "ah," but it was just to do the quality of testing that I thought we needed to do.
Okay. Thank you.
DR. YOUNGMAN: Thank you very much, Dr. Walker. The next thing on our agenda is a 15-minute break. We are going to try to keep on time as best we can, but I imagine we can’t really do a 10-minute. So, if we could come back in 15 minutes, at about 10:35 or thereabouts, please, and we will start again. Thank you.
(Whereupon, at 10:21 a.m., a recess was taken.)
DR. YOUNGMAN: In the interest of trying to keep on time, if we could start again, please. I just want to remind everyone that the main purpose of our meeting today and tomorrow is to try and address the questions that are in the booklets that you have been provided that are headed with discussion points.
So, as the talks are occurring today, if you could consider those questions, those are the things that we would really like to hear your individual responses to. And if we could keep the focus on that, that would be really, really helpful.
Our next speaker is Dr. David White, who is going to be talking about the Retail Meat Arm of NARMS.
DR. McEWEN: In a quick leaf through here, I am not sure the questions are in here.
(Pause.)
DR. YOUNGMAN: I’m sorry for the confusion. There were two different booklets that were passed out. The panelists’ booklets do not have the discussion points in the front. The booklet that I got does have the discussion points in the front. We will make sure that the panelists get copies of the discussion points.
(Pause.)
DR. YOUNGMAN: I apologize. We will make sure that the panelists get copies of the discussion points. They were in the larger booklets that I had seen that the participants got. I apologize for that, but we will make sure that you get them.
DR. McEWEN: One other procedural point. Do you suggest going through these question by question at some point? You know, the panel member offering their perspective? Or do you see us interjecting as we go along?
DR. YOUNGMAN: Well, if you check the agenda, those are points that are going to come up later in the day. Each of the questions that are part of the discussion points will be raised later in the day.
Okay. And if I can, I will turn it over to Dr. White.
DR. CHILLER: Dave, I just wanted to say one thing. Tom Chiller. I passed out to each of you guys CDC’s external review that was conducted last year. It has the reviewer’s summary comments to the different questions we addressed.
Also, highlighted is sort of CDC’s response.
It is just an internal document that we put together, but I thought it might be helpful at least this evening, when you are sort of digesting all of this information, to see what we had done last year. Obviously I will be talking about that in just a couple of talks from now too.
Retail Meat Arm
by Dr. David White
DR. WHITE: I am going to take, hopefully, the next 20 to 25 minutes to talk about my real job. I am a team leader for the NARMS retail part, and I would like to share with you how it started, the history of it and some pre-work we did before we went into the NARMS to become the third arm and end with a little of data.
(Slide)
Of course, as background, the food supply, including meat and poultry, is an important source of enteric bacteria, including Salmonella, Campylobacter, E. coli and possibly enterococci. I think the debate is still on enterococci coming from foods as pathogens.
Antimicrobial resistance among these foodborne bacteria is not uncommon and is often associated with the use of antimicrobial agents in food animals. Retail food represents a point of exposure close to the consumer, and when combined with data from slaughter plants and on-farm studies, may provide an indication of the prevalence of resistance in foodborne pathogens.
(Slide)
So, I would like to start with the history of microbiological surveys, focusing on animal derived meats from CVM’s perspective, and our primary objective has always been to characterize antimicrobial resistance among isolated from retail meats of animal origin in meat and poultry.
I will talk about three studies in particular. The first one was our collaborations with the University of Maryland. In particular, Dr. Jianghong Meng, looking at the greater Washington, D.C. area as our sampling areas.
We will talk a little bit about the Iowa project, which was our pilot project for NARMS retail, and then how we expanding into retail meats as part of the NARMS program.
(Slide)
So, we started off with back in 1998/1999, partnering with Dr. Jianghong Meng, and we initiated a study looking at retail raw meats in the greater Washington, D.C. area. We tested 825 samples of retail raw meats for E. coli and Salmonella, 719 for Campy and these were random samples taken from 59 stores, published in the Applied Environmental Microbiology in 2001.
Of course, one of the things that catches your eye, in terms of poultry, we had 71 percent positive for Campylobacter. So, it was one of the first indications that Campylobacter was highly prevalent in chicken breast. Now, this is pre-HACCP. Okay? These were samples pre-HACCP.
(Slide)
We followed up with another study with Dr. Jianghong Meng that we were fortunate enough to publish in the New England Journal of Medicine looking at ground meat samples from the same area, and we looked at chicken, beef, poultry and pork, and these were ground samples. We were able to go back and find out that there were four poultry processes, one pork processor and beef was ground in the store.
Again, we found Salmonella. In this case from 20 percent of the ground samples. We found a variety of serotypes, most frequently isolated from poultry meats, and we found eight isolates of Typhimurium. The majority of those were DT104.
(Slide)
And just quickly. I don’t know if we can see this. This is what caught our interest. When we looked at those Salmonella isolates we did recover from the ground meats, we found the multi drug resistant to several antimicrobials. In particular, S. agona. We saw a lot of beta lactam resistance, strep-sul-tet and trimethoprim. We did find some DT208s which were resistant to about nine to 12 drugs. Very interesting.
These appear to be all plasmid mediated as well.
(Slide)
So, these two studies led us to think that we needed to investigate further the role of retail meats as reservoirs of resistance, and we initiated a study called the Iowa Retail Meat Pilot Study in 2001 and 2002, and then we segued it into NARMS, the FoodNet -- I will go over the FoodNet Retail Meat Surveillance. The first year was 2002.
(Slide)
In terms of the Iowa Study, this was started in 2001 and goes into 2002. We went to 24 samples a week, and
Dr. Terry Proescholdt will talk about sampling later on and how we developed the study.
But we picked four meats, much like we had done in the other studies in D.C. Ground turkey, pork chops, ground beef and chicken breasts; 24 samples a week. They went to six grocery stores and they picked four meats of each for 24 samples per week.
And ideally, what we were trying to determine was the prevalence and susceptibility patterns of Enterococcus, Salmonella, Campylobacter and suspect extended spectrum beta lactamase producing gram negative bacteria. This goes back to Dan’s questions.
As part of our study we are trying to determine if we could find ESBLs on foods, and we did a whole screen with ceftazidime and cefotaxime with one microgram per ml. The Salmonella and enterococcal plates were -- we used the NARMS plates to determine susceptibility, and for Campylobacter we used agar dilution and E-test methods.
(Slide)
So, I will just give you some brief data. The studies I mentioned went on for 15 months. We had 24 samples per week. We ended up sampling about 263 grocery stores; 209 for Campylobacter. It took us several months to optimize the Campylobacter methods. Overall though we had almost 1,000 meats analyzed, and as I mentioned, we looked for Salmonella, Campy, Enterococcus and ESBL-producing gram negative bacteria.
Susceptibility testing was done using the NARMS panel or agar dilution. All Campylobacter and Salmonella were subjected to Pulsed Field Gel Electrophoresis as well using two enzymes, and we just finished and it has taken us two years to do that. For those of you that do pulsed field, doing one enzyme is hard enough. When you have to do two enzymes, you double your amount of work.
We were also able to get the human isolates from that same time from the State of Iowa. So we were able to have comparisons from what we recovered on food to what was submitted to the clinical diagnostic lab at the University of Iowa as well.
(Slide)
So, some quick data on that. Of the 981 samples, 126 were positive for Salmonella. So, about 13 percent overall. Ground turkey and chicken breast accounted for the majority of those. Forty-one percent of ground turkey samples were positive for Salmonella and 12 percent on chicken breasts.
Overall, 21 percent of samples were contaminated with Campylobacter, the majority being chicken with 75 percent. So, if you remember back to our original study in Washington, D.C., it was about 71.7 or pretty similar. We are talking almost three quarters.
(Slide)
And in terms of susceptibility, one thing I didn’t point out is that we did not recover Salmonella from any pork chop sampled. They were completely free of Salmonella. And all of our Salmonella from beef, there were five of them, were completely susceptible to all antimicrobials tested.
We saw the majority of resistance to isolates from either poultry, ground turkey or chicken breasts, and all isolates were susceptible to ciprofloxacin, amikacin and imipenem.
(Slide)
In terms of pulsed fields, we just want to show you a couple of things where we did do Pulsed Field Gel Electrophoresis. This was on Salmonella Heidelbergs that we found. We did get several hits in terms of indistinguishable pulsed field patterns between those recovered from ground meats in Iowa and also those recovered from human illness.
(Slide)
In terms of Campylobacter, this is the breakdown of what we found. We found primarily the Campylobacter in the chicken breast, and we also had about 181 isolates that we had from humans that we were able to compare as well.
(Slide)
This is just a -- I will give you quickly some ciprofloxacin resistance. This is based on agar dilution in an interpretative resistance breakpoint of four µg/mL. As you can see, that is supposed to be a micro.
But just to show you overall, 19 percent of the C. jejuni from chicken breast were cipro resistant at four microgram per mil or greater, 22 percent of C. coli. None of the other C. jejuni were resistant to cipro from any other meats. And overall, Campylobacter recovered from humans were about 12 percent resistant. So, we are talking in the teens for ciprofloxacin resistance.
(Slide)
Again, we did pulsed field on all Campylobacter jejuni and on several occasions we got identical pattern matches between those Campylobacter jejuni from retail meats and from human illness in Iowa.
(Slide)
Now, during the time of this study the WHO recommended a tripartite approach to include isolates from human clinical cases, food animals and retail meats when conducting antimicrobial resistance surveillance and monitoring for foodborne pathogens. So they recommend this three-prong system of surveillance, if you are looking at foodborne pathogens of on-farm, slaughter, retail, slaughter/retail and human isolates.
(Slide)
So therefore, in 2002 NARMS teamed up with FoodNet out of the Centers for Disease Control and Prevention in developing a program aimed at determining susceptibility of bacteria isolated from retail foods of animal origins, and this has become the third part of NARMS and the most recent. It is located at the Office of Research in Laurel, Maryland.
(Slide)
So, the overview. We started in 2002 with six of the 10 FoodNet sites. We had initially Connecticut, Georgia, Maryland, Georgia, Maryland, Minnesota and Tennessee started in January 2002. Oregon joined us much later in the year, in September. In January of 2003 we added New York and California, and in January of 2004 we added Colorado and New Mexico.
And currently we have 10 of the 11 sites. I think the 11th site is Texas. Correct? And they have yet to join our surveillance system yet, but they are still in their first year of FoodNet as well. So, we have the 10 major players.
Each of the 10 sites has a similar sampling scheme where the sites visit at least one grocery store per month. In the past, between 2002 and 2004, they did this. In 2005 we introduced a random sampling scheme. Again, Terry Proescholdt will talk about that later on.
Each site purchases 40 meats per month. Ten packages each of chicken breast, pork chops, ground turkey and ground beef. All 10 sites culture the rinsates for Salmonella and Campylobacter, and due to the large numbers we recovered of Enterococcus and E. coli, we only have four sites culture the rinsates for E. coli and Enterococcus.
And I may have a slide to show you that. The reason why is our Enterococcus were close to 95% contamination. So, if we had all 10 sites doing that, we are talking 5,000 Enterococcus isolates per year. It would quickly overwhelm us.
(Slide)
This just gives you a picture of the laboratories; where they are disbursed throughout the United States. You can see we have Oregon and California on the West Coast, Colorado and New Mexico, Minnesota, Tennessee, Georgia, Maryland, Connecticut and New York. So, these are the sites that are participating with us currently in the sampling.
(Slide)
As I mentioned, we used similar standardized methods at each of the sites to isolate the respective bacteria. They are adapted from the FDA BAM Manual. All of the meats are initially rinsed in peptone broth and then the peptone broth is then put into the selective broth, depending on what isolate we are trying to recover.
And for Salmonella there are several broths. Lactose broth, RVR10 and the M Broth, and we use a Tekra EIA Kit. For Campylobacter we use Bolton broth. For E. coli, MacConkey broth. Enterococcus is enterococcosel broth. All of these broths have been incubated at appropriate incubation temperatures for an appropriate amount of time and then plated onto selective agars to isolate the respective bacteria. XLD for Salmonella, CCA Agar for Campy, EMB for E. coli and Enterococcosel agar for Enterococcus.
The first two years we provided training where, we had the sites come out to our facility, and we had a week-long training to show them how we were doing it, because we wanted to make sure that all of the sites followed the same methods. It is critical.
(Slide)
So, the sites are the ones that isolate the bacteria, and once they isolate it, they presumptively identify it. Then they send it to us at the Office of Research for confirmation of identification and antimicrobial susceptibility testing.
We confirm all bacterial species by biochemical identifications, primarily using the Vitek, and we also have API if we have some disparate results coming from the Vitek. We have PCR capabilities for Campylobacter speciation using several genes that are specific for lari*, jejuni and coli.
We have our own serotyping laboratory that serotypes all of the Salmonella isolates that come again. And again, we do susceptibility testing, which is our bread and butter. We use broth microdilution/agar dilution, when appropriate, and as I mentioned before, we follow CLSI/NCCLS standards where available.
(Slide)
And just to give you an idea what happens in this whole scheme of collection and reporting, the sites go out and buy the meat. They isolate the bacterium from the different meats. Again, on the selected medias. There is a log sheet. Dr. Elvira Hall-Robinson will talk about this later on. It goes into our database. It is an access database. Again, that will be talked about later on by
Dr. Hall-Robinson.
(Slide)
As part of the retail arm, all Salmonella and Campylobacter isolates are subjected to PFGE, which is a big burden on us, but we do them all. I think it is very important information.
We follow CDC guidelines for PFGE analysis and
CVM-OR is a PulseNet certified laboratory. Our PulseNet unit is headed up by Dr. Shaohua Zhao, who will talk about that later on.
All of our data goes into the PulseNet program, and our isolates can be used for future research projects for anyone who is interested in these isolates as well. We have been involved in biosource tracking experiments, virulence studies and, of course, resistance studies when we try to find particular genotypes associated with particular phenotypes.
Enterococcus and E. coli. Right now a lot of these should have question marks next to it because we have these isolates. We are trying to determine what to do with them in terms of follow up studies.
Some questions that have come up are what virulence factors are present in Enterococcus isolates and are they present on the ones we see in foods? Streptogramin resistance, of course, is a big interest to CVM, and we have initiated some studies to determine what streptogramin resistance genes are there.
We find that in a lot of our Enterococcus isolates that show high levels of streptogramin resistance do not have the typical genes that people have found in the literature. So, there are genes yet to be identified, I believe, in these isolates.
E. coli. We are going after generic E. coli. We are not going after 0157 or shiga-toxin producing E. coli. So, it is important to serotype these isolates? Jim Johnson’s work out of Minnesota is showing that some of these E. coli from foods are potentially urinary tract pathogens. We don’t know that. And we do see fluoroquinolone resistance and ESBL phenotypes in our E. coli, and we are working those up via PCRs for mutation analysis and gyrase* and so forth and what type of beta lactamases are present.
(Slide)
Just to give you some data on the three years, what I wanted to point out here is the number of meats that have increased from 2002 to 2004. Our first year we had approximately 2,500 meats under analysis. As of 2004 we were almost up to 5,000 meats. So we are talking 4,600 and 4,700 meats in terms of our preliminary data, and the numbers are pretty stable across the board for Salmonella.
So, chicken breast was about 10 percent to 13 percent over the three years. For ground turkey it is about 11 percent to 13 percent again down to 12 percent. Our ground beef is almost identical over the years in terms of percent positive, and our pork chop is pretty similar as well.
But it is interesting. You can see it is mostly coming from poultry products.
(Slide)
This is to compare two years of data for Salmonella, 2002 versus 2003, and I tried to group the beta lactams together on the left, then your aminoglycosides and then our other drugs. You can see where we see a little more resistance in 2003 to several drugs, with the exception of TET where we did see a decrease. So, we don’t have 2004 data yet, but I would be curious to see what we get.
(Slide)
Again, I just wanted to show you that we are doing pulsed field, and Dr. Zhao will talk a little bit about this. But we do see some hits between clinical isolates and what is getting through into the ground products. Where it says turkey, they are actually clinical isolates from the diagnostic lab.
Again, we see another hit here. We see three indistinguishable clusters here, and this big cluster is mostly ground turkey. You can see from multiple states. Tennessee, Connecticut, Minnesota and different sampling times as well. So, we are seeing the same clonal isolates out there in the food supply.
(Slide)
In terms of Campy for the three years, it does appear like we have an increased rate of isolation of Campylobacter. I think a lot of this is due to the laboratories improving their methods of isolation. Our first year we had a lot of isolates sent to us from the laboratories that were indeed not Campylobacter.
Remember, we confirm everything at OR. So we had to throw a lot out, because our initial rates were pretty similar to what we see here in 2003 and 2004. But again, you can see we are not seeing much Campy coming from any other of the three meats sampled. Primarily, when we recover it, it is coming from chicken breasts.
(Slide)
And here is two years of data on resistance. This is agar dilution data. In 2002 we had approximately 300 Campy isolates. In 2003, 464. Our ciprofloxacin is pretty stable. About 14 percent to 15 percent. Erythromycin went down from six percent to about three percent. Doxycycline is fairly high, about 27 percent to 28 percent. Gentamicin we -- and remember, these are interpretive criteria or breakpoints that we have determined. They are not CLSI/NCCLS interpretative criteria.
We did see our first Gentamicin resistant Campylobacter this year, and it is at an MIC of 32 microgram per ml. And interestingly, in erythromycin here these are all C. coli’s. We don’t have a single C. jejuni that is erythromycin resistant.
(Slide)
On E. coli, I will quickly go over this. This is, again, three years worth of data. The numbers, remember, are lower because we only have four sites doing E. coli, and enterococci versus the 10 sites for Salmonella and Campylobacter. But your numbers are pretty steady for the three years.
We do seem to see this reproducible phenomenon with pork chops. They seem to be less contaminated with E. coli than the other three meat types.
(Slide)
And again, this is comparing two years of data. You can see almost the E. coli from 2002 and 2003; they are almost like mirror images across the board in terms of percent resistance to the drugs under surveillance of NARMS.
(Slide)
One thing we are trying to do now is to pull out to see if we see certain phenotypes associated with certain meat products, and this is an example of what we can do. If you look at gentamicin and nalidixic Acid, among those isolates that were GEN resistant and NAL resistant, the majority of them are coming from poultry products.
We see very little GEN or NAL resistance in other ground beef or pork chops, and they are primarily coming from chicken breast and ground turkey. So, we can do this for all of the drugs under surveillance at NARMS. At least for the retail arm.
(Slide)
In summary, since its inception in 2002 NARMS retail has increased the number of sampling sites and retail samples analyzed and improved its sampling scheme. For example, six sites in 2002 to 10 sites now in 2005. We have almost doubled the number of meat and poultry samples under surveillance, about 2,500 to almost 5,000, and we introduced random sampling strategy in January 2005 that will be discussed later on.
Its primary purpose is to determine the prevalence of antimicrobial resistance among foodborne pathogens and commensal organisms, and the commensals that we are looking at, of course, are Enterococcus and E. coli as potential reservoirs of resistance from retail foods.
We hope the results will generate baseline data and establish a reference point for analyzing trends of resistance among these foodborne bacteria at a point closets to the consumer. As I mentioned earlier, when combined with data from the rest of the NARMS program, it can give us an idea of what is happening and where we can target intervention and mitigation strategies.
(Slide)
With that, I would like to thank -- there are a lot of people involved in this project and there are certainly many more players, but this is a good collaboration between CVM and primarily CDC, because without their help we can’t work with the FoodNet sites as well. So, thanks. I will take any questions.
DR. VOGEL: I have got several questions. I have always been curious why you chose ground turkey as one of your products to sample. It doesn’t seem to me that per capita consumption is very high of ground turkey.
DR. WHITE: That is a great question, Lyle. During the Iowa Pilot Project we tried to determine what would be the most appropriate and the best handled in the laboratories, and we found out that buying the big carcasses was not the best way to go, because they were Fed Ex’ing them to us. So we tried to go with a product that was easily obtainable.
I think Terry will talk about it later. Ground turkey is actually the fastest growing commodity in the turkey industry being sold in grocery stores. So, that is why we went with ground turkey. But we could have easily gone with turkey legs and turkey thighs. Absolutely.
The thing to remember too is that USDA has performance standards for ground turkey. So that is one other thing we were looking at as well; to try to use that data to maybe help some other agencies when we get some data.
DR. SAHM: Thanks, Dan Sahm. Just in terms of trying to keep questions on target for the discussion points, with regard to sampling, now that what you have learned this data is sort of what we talked about at break. In looking at this data, if I only had a dime to spend I would put it on poultry.
How much information are you getting out of the other meat sections and would you not concentrate on poultry as being the highest risk of exposure to antibiotic resistant organisms for this program?
DR. WHITE: I think for the overt pathogens. Yes. They are primarily coming from poultry products. We are not getting that many isolates from either pork chops or ground beef. Absolutely. So in terms of bang for the buck, yes. We are not getting that much for those meats.
DR. SAHM: What would the risk be of going forward and not pursuing those other meats at this point if you wanted to concentrate more thoroughly on -- if people would agree that is where the risk is, to put your efforts in that area.
DR. WHITE: That is a great question. I think the major risk would be we would want to have information from those particular food commodities, and as we approve drugs in numerous animal classes we would only have information then from poultry. We would not have that information from say swine and from the resulting meats that are obtained from the swine, as well as cattle as well. So, we wouldn’t have that information, and we are trying to look at drug use in certain animals with what might be happening.
We wouldn’t be able to do that connect between say approval -- not to point out Sue’s drug, but ceftiofur is approved in numerous animals species. Without looking at the different foods, we would not be able to see if there is anything going on in terms of decreased susceptibility or what have you.
DR. SAHM: But in our surveillance efforts -- I just want to pursue this a little more to think through it. You learn from your baseline data and you redesign. It is analogous to your decision not to include imipenem anymore.
I just put that out as a point to consider based on the progress of surveillance of learning what you know and where you put your efforts going forward.
DR. WHITE: I think it is a good point. Thank you. I agree.
DR. ALTEKRUSE: I would just like to say from the FSIS standpoint we believe that also the greatest risk is for poultry and Salmonella. Our programs are being sort of reconfigured to address that. So we are going to put greater emphasis on the testing of poultry products. That will have some implications for sampling issues later related to the FSIS isolates.
But I am also very intrigued in the finding of matches with the PFG patterns between human and food isolates, and I would like to hear your opinion on the potential of that to actually detect outbreaks or identify sources of infection.
DR. WHITE: I think that is probably why we do it. In terms of outbreak assistance I doubt that will be of much help at this point because we are not real time. Our isolates are going in probably two years from when they were collected. So really, what we are trying to do is expand the data at PulseNet so that if there is an outbreak and they submit a pattern and it does have a hit with one of ours, maybe they can start focusing their efforts on a particular food.
So, if they say I have a Salmonella Heidelberg and it is a match with a Heidelberg from our retail foods from ground beef, maybe that will help the health personnel to start focusing on where it is going to go.
However, we do have numerous pulse field patterns that are identical among the different meats, like Newport or Typhimurium. So, it depends on the particular organism as well.
DR. ALTEKRUSE: Just to take it one step further, the reason I am interested in that is because one of the triggers that FSIS that FSIS is likely to use for going into an establish and doing a really intensive assessment of their food safety system is the finding of some epidemiologic evidence, like a pulsed field match between human isolates and isolates coming from that plant.
DR. WHITE: A lot of it too -- when people do those comparisons without, I think, a good epidemiological study to back that up, it is -- it just tells you there is a hit, but we need to do a lot more in between to see what is going on. So that is why we do the pulsed field.
I think it is very important to expand that database. They don’t have much in their database from actual foods. Now we are expanding with Paula’s isolates and CDC’s isolates as well. Hopefully, we will get all of those in the system, and when we do have outbreaks they may be able to better target their intervention strategies.
DR. ALTEKRUSE: I would like to ask you how -- what were the criteria to select the 10 sites that you include?
DR. WHITE: Okay. That is a great question. The 10 sites were already existing as part of the FoodNet system. So, they were already in existence.
DR. VOGEL: In one of your slides you mentioned that it was a samples from a diagnostic laboratory. Are you obtaining samples from diagnostic laboratories yourself? Or is that a sample that was obtained from USDA and then shared? If you are doing it yourself, it seems like it is duplicative use of sources of resources here.
DR. WHITE: Yes, I agree. That was a diagnostic isolate from another study. It is not from a NARMS study.
Remember, a lot of us are also Office of Research principle investigators. We have our own studies underway as well besides NARMS. That was a study that Dr. Zhao and Dr. Walker are PIs and were working with diagnostic labs. But we do the same thing. We do susceptibility testing and pulsed field.
So then, when we run it through the system and we get hits, it would just happen to come out as one of those.
DR. VOGEL: Can I ask another question?
DR. WHITE: Sure.
DR. VOGEL: On Campylobacter you were testing for Doxycycline why did you choose that instead of Tetracycline like the other arms?
DR. WHITE: Yes. Doxycycline I believe would be the actual drug that would be used to treat human illness. I think more so than tetracycline. But we have gone back to tetracycline for the microdilution.
DR. KOTARSKI: Yes. I come back to a comment that I made earlier in understanding the purposes of the sampling strategies. Currently it looks like you are consisting sampling the same number from different -- getting the same number of meats from each store.
So, on the surface it looks at your sampling strategies are designed to look at prevalence of Salmonella or Campylobacter of meats in stores. It is not set up exactly to examine exposure to humans.
DR. WHITE: I agree.
DR. KOTARSKI: Okay. So that is understood. Now you have generated data to show that the prevalence of Salmonella actually is very low in certain meats and that point has been made earlier. I haven’t looked in detail at years 2003 and 2004, but I do recall, for example, your ground beef data for 2002.
You had very few isolates. On the order of nine Salmonella isolates, as compared to hundreds maybe of isolates in poultry. That being the case, to look at resistance trends of nine isolates in ground beef from one year to the next, probably your sampling error associated with only obtaining nine isolates of Salmonella, for example, in ground beef does not permit you to look at temporal trends that you would like to do without a lot of variability, just because of the number of isolates that you get.
So, I come back to the original point. What is your objective in your sampling strategy for the retail meats? Is it to understand the prevalence in meats, per se? Understand resistance prevalence in retail stores? Or is it to understand the prevalence in food consumption?
I think that those objectives should be clearly stated and then select your sampling strategy accordingly.
DR. WHITE: Okay.
DR. ALTEKRUSE: Many of the serotypes that you are isolating, for example, from poultry aren’t serotypes that are associated with human illness, and I wonder if those get the same priority or perhaps they are tested later. Have you thought about it?
DR. WHITE: In our system?
DR. ALTEKRUSE: Right.
DR. WHITE: Well, the first thing is serotyping takes the longest amount of time, besides pulsed field. So actually, as soon as we get something in it is susceptibility tested. So, they are done right away before we do serotyping. You know what I mean?
We have different groups working on the bugs at the same time. That is the best way to get things done the fastest, not wait until they are done. Otherwise it would take years for us to get each year done. But the majority of our Salmonella, as we see it, are the top five that CDC sees in human illness.
We do see some other ones that we don’t traditionally see in human illness, but across the board they are typical household names that most people know. Heidelberg, Newport, Typhimurium and so forth.
DR. VOGEL: You mentioned that you are sampling E. coli and Enterococci from only four of the FoodNet sites, and I understand why, because of the number of isolates. Have you looked to see if there is any geographic variation in E. coli or Enterococci? If so, might you be better doing a systematic sampling from all 10 sites or all 11 sites instead of restricting yourself to four sites?
DR. WHITE: Yes. In our first annual report we tried to break it down by state, and I think at least with Enterococcus we do see a species distribution with -- Maryland sees more faecalis than faecium compared to the other three sites.
We haven’t look at the 2003 data yet to see if that holds up in terms of a trend. So, it is definitely something to throw out and look at the data.
E. coli seems to be different, we don’t see resistance associated with one state more so than another. They are almost mirror images for the two years. We try to pull out those things to see if there are differences between the states as well.
We did that with our Campy. If you look at Campylobacter isolation, you see large variations between the states in terms of what they isolate over the year. Okay. Thank you.
DR. YOUNGMAN: Thanks, Dave.
In the interest of time, if we can press ahead, our next presenter is Dr. Paula Cray, who is going to be talking about the animal arm of NARMS.
Animal Arm
by Dr. Paula Fedorka-Cray
DR. CRAY: I actually did these slides so they were done yesterday morning, as per Dave. I called him. I said I was sending them, and my father has a dial-up connection. I dropped my son off at my parents’ house, and I tried to download a 10 megabyte file over a dial-up connection. It was a rather painful experience for all concerned.
I thank Bob Walker, Linda English, Dave and Pat for staying later in the evening to do that. I could have driven here faster it turned out and xeroxed them and did what I did.
(Slide)
Nonetheless, this is the animal arm of NARMS. Dave went over some of this, but I will reiterate and give you a little bit of a different perspective on some aspects. It began as a research project, the animal arm actually did, with Dr. Leanne Thomas and myself. Dr. Leanne Thomas was ta the National Veterinary Services Laboratories and I was a scientist at the USDA National Animal Disease Center in Iowa.
We got together and we decided that at the time, in 1994 and 1995, there was an increased interest in antimicrobial resistance. So we actually bought panels that just had breakpoints on them, and we were doing the testing when we received a call from Dr. Tollefson, who had heard what we were doing, and asking us if we would be interested in joining NARMS.
And we were certainly interested in doing that and NARMS officially launched in 1996, and we used up our first 1,500 plates of just breakpoint and then we went to the MIC format. Although we didn’t have comparable plates until 1997/1998.
(Slide)
The evolution is that NARMS is really a passive system and the original goals and objectives were to provide descriptive data on the extent and temporal trends of antimicrobial susceptibility in Salmonella and zoonotic enteric organisms from human and animal populations. To identify resistance that arise. To provide timely information. To prolong the life span of approved drugs. To identify more areas for more detailed investigation and guide responsibility.
It is actually a passive system and it relies primarily on the submission of isolates. And really, all three arms either relies on the submission of isolates or the submission of some type of samples. This isn’t an active system that we have gone out and actually designed ourselves.
We do believe it does provide a comprehensive snapshot, and that really is what it is. It is a one time point snapshot of food animal production through the U.S. We have all animal species that are representative in some way, shape or form.
We look at small, medium and large slaughter plants now, and Dr. Anandaraman will talk about it this afternoon. This did not start until a later date in time. We had the plants as they were coming onboard. So the system was not as robust as it is now until 2000.
We also have isolates from ill animals. There has been a lot of discussion about the necessity of having diagnostic isolates, but we believe that these can provide information on isolates that may be emerging or that we may see in the food chain over time, because these animals are presumed to have undergone some type of treatment over time, and while they should not be part of the actual food that is being consumed, at least in the raw way, shape or form, they may, in fact, be representative of what is to come.
We can see in that some respect with Salmonella Uganda, which is first emerged as a diagnostic isolate and now we are trying to see more prevalence in some of our other samples.
Our isolates from on-farm samples are limited. They are limited to the NAHMS studies, the National Animal Health Monitoring System studies that occur, and they are also limited to epidemiologic studies that we might conduct or our collaborators might conduct over time.
However, at some point in time, and for the most part, during the year we can receive isolates representative of all geographic regions in the United States. So we have these three sources of isolates, on-farm, diagnostic and slaughter, and we looking for Salmonella and Salmonella is our sentinel organism, E. coli, Campylobacter and Enterococci.
(Slide)
This just illustrates that and shows the differences between the human and the retail. We have tested Listeria. The question was asked earlier this morning. We did a snapshot a couple of years ago on about 600 hot dog isolates. We occasionally have some coming in on various other -- from other sources now. We actually have one student who we acquired through a reorganization who is looking at Listeria for part of his project, which is not part of NARMS.
(Slide)
Again, our isolates come from diagnostic and
non-diagnostic sources. These diagnostic isolates are presumed to be associated with clinical illness. The animals are not likely to enter the slaughter facility. Right now we have 12 veterinary diagnostic labs. California, Colorado, Iowa, Indiana, Kentucky, Michigan, Nebraska, New York, Oklahoma, Texas, Washington and Wisconsin who have submitted isolates over time.
We have approached Minnesota about submitting isolates, and Pennsylvania will actually join us, the University of Penn, later on this year.
Then we also go up twice a year and we collect isolates from the National Veterinary Services Laboratories, and what we are doing is we are collecting the diagnostic isolates from there that were associated with either a primary or a secondary etiologic outcome. So, Salmonella has to be identified as the main cause of disease.
However, we exclude selection from the sentinel sites so we are not duplicating isolates. So this way we then can saturate the rest of the United States, as far as the isolates coming from other states and the various regions.
For our non-diagnostic isolates we presume that all of these are coming from healthy animals. Our on-farm isolates are part of the NAHMS studies, as I said; however, there is a five-year rotation between commodity. So, for instance, there was a swine study in 1995. There was a swine study in 2000. There is scheduled to be a swine study coming up within this next year.
But when we look at these, again, they are snapshots of time, although they do provide a statistical representation of greater than 95 percent of animal production on-farm in the United States for that particular time period. So, they tend to be very invaluable as far as resources because they also collect management information at the same time, which gives us an idea of what antimicrobials may have been used in practice that may be affecting outcome of the resistance patterns that we may see.
I regret that our APHIS partners were not invited to the meeting, because I think that they could have provided some important additional information.
The slaughter isolates then are rinsates, carcass swabs and ground product. Now we are receiving ready-to-eat products and isolates from eggs. This is a comprehensive snapshot based on the FSIS compliance. It is related to retail. It is not statistically significant, but
Dr. Anandaraman will address that this afternoon.
(Slide)
I think it is important for you to realize for you that for the diagnostic isolates what we are just getting are NVSL isolates in the sentinel sites, and we receive anywhere from about 75 to 500 isolates from these various sentinel sites. So, the larger the sentinel sites, like Michigan and New York, we tend to get about 500 of their diagnostic isolates because that is about what they get per year. So we get all of them.
There are some smaller sites, and we actually get all of those too, like Washington State. For actual diagnostic submissions we tend to get about 75 to 125 different isolates.
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All of this information is collected and reports are generated and sent back to the sentinel sites, and we give them free range to publish their own information, either including us as co-authors or citing the NARMS system as providing the information to begin with.
The compilation of data that you see on the reports is usually all of the sentinel sites together.
E. coli right now only comes from our sentinel sites, as far as diagnostic lab goes. We get very few Enterococci, too little to really count, and we do get a few Campylobacter and they tend to be sporadic. Those are available too in ancillary reports.
For our non-diagnostic isolates then all of the E. coli, Salmonella, Campy and Enterococci come from the raw product, federally inspected slaughter plants and from focus studies, but there is a caveat. Only the Salmonella comes from the actual FSIS compliance testing.
For E. coli, Campylobacter and enterococci then we actually take the spent carcass rinsates that are sent to the eastern laboratory for FSIS. Once they take their aliquot for Salmonella testing, anything that is left over that is greater than 10 mils is sent to our laboratory, and from that then we can isolate E. coli, Campylobacter and Enterococci.
Now, the FSIS laboratories, the eastern laboratory, doesn’t just receive samples from the eastern part of the United States. The distribution is randomized so they are receiving them from all different -- from through the U.S. on a rotational basis. But it is important to realize that these carcass rinsates are also only coming from chickens.
So, the information we get for Campy, E. coli and Enterococci through non-diagnostic slaughter isolates are limited to chicken carcass rinsates from spent Salmonella samples.
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This is what I see as one of the weaknesses of NARMS when we look at critiquing our own program and how we can improve the robustness of the program. But farm sampling and diagnostic isolates are independent of the slaughter program. So, we don’t have a flow through.
We have farm isolates that are coming because of NAHMS related studies, we have diagnostic isolates coming from the diagnostic labs and then we have the compliance testing out here. And we really don’t have an association of what is happening between the farm then and the plant.
We believe that while you can say that, yes, there is some association -- and, of course, what we see on the farm is what we see, in part, of what is coming in the plant. But we can’t say a lot of times what the use is associated at a plant, particularly with antimicrobials, and what is occurring then in the slaughter plant.
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If we move to looking at the individual organisms, Salmonella is our sentinel organism. We have all sources. We receive the isolates primarily. We are not doing a lot of the isolation.
These serotypes will vary over time and vary by source, and we believe it is absolutely critical to take the information and break it down by serotype and by source because we see distinctly different patterns associated with these various sources and serotypes.
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When we look at our sampling scheme, what we were doing up until just recently took us several months to complete when we would receive an isolate, either slaughter isolates, which are the actual isolates themselves received weekly, or diagnostic isolate, which is the actual isolate.
On farm feces we have always been the testing laboratory for NAHMS studies. So we actually receive the feces or farm sample, and we have to go through a seven to 10-day confirmation and isolation procedure. For this we have used our published methods, which have been compared and contrasted and now are considered to be one of the gold standards in the industry.
Once we had the isolate then it went for serotyping, if we were doing the isolation, or it came with the serotype if they were slaughter and diagnostic isolates. They were susceptibility tested. Then we freeze and store all of our isolates. So we have every isolate that we have tested since 1995 in triplicate frozen in the building.
And right now, we are lyophilizing everything, and one isolate will be sent to the National Germ Plasma Repository in Ft. Collins for, hopefully, lifetime storage if anything happens to the Russell Building.
If we don’t have a phage type, then we have to send it back out to NVSL for phagetyping, and because of our involvement now with USDA VetNet, we send all of our isolates to our pulsed field lab where they undergo Pulsed Field Gel Electrophoresis. And then it goes on to preliminary data and then year-end analysis.
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At the request of FSIS, who has a need now to send information back to the plant on a regular basis, we have looked at how we can compress our time frame in getting this information back to FSIS that they might be able to use. So, particularly for our slaughter isolates now, we are looking at if everything works right in life and we have perfect gels and perfectly susceptibility tests, about a two-week turnaround time from receipt of isolates to a susceptibility pattern and a PFGE gel picture that we could send back to FSIS which they, in turn, we understand, will send back to the plants.
Now, in an testament to the compliance testing success, we see that the number of isolates from 2000 to 2004 has gone down from about 3,500 to 2,500 or so that we receive for a year from FSIS. The number of isolates in toto that we receive for diagnostic are on-farm is also about another 2,000 to 3,000 isolates for any particular year.
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For generic E. coli this did not start for us until 2001. We do have some diagnostic sources. We have on-farm sources, which we have not included as part of NARMS. But we have those isolates available that are for our own independent tests, and again, these are from the spent FSIS rinsates and only represent poultry. Particularly, chicken.
There are thought to be reservoirs of resistance genes, which is why we are still testing them as part of the program.
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And the isolation procedure is almost identical to Salmonella, except that we use com-agar* for our isolation. I will put a caveat in here; that we have done extensive and exhaustive testing of the different medias that you use for isolation and we can say that you will have different results on occasion, dependent upon the isolation methods that you are using.
So, for instance, if you use Campy line agar for a Campylobacter as opposed to cephex, you will get different populations than you will get of Campylobacter jejuni or coli. The ratios will be different, and within those different populations from those different plates you will have different antimicrobial susceptibility patterns, and I think that is important to remember.
From 1998, which is our Campy started, through 2000 we had Campy obtained as an actual isolate, because FSIS was taking care of the methods. The isolation. They were using nalidixic acid in their screening and at that time we believe that it probably under represented the percent resistance to quinolones and fluoroquinolones.
We changed the method in 2001 when our baselines actually stopped. Again, these isolates originate from spent FSIS rinsates and we believe that now at least the susceptibility testing more accurately reflects the resistance of the total population.
(Slide)
It is very similar, although there is a difference in the amount of manipulation that has to go on with Campylobacter, and again, it depends on if we get them on-farm and we actually have to do the isolation too if we get the isolates from diagnostic sources. And now we are doing all of the enriching for slaughter. So, the time from receipt of a sample is several months before we are ready to have some preliminary data.
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For Enterococci this started in 2001 also. Again, we are at the FSIS rinsate origin. Our methods were tested and developed in 2003, and dependent upon which incubation temperature and media that you use, you can obtain different populations of Enterococci. We will look at some of this. We do have a paper that has been published on it. Like E. coli, they are thought to be reservoirs of resistance genes.
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I will say that USDA started USDA VetNet in 2003 and this was established in collaboration with CDC and FDA and is essentially the animal arm of PulseNet. It was originally known as PulseVet, VetNet-Animal and then the under secretary decided they liked USDA VetNet and that is what we went with, because they were doing the blessing.
All of our personnel are trained and certified at CDC, but the program actually resides in Athens, Georgia.
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We aim to capture PFGE patterns of Salmonella isolates submitted to NARMS, and we also have a branch for our CAHFSE program. We compare VetNet and PulseNet PFGE patterns and we would like to say that eventually what we would like to do is to be able to use this data to assist in surveillance and then the study of the ecology of organisms along the food chain and for us to be able to assist in the investigation of animal illness outbreaks on-farm, as well as foodborne outbreaks when CDC has them.
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This is modeled after PulseNet. We have two independent databases because we have two independent programs, the CAHFSE and the NARMS Program. These databases by the end of this year will be moved to RRC and they will be able to be accessed much like PulseNet does now through an online server. Currently the gels are being sent -- currently everything is being run and analyzed by USDA-ARS, but that will change with the submission of gels from the University of Penn Lab.
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So, as of May 5th we had 82 serotypes. We have 3,596 isolates. We had 1,452 unique patterns, all done with just one enzyme, XbaI. That has since gone up to well over 4,000 isolates now.
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One of the things, when we look at our PFGE distribution, is these are the top serotypes that we have, and you will see that in the rank, 1, 2, 3 through 10, the "R" stands for isolates that are also seen from the retail arm of NARMS.
Each is indicative of isolates that are serotypes that are also seen from the human arm of NARMS. These are the numbers that we have so far for these top 10 in our database, and you can see the numbers with unique patterns. One of the things that we have been able to do in comparison of the VetNet and the PulseNet databases already is to see that there is much more heterogeneity amongst the animal isolates than there are amongst the human isolates. So, we have many, many, many diverse patterns in the animal arm.
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When we look at the top 10 patterns that we have to date, we can see that, in fact, there is an overlap with particularly four of these, and they are coming from Enteritidis, Heidelberg and Typhimurium. And you can see the PulseNet on the right and the VetNet on the left.
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And we can generate patterns much like this, and we intend to be putting this on for our reports in the coming years too so that we will be able to actually have available what the pattern looks like and what the serotype is; how many isolates we are generating from that and what the XbaI pattern is.
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Now, we go through this debate and I know that Dave just put up the PFGE pattern, and you can see that this is for Salmonella Newport, and I am not sophisticated enough like Dave to draw up those nice little boxes that come up. But you can see that we have significant clustering of one particular clonal type and then we have some independent.
I always have this debate with Pat and Dave. If you look particularly at 44 and 42, they both came from cattle. They are both in the same cluster, but they have different antibiograms*. And so, yes, they have similar PFGE patterns, but are they, in fact, the same isolate because they have -- are expressing different resistance patterns?
And depending upon how many microbiologists you have in a room and how many molecular biologists you have in the room, that is dependent upon what the opinion is of the day. So, whether you can actually use it for an analysis to say this came from a human versus this came from an actual animal source, I think that we can generate some interesting discussion about that.
Certainly, when you have matches that are all the way through and you can identify the same genes, the same resistance patterns, the same PFGE pattern is the same, then that would be an indistinguishable and a confirmation.
(Slide)
When we look at VetNet, we see NARMS, we see CAHFSE. We have -- NVSL is interested in submitting, and they are going to begin later on this year submitting diagnostic TET files to VetNet. The American Meat arm of USDA, from their feeding programs for the food school lunch program and the military, will start submitting their files.
Veterinary diagnostic labs, as in Shelley Rankin, will begin submitting her files, and then we hope to have -- EPA has expressed some interest; other government related agencies. Perhaps some university labs and some Ag State labs at some point in time, making this a very comprehensive system.
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So, when we look at what NARMS has evolved to over time, these colored regions are actually representative of the FSIS regions where we have isolates coming from the compliance testing. When we see -- and that would be in region one, two, three, four and five.
When we look at Dn and the diagnostic laboratories, the red Dns now are labs that have not submitted isolates in 2005, but we have isolates from the past. The blue is where we are in discussion for getting isolates. Vn is the VetNet lab. This is the VetNet hub. This is where we will begin getting isolates for the VetNet program.
And "C" overlays our CAHFSE and our future CAHFSE site. I think that what this shows is that -- the point that I wanted to make is that with NARMS we have a very comprehensive look at what is going on in animal production.
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One of our most exciting things, and this was the killer last night that caused the major snafu over the dial up, is the advent and development of microarray technology. We now have a chip that has about 150 resistance genes on it, and you can use any bug. Campy, Salmonella, Enterococci and E. coli. It will pick up all of the resistance genes.
We also have about 900 virulence genes that are also placed on this. So, we hope to be able to use this, particularly when we have questions about isolates that may be part of an outbreak so we can tell, with a higher degree of certainty, what their degree of relatedness is over time.
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Other studies that we undertake as part of NARMS. We have optimized isolation protocols between labs, and we have, just about every 18 months, a methods meeting. Either CDC comes up and FDA comes down or we go someplace where we talk about how we are doing everything and what the optimized method is and how it might be different between labs and effect our results.
Campy isolation protocols for Enterococci in particular and E. coli. We also conduct additional molecular studies, like the retail arm does in CDC on plasmids. Mechanisms of resistance. Dr. Charlene Jackson has identified several new mechanisms of resistance in Enterococci, we look for integrons and we have developed some multiplex PCR assays.
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When we go to reporting, I think a soft critique is that this the part of NARMS that I am least happy with. Our reports are posted to the website now. We don’t do a hard report. A big part of this is because our -- one, we are short staffed, as far as any people spending time to get this done.
But our reports now, if we put out just what is on the website right now, you would be printing about 329 pages of must Salmonella information and then another 100 pages of all of the rest. Since 2003 we have worked diligently to try to update our reporting so that it is more timely.
One of the things is that as soon as our reports now are completed or as soon as our data is completed and cross checked, then it starts going up. Within the next two weeks you will start seeing tables and graphs going back up for 2004. So, I think that we don’t have text. That is one of the big lacks. We don’t do an interpretation, but the data is essentially all there.
The biggest problem with the data for all of the arms that I see right now is the lack of flexibility in the way that you look at this data.
John Stelling and Terrell Miller have been working diligently to have a unified reporting database and this I am really excited about. Tom Chiller and I spent a lot of time kidding each other about this, but we have had many meetings with -- the CPAR group has come down now, CDC has some over; FDA has come down.
We have gotten together so that we have -- the goal is to have similar reports so that if you click on a table one for CDC and look at Salmonella, you should be able to click on the same table for all of the other arms and get a similar report up. I think that is spectacular, and that is where we need to go.
However, given enough money and time, what I would really like to have is a dynamic database, much like they have for TSN and some of the others where we have drop down menus so you can just go in, if you want to look at Salmonella or Typhimurium, a particular region, a particular drug, a particular year, you just go in and drop down all of that, click go and it gives it to you.
I think that that, in fact, will be when we have reached our pinnacle of success. So that is what we are working toward, and I see that particular drop down menu option as probably being two years away.
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If you go to our website, this is the first thing that comes up. ARS, in its infinite wisdom, said that they were going to consolidate and make things easier. No one can find anything now.
But if you go to our website, what we did get them to do was allow us to put a separate box up and you will see that there are NARMS reports available in that box.
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If you click on that, then you get the report screen that comes up, and you can click on each individual report that you want for every year.
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One of the things that the report doesn’t do is it doesn’t give the type of presentations or it doesn’t the give the summary of information that we can give when we give presentations. One of the other things that we are working toward is before the end of the year we will have a presentation where everything will be converted to PDX and every presentation that anyone in the lab gives associated with NARMS will be up there so that people can look at the way we have manipulated the data in a different way, which I think will provide an extra layer of information.
But you can see that this is the distribution. All sources, all clinical status for these particular serotypes. Again, the "R" is retail or human sources that they show up on their particular reports. You can see the frequency. Salmonella Kentucky is, by far, our most serotype; 9.2 percent of our total number of isolates, and a majority of them tend to be pan susceptible. Then there is about five percent that is resistant to five or more antimicrobial.
This is the type of information that people request. One of the things that I have always tried to encourage, and people have made use of this, is if they want information presented a different way, they just call and we get that information to them. So, it is, by no means, a static system.
(Slide)
We can look at our isolates and this is part of our report where we actually go by source. These are from slaughter: Cattle, chick and swine and turkey. You can see the top 10 serotypes within each, cumulative for 1997 through 2003, and the blue highlights will be indicative of those serotypes that also show up in the human arm.
(Slide)
We can compare now with the human serotypes that are coming up. This is an example of dairy cattle serotypes. We think it is absolutely critical. Here is diagnostic, here is on-farm, here is the human, because you can see that there will be some differences, particularly whether we are getting them on-farm versus diagnostic. This gives us a much better handle on what might be happening over time.
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And you can see then that it changes for each source, and that is why it is absolutely critical. You have to look at cattle isolates separate from swine isolates, separate from chicken isolates and by each of the different serotypes.
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We had two post-docs and they have, cumulative, six publications that will be coming out this year. We had the first ciprofloxacin resistant isolate identified in 2003. I’m sorry. In 2000. And you can see that Salmonella Niakar* was in 2000. We had Newport and Typhimurium in 2002 in Kentucky, but we have very little fluoroquinolone resistance amongst our Salmonella isolates.
(Slide)
We can look at our Salmonella Newport isolates, and I think that is what this is -- particularly important to look at is these are the years when we didn’t have any farm isolates. So it is not necessarily that there weren’t any isolates. It is just that we didn’t have any studies conducted by APHIS during those years, and those are critical gaps that we are missing.
But you can see compared non-diagnostic, which would be slaughter sources to diagnostic. The majority of our Newport comes from diagnostic sources.
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Dr. Jenetta Tankson then has characterized a number of these Salmonella Newport and we can look at the presence of entigrons, plasmids in the CMY2 gene, and we find that most of the multi drug resistant Newport harbor the plasmids and CMY2 genes.
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Our reports also contain information on DT104. Quite interestingly, our isolates primarily originate from swine for DT104 and not cattle. Cattle is in this green and this purple. Forty-three percent are from swine, versus about 38 percent from cattle total.
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We can look at the various sources or sites then, whether they are diagnostic, slaughter or on-farm, and you can see that particularly in dairy. They are primarily diagnostic isolates.
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We then have the ability now to look at phage types. This is not included as part of our report in the past. These are being done for all of the various species, and they will be presented this coming year as an ancillary box that you can click on in our report page so you can see how, in fact, the various serotypes emerge, stay and then seem to disappear over times in the various years amongst sources.
We think that this will help also with the comparison between human outbreaks.
(Slide)
For Campylobacter this is the type of information that we can generate now, and you can see for Campy and coli we have more resistance there for more drugs than we see for C. jejuni. In our other talks that we have given this is where our methods change. Up to this point in time was when we were receiving isolates where FSIS was using nalidixic acid in the screening. After it changed then you saw an increase in our resistance to the fluoroquinolones.
(Slide)
We can look at E. coli now, and we have this broken down by year. You can see that in 2004 we did several thousand isolates, and we have seen very little change over time, except in 2004. We see our first ciprofloxacin resistant E. coli.
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We also have E. coli in cats, dogs, eggs, dairy cattle and chicken in 2003, and we can see the differences in resistance patterns amongst the various animal sources.
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When we look at Enterococci, by far faecalis is our primary serotype, although we also have a significant number of faecium, durans and hirae and gallinarium, casseliflavus and then it decreases from there.
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When we look at resistance for all of them, we get a snapshot that is indicative of bacitracin, tetracycline being prominent resistant patterns.
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But when you look by serotype, you get different results and this is why -- for species this is why it is important to have all of this broken down over time.
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So, what are our needs? We have tested, as part of NARMS, almost in the 11 years that we have been around I think, we are close to 80,000 isolates now, and where do we see our greatest needs? Well, our greatest needs I think are in permanent staff.
With the receipt of the interagency agreement the USDA does not recognize this as permanent dollars, and what we really need is to have some type of language or have some type of agreement between the agencies themselves that says that this money is going to be stable, at least at a particular level. So that would enable us to hire the staff permanently.
Unfortunately, at USDA staff have to be rotated after four years, and if we lose that continuity, particularly from the people that we have now, we lose a lot of ability to maintain the numbers of isolates that we are able to analyze right now.
Permanent staff is also required for the computer end of things. We know that CDC has gone through several programmers. We have John Stelling, and John is only paid on a part-time basis because he is off and about on his many other journeys across the globe. I think it would be important for the system to have someone looks at the computer and the reporting and is able to meet all of our needs comprehensively over time.
Funding again and the coordination between the three agencies. Continual reviews. I think that we have our own annual reviews that we do internally, but it would be important for us to have annual reviews.
Right now we have 18 month where we have a NARMS get-together and we have presentations, but I think that we really need to have these reviews where we all, the people who are doing all of the work, get in a room, say this is how we are doing it and then have somebody just ask us the hard questions; so that we are constantly then asked how we can make this better, how we can change to meet everybody’s needs.
We have, for the past four years, for some outside oversight, and we have been told that it is difficult because of regulatory agencies being involved, but I really think that it would be important to have many stakeholders from many different areas who can give us input on a regular basis. A core panel of people who can look at the program, who will be able to accurately and adequately analyze and discuss the needs, both within and without of the industry and internationally to tell us how we are doing.
We have lost Dr. Marcia Hedrick, who was the coordinator, and I think it is absolutely critical that we have a coordinator. This coordinator has to have the trust of all three agencies.
In my opinion, I believe that we, FDA, CDC and USDA, should be the people who select this coordinator, because this would be somebody who would be our spokesperson and who would be moving the system forward and meeting all of our needs, because we also have individual needs within each of the agencies too. And they should be able to have freedom to make decisions regarding both monies and studies that need to be done.
(Slide)
There are many, many people who are involved with this, and I just want to acknowledge the staff. This is just a mere snapshot of the staff that is back at USDA.
(Slide)
Again, this is an interagency. The animal arm of NARMS is only as good as the human and the retail arms are too. This is the best example of a three-legged dog that makes it in veterinary medicine. So, with that, I’ll entertain questions.
DR. SAHM: I want to get back to something that helps us formulate the questions around these points of discussions, and it is something that Sue brought up earlier regarding the goal of the NARMS program.
When this was started we weren’t quite sure to what extent antimicrobial use in animals presented a human health threat. Is that true?
DR. FEDORKA-CRAY: That is right.
DR. SAHM: And we haven’t done the analysis yet to completely show what the extent of that threat is. Correct?
DR. FEDORKA-CRAY: That is correct.
DR. SAHM: And then, with NARMS -- what I am getting to is if that threat or link was established, then there would be a better case for maintaining ongoing surveillance or what have you. Right?
DR. FEDORKA-CRAY: Yes.
DR. SAHM: So the question is are these studies eventually going to be -- when are we going to get to finding out what this data tells us in terms of the threat to the human population, because that will help make the case as to whether or not it is worth it. Do you continually survey these animals and at what stage, which types or what have you?
So, that is one question as to what is the staging of the NARMS goals.
DR. FEDORKA-CRAY: Well, I think that is a lofty goal, and that is one that we have all wanted to work towards. With the limited resources that we have had with the NARMS program and with the monies that we have been able to obtain from the interagency agreement, you have to go with what you have.
There isn’t in the U.S. a comprehensive program
on-farm that allows us to look at on-farm use of antimicrobials, the isolates that are obtained there over a period of time, follow those through, those same animals to the slaughter and follow that then with what may end up in retail.
Because of this and -- you know, this is the NARMS review, but USDA -- I will just mention that USDA underwent some soul searching and came up with the CAHFSE program that I discussed, which sets out to meet those missing needs. Until the CAHFSE program though goes from its infancy to a fully mature system, we have to continue with the isolates that we have available to us.
Right now the only isolates from the veterinary side, in fact, are the diagnostic isolates that we are getting now, the slaughter isolates from the compliance testing and any on-farm studies that may be conducted either through NARMS or other epidemiologic studies. I think it requires more of a discussion than we have time for here.
DR. SAHM: Right. I agree. The reason why I pose that question that way, Paula, is because from Dave’s previous presentation I am wondering what you have learned with all of this extensive work already can be used to start weeding out and narrowing the focus of the program.
For example, one could make the argument that the poultry that was brought up earlier is the key. The other thing I noticed about your data is on the human side 70 percent of enterococcis faecium in the U.S. population among humans are resistant to vancomycin and about three percent faecalis.
Of all the enterococci you are isolating from these animals you are not seeing vancomycin resistance?
DR. FEDORKA-CRAY: No.
DR. SAHM: So, is there a plan to take that kind of information and say, okay, this is probably not the level of threat and let’s turn more of our attention to Campylobacter in poultry or something like that and start to narrow the focus and redesign the focus, given the data that you have colleted and analyzed already?
DR. FEDORKA-CRAY: I think that discussions that Dave White, Tom Chiller and myself have had would go along the lines of being able to sit down and do a self-critique of a lot of what we are testing now. We haven’t done it up to this point in time.
A lot of us were waiting for this review to see what would come out of this. I think that that is a very appropriate task. Do we need that level of testing for, particularly, the commensal organisms? That could be something that could be addressed more along our research lines and less of an emphasis on our NARMS testing where, in fact, then we would look at either getting more on farm isolates and following that through or changing the dynamics of the system.
DR. SAHM: Right. I agree. I think you all have learned a very -- we have learned a lot from the NARMS efforts, and it is building on that rather than continuing it in all aspects as it was originally designed.
DR. FEDORKA-CRAY: Right. Exactly. It needs to be more robust and it needs -- I think that the biggest problem is -- like I said, it is all independent. So, you make an inference here, you make an inference there.
If you see an isolate, N = 1 or 2 or 3 for a particular year, you could say it is emerging, but you don’t see it again for another year. So yes. I think we can do it better. I agree.
DR. MILLER: Jumping along what Dan was saying, one way you might be able to focus down is by looking at the commonalities across the three surveillance systems. And if your ultimate goal is to link to human health, that is another way to limit down.
I just ask you, Paula, -- I mean, from the pet species, the cats and dogs, has that work led to any sort of hypotheses about human disease at all that have been followed up? Or is that something necessary to continue on?
DR. FEDORKA-CRAY: When we go to NVSL, you can see that our numbers of pet and dog isolates are very small. We take those mostly because they don’t get very many. We have some years where there is 25 isolates or 50 isolates.
So, we take those as more of an interest, and they are a very, very small percentage. I can’t tell you the percentage exactly off of the top of my head, but they are small numbers.
When we look at saying -- I think there have been a number of studies that have come out now, particularly recently. For instance, the pocket pets have been a problem. There have been some reports with dogs harboring some of the Salmonellas.
There are some, but that is such a small part of what we have here. It is there as a piece of information for people, but the focus of NARMS is not on that number of isolates whatsoever, and if we didn’t do that number of isolates, that is something that we would pick up as just a research type of look-see. But that is not the major focus of the program
DR. ALTEKRUSE: In addition to this overlay of the public health impact where we talked poultry isolates perhaps having more importance, there is another -- it is not a scientific consideration as much as it a policy consideration. I think it is important to point out that the FSIS slaughter isolates are going to be used in a very specific way in the future.
Rather than just counting the number of positives in a Salmonella performance set, we intend to provide that information back to the establishments so they can make some adjustments in terms of their HACCP plans about antibiotic resistance and serotypes.
And so, the timeliness of getting that information is important. You mentioned that, that we are going to try to improve timeliness. But also, those isolates have the potential to have to be used for enforcement purposes, in which case chain of command -- chain of custody issues are essential for those isolates.
They really do rise to a high level of priority in terms of what needs to be done and how they are handled.
DR. FEDORKA-CRAY: The FSIS isolates are always our priority in NARMS. I mean those are isolates that historically are always done first. Diagnostic isolates are filled in on the side. NVS isolates are only obtained twice a year. It is only from NVSL that we get the small number of dog and cat isolates that we do.
We have discussed with Dr. Pat McKaskey all of your needs, and we will be implementing and undergoing chain of custody training so that we can provide the appropriate paperwork to you. We also have all of these isolates that are available and that have been stored for the past 11 years. We have worked with you and with the lab to take this information and compress it from months down to several weeks.
We think that if we are providing that information probably back to you, that that would be -- we will have to look at ways that we can also probably put that on the website on a more timely manner too so that people are seeing all of the information as it is coming out more real time. And that is the whole key, is the real time.
DR. ALTEKRUSE: I just really want you to know that we really appreciate the value of the collaboration too.
DR. FEDORKA-CRAY: It will cost you a lot of pizza over time.
DR. YOUNGMAN: If we can hold the rest of the questions, we need to press ahead, because lunch is only during a certain time. So, we want to hold Tom Chiller’s talk until after lunch, and if we can, continue with questions afterward.
Otherwise, you might not get lunch. So, we can break for lunch now. Thank you, Paula.
DR. FEDORKA-CRAY: Thank you.
DR. YOUNGMAN: And Aleta will give you some information about lunch.
(Whereupon, at 12:12 p.m.,a lunch recess was taken.)
A F T E R N O O N S E S S I O N
(1:14 p.m.)
DR. YOUNGMAN: In the interest of time, are there any questions that people had from before we went to lunch, Dr. Cray question would be happy to answer them.
DR. VOGEL: I guess I raised my hand before, so I will start off here.
I guess first a compliment. Even though you criticized yourself for the lack of timeliness of the reporting, I note that the USDA report is one year more recent than the retail meat or the human arm. So, you have got 2003 on your website, whereas the other two only have 2002. So, I compliment you for that.
And I also thank you for your offer of allowing us to give you a call and ask you for a presentation of data in a different format, because I have tried to put data together that is the same for all three arms and I find it difficult. So, I will be calling you to ask for some of that data.
thirdly, just a minor kind of question I guess in the tone of uniformity of reporting. I notice on the USDA reporting you differentiate the Salmonella Typhimurium variety of Copenhagen from Salmonella Typhimurium, which is different than the other two arms.
Is there a reason why you do that and do we need to continue that? Or can we consolidate it and report it the same?
DR. FEDORKA-CRAY: We have the -- well, we receive our serotype information from NVSL, and NVSL differentiates between the two. Historically, if you look at a comparison of data between Typhimurium versus Typhimurium Copenhagen, you will notice a tendency toward more multi drug resistance and a higher resistance levels amongst the Copenhagen.
Additionally, there are different PFGE patterns associated with the Copenhagens as opposed to the Typhimuriums. I would be less in favor of consolidating them than I would in maintaining their separate, and I would encourage the other two arms to look at differentiating theirs.
Additionally, on a number of our Copenhagens, more than so the Typhimuriums, end up being the DT104. My understanding from NVSL is that this requires one additional o-antigen differentiation and it could be done. So, I would be less inclined to combine them than I would be to keep them together or to keep them separate.
DR. WHITE: I think it is something we have to talk about because I am more inclined to do the opposite, because our pulse field shows that they are indeed the same when you look at Copenhagens and Typhimuriums. But I think it is something that we can talk about. We speciate down to Copenhagen as well, but we combine them together. But I think that is something the three arms can together and agree on.
DR. BARRETT: This is Tim Barrett, from CDC. We have had the same experience. There are two issues for us. One is that we see them the same by pulse field. The other issue is that we get data from the state health departments and they don’t all test with the 05 antigen. We don’t always know whether they have tested or not. It is not clear.
We don’t feel that we can necessarily trust that information to separate them. But Paula is another situation obviously.
DR. FEDORKA-CRAY: The other thing that I should make clear is that the slides that I present are less in number than you received as your handout. This was due -- I gave you additional information and additional examples of the way we break some data out by species and sources.
So, the addition that you are going to see is a data one. Additionally, FDA gave us some questions to consider at one point in time and you will also see those on there also, and we have tried to address those over time.
The last thing that I would like to do is that I brought with me a -- what I call our unit presentation package, which is information that deals with our activities from our unit. Our accomplishments, the publications, whether they have been submitted or whether they are actually in press with page numbers and complete journal citations and then some of our other program information and three papers on NARMS, VetNet and the CAHFSE program. I will provide that to each of the panelists.
DR. MILLER: Paula, I just want to thank you for all of the work that you have done. It is an impressive nine plus years of data collection and toughing it out and making the connections with the diagnostic labs and through NAHMS and so forth.
Perhaps some of what I am asking is in the blue folder that you are about to hand out, but maybe you can summarize since a mature surveillance system really should have some impact on public policy and public health practice. And maybe, could you reflect for us what you have done and even the other parts of NARMS and any kind of public health outcome decisions, secondary questions that have been asked that have been important with public health follow up? That sort of thing.
DR. FEDORKA-CRAY: I think that some of our endeavors have certainly been with the commodity groups in the development and publication and acceptance and putting into practice actually judicious guidelines. We have done a lot of work with the industries, with the particular commodity groups, providing information back to them to develop these guidelines that they would go back and use.
We have had probably not as much of an impact on policy and that -- I am a little puzzled by that from the fact when -- for instance, when the NLOH was being developed, we provided all of the information to FDA for use from the animal arm, and the animal arm was actually excluded as part of the package. The CDC human arm was used in a lot of the decision making process at the time.
I think where we have significant inroads is in our relationship with other countries, both in an international level and with other programs within the United States. We have had the CIPARS group down, and we actually had them down four or five years ago when they were going to launch their CIPARS program.
And Rebecca Irwin said that rather than reinvent the wheel, she would come down and improve the wheel, and we agreed. I thought that was very good on her part, because we were able to share all of our mistakes and some of where we would go, and I think that you see some of that reflected in the completeness of their reports.
Rebecca has had tremendous success in integrating all of the programs, primarily because she is the head of all of that up there. So they have the ability to look more at their data. They know some of our frustrations that we have undergone and they were able to go back and talk to some of their commodity groups about that.
They recognize the importance of having on-farm antimicrobial use data from on-farm as part of the big picture. They came down and spent -- on several occasions, Anne Muckle and Anne Decker came down and then now -- I forget her name. Lucy. Yes. Comes down with the data analysis.
They looked at how we were doing our database, and we talked about what would be changed in it, and they have gone ahead and done that. By the same token then, we have learned from them. I mean, we now have reports like -- we can actually punch out some of the MIC data the way they report it, and we are excited to be able to put that up, hopefully, in the 2000 report so it can reflect; you can look then at the CIPARS data and our data.
By the same token then, we were instrumental in getting the Resistvet Project started in Mexico. It was our lab that did all of the training, like for CIPARS. We trained people and they needed training in micro broth dilution and other methodologies. We shared all of that.
We did the same thing for the Mexico project and brought that lab up, trained them; went down on several occasions. So, I believe that we have had that type of impact. We have had interaction and we provided our protocols to numerous labs, veterinary diagnostic labs in the U.S., who now follow our protocols for both isolation and testing.
All of this information then is used to make decisions in some way. So, it is more of a subtle impact than a -- I think one of the things that we haven’t been good at is touting our own horn. I think we have had tremendous impact, except when it has actually come down to using data for policy.
DR. SAHM: Paula, could you briefly describe the interconnections between PulseNet for the retail food and the human isolates in VetNet? Is it 100 percent comparisons across the two systems? Or is it somewhat selective?
DR. FEDORKA-CRAY: No. Our system is 100 percent comparable to PulseNet, in that the training is the same. The protocol is the same. One of our agreements with Bali Swami Nathan, who is the head of PulseNet, was that we would be certified by CDC to run gels. So we cannot submit gels even to our own database until we are certified and receive that certificate from CDC.
The University of Penn, who will be our first sentinel site, and NVSL, who will be a second sentinel site, will need to be certified by CDC to be able to submit TET files for inclusion in our database. Our databases are set up exactly the same way, with the exception that we have also learned from their beginning history.
So we have now started naming all of our isolates a certain way and could go back then and match them with PulseNet’s isolates. But our isolate I.D. numbers will differ from PulseNet’s. We have a mechanism by which we will be able to have cross talk between those so we can identify human and animal.
I think some of the differences is that -- well, I think the retail arm does all of their isolates for PFGE, and we will do all of the slaughter isolates initially because of monies, but right now limit it to all of the slaughter isolates. And pending monies and personnel increases, then we will do diagnostic isolates, although we have done those already on special requests. Particularly from FSIS as an interest in SE or Kentucky or some other serotype.
So, there is complete comparability between the two systems, and we would like to be able to have the same type of program where you could scan -- you know, there will be different levels of access. So, there will be administrative level of access, then there will be a collaborator level of access and then there will be a public level of access.
So all of the NARMS participants will have a collaborator level of access, internal then to USDA we will have an administrator level of access and then the rest of the public will have access to it too. They should be able
-- the hope is that they should be able to look for a pattern on ours; look for similar patterns on PulseNet’s.
What we hope to be able to do though and what we are already working towards is linking all of our isolates with all of the resistance data. So you should be able to pull up all of the resistance data, you should be able to pull up all of the demographic data, species, source, region; all of the descriptors like that from a public level.
DR. KOTARSKI: Paula, I am impressed with the breadth of this program. I wanted to ask you. If I understand correctly, the diagnostic sampling program is passive surveillance. What I would like to ask from you is in terms of that sampling strategy how does it meet the objectives for your program and what would you do to change the current system to meet the objectives and does it meet the objectives of the program?
DR. FEDORKA-CRAY: Thank you for the question. I think that the diagnostic isolates provide one level of information to us in meeting our objectives in how resistance might be arising, because those isolates from the diagnostic lab tend to be more resistant than we see from slaughter. So, if you are looking at resistance emerging, you would want to look at diagnostic isolates first I think, because that is where we see most of the multi resistance especially.
The other thing is if we talk about the concern about transfer of genetic elements between organisms, I think that is also going to be one of the first places that we probably catch these multi drug resistant plasmids that might be moving, and simply because when you use -- when you treat animals, then you know that you have the ability to maintain a resistance population more so than in healthy animals.
So, I think that on some level it does meet. What we did when we started that is we sent out invitation letters to all of the veterinary diagnostic labs, and in my discussions we specifically targeted some labs. We have had -- everybody has had the opportunity to participate.
We pay $10 an isolate actually to receive those isolates, and we give them all the data back. Some states are reluctant to do that because they fear some type of regulatory or punitive action being taken against them from different places.
Some diagnostic labs don’t have the personnel to be able to participate, and some diagnostic labs have chosen to participate with other people because of friendships that may have been developed or -- and so, they don’t send isolates to us. They will send isolates elsewhere. And some have decided to just keep everything in-house and do their own work.
We have talked with Chin-Chin Woo at Purdue. Again, it all comes down to money, but having enough money to do everything, we would move toward integrating all of the diagnostic labs so that we actually wouldn’t do any testing. What we would do is compile data. We would ask them to either switch their testing methodologies to Sensititre and try to facilitate some way getting that equipment into them so that they could then move to the same type of testing so that we have continuity of testing between all of the protocols.
Or we would ask them to send us their data or send it to a central repository where we can then access it and then we can do an analysis. If they are still doing
Kirby-Bower, we know how that compares to broth microdilution or to agar dilution or to E-Test and then do an analysis that way.
Under the idea situation APHIS would facilitate that, and we actually have a list with Chin-Chin and Dave Dargantz at APHIS. That has all been compiled. We have methodology complied from the majority of these. We have a lack of funds to implement something like that at this particular time, but that is what I would like to implement.
DR. KOTARSKI: A follow up question is between the sentinel sites from which you get diagnostics and the NVSL isolates that you receive, is there a possibility of duplication? Do those laboratories also contribute to NVSL?
DR. FEDORKA-CRAY: Those laboratories do contribute to NVSL, and what we do is we do not take isolates from those states. For instance, within New York, New York has all of the New York isolates. They will have some isolates that come from Pennsylvania, if they don’t go to Penn. From the northern region of Pennsylvania. They will have some Vermont isolates that come down there too, particularly from the state public health lab. We have some codes that we can look so that we try not to get those at all. From New York in particular, because that is the one state where we get 500 isolates from. They get about 500 isolates a year from their diagnostic lab.
Otherwise, we do not -- like we would never go to NVSL and take an isolate -- we can actually see what state they are from. We would never take an isolate from New York.
Now, what that might do is preclude us from taking an isolate from a smaller diagnostic lab in New York that might be submitting isolates. We are trying to exclude all of the Cornell isolates. This way we are also excluding some other isolates from New York too, but we feel that is safer than get duplication.
So we believe, to the best of our ability, we have voided duplication of isolates from the diagnostic labs.
DR. KOTARSKI: One more question. For the diagnostic isolates then are you getting samplings that is more representative of animal populations? Are you getting sampling that reflects outbreaks? Are you getting sampling strategy that ultimate reflects human population dense areas? Or do we know?
DR. FEDORKA-CRAY: I don’t think that I’ve done -- I mean, I know I haven’t done that kind of an analysis and I don’t think that we can say. What we do try to do is that -- we know that NVSL gets in "x" thousands of isolates. We actually get these in printouts. We don’t get it electronic. We get stacks of paper like this.
We go through and cross out all of the states that we don’t -- we aren’t going to take isolates from at all. Then we look at how many isolates are left and then from there we try to do a random, every fifth. And then we also look down in there, because you will noticed that some diagnostic labs will send NVSL.
For instance, 50 isolates on one day that may all come from the same herd, and you can actually see the accession number on there and you can see the accession date. What we will do in that instance is that instead of going every fifth or every 10th isolate or however our selection is for that particular commodity, we will actually look down through those 50 then.
We will see if any of the serotypes deviate. If none of the serotypes deviate, we will take no more than two from that one submission, and we do that simply to make sure that we don’t see differences between the isolates because we know that just because the serotypes are the same it doesn’t necessarily mean that the isolates are the same.
Then we will go to the end of that submission and then start again our every fifth or 10th or 20th isolate that we are taking. So, I think we got to some fairly extensive and extraordinary manual means to insure that we don’t over represent one submission or over represent a particular state or an outbreak.
DR. MILLER: Along those same lines of the diagnostic samples, you mentioned the rationale for continuing to collect and analyze them is they can be an early warning of emergence of resistance that will later show up in the food chain. Have you actually had examples of that where you have seen it or you were suspecting something was emerging and then you saw it later in the food?
DR. FEDORKA-CRAY: I will say that we have not used that as much as we could have in the past, but we are starting to use that and now paying more attention to what the serotypes are that are coming out. In addition, we are on a list serve for the American Veterinary Microbiologists, and they will send around from the diagnostic labs that, gee, they are starting to see, for instance, a significant increase in Uganda and are we seeing this.
And we will go back and take a look at it and see if we are starting to see this too. We really need to pay more attention to that over time, because when we go back and do retrospective analyses now, we could say, yeah, it was a good question. But yes. It is something that we need to improve.
Sue, did you have another question?
DR. KOTARSKI: No. That is fine.
DR. YOUNGMAN: Thank you very much.
DR. FEDORKA-CRAY: Thank you. And I will pass out these blue folders for each of you. I have a few extra if someone wants one.
DR. YOUNGMAN: I would now like to turn the podium over to Dr. Tom Chiller, from the Centers for Disease Control, who is going to be talking about the human arm of NARMS.
Human Arm
by Dr. Tom Chiller
DR. CHILLER: Thanks, Linda. It is great to be here to summarize a little bit about what we are doing at CDC. For those of you who don’t me, I am Tom Chiller, and I run the epidemiology side of the human arm of NARMS at the CDC.
I’m an M.D. I don’t think there are any other M.D.s in the room, which is a first for me I have to admit, because usually at these kinds of things there are lots of M.D.s. This is not about getting on soap boxes and preaching. This is about reviewing the entire NARMS system.
My only soap box issue that I do want to say is that we are here because of human health, and that is what I do. I deal with sick people, sick kids, sick adults, and so, we always need to be thinking about that in public health and why we are doing surveillance, and I think that has been brought up a few times here.
I have only been with the NARMS system now for two years, and as you have all heard, NARMS has been in existence for quite some time. In the two years that I have been here my sole goal in NARMS has been to integrate the surveillance reporting, which has not been done in 11 years. We have separate reports. We have separate systems. We don’t look at the data together.
That has been very frustrating to me, and that has really been my primary goal for the last two years; is trying to integrate and trying to understand. I think that is the only way we are going to be able to get something out of this surveillance system, is when we have some sort of integrated reporting.
So, now I am off of my soap box. What I am going to do is I am just going to tell you a little bit about the human arm. The review questions and the discussion focus more on the animal retail stuff as I looked through them. We conducted an external review. Two of you were on the panel, Lyle and Scott. So I am going to sort of review that.
As I mentioned, I handed that out to you; what the reviewers said about what we did, and also, some of our internal responses to some of the external review’s comments. So, I will rush through the background about the human arm. I am not going to spend a lot of time, and I am not going to talk about data at all.
I am just more going to give you a little bit about what we sample, what we sample and then highlight a few of the external review issues, and then turn it over to Tim Barrett, who is here and who is the leader of our NARMS lab to talk about more laboratory based molecular characterization issues that we deal with.
(Slide)
So, NARMS is located within the Foodborne and Diarrheal Diseases Branch, and we have some overriding missions in our branch. Obviously, to reduce the burden of foodborne diseases is one, and that includes diseases caused by resistant bacteria.
We do through surveillance, epidemiological investigations and applied research, and I will talk to you a little bit about all of those. And we have numerous partnerships, but our major partners in all of our activities are our state public health departments. Both from the lab and from the epidemiology side.
(Slide)
Just to see where we are organized, and I have to admit that CDC organization seems to changing daily, but for now, just within our branch, this is sort of how we are organized within the overall structure. You can see that there are -- in our branch there are three sections. Foodborne diseases, diarrheal diseases and a lab section, and within the foodborne diseases section there is an outbreak unit and a FoodNet/NARMS unit, and obviously within the FoodNet and NARMS unit there is NARMS and there is FoodNet.
As you have heard a little bit, and you will hear about it more tomorrow, Global Salm Survey within actually the NARMS group.
The lab though is within the laboratory section, and so we actually are -- NARMS is actually within two different sections, and we have managed to continue to work as one big NARMS group, despite the fact that sometimes it is difficult to involve epi and lab together.
(Slide)
Just to show you our group, this is the folks on the epi side. We have got a couple of veterinarians, a couple of M.D.s and a group of MPH epidemiologists, as well as data analysis. Terrell Miller, who was mentioned before.
(Slide)
Similarly, on the lab side we have a couple of veterinary microbiologists and then several microbiologists and technicians that do all of the hard work that you are hearing about.
(Slide)
So, public health surveillance. I like to define it as ongoing, systematic, collection analysis, interpretation and dissemination. We feel very strongly that surveillance is data for decision making, as was mentioned, or information for action. Something needs to come about as a result of surveillance.
(Slide)
I like to show this slide. Anyone who has ever seen me talk -- or probably anyone from the CDC talk, we always show this slide. We love this slide. This could be applied to a different diseases, but here I apply to the cycle of foodborne disease control and prevention.
Starting with surveillance and, as you can see, moving to epidemiologic investigations, applied research, both of which could lead to prevention measures, which you then to reevaluate your prevention measures by doing more surveillance.
(Slide)
So, let me talk about how NARMS fits into that cycle from the human side. As we mentioned, we do routine surveillance of susceptibility and resistance. We look at non-typhi Salmonella, O157, Shigella. We also look at some traditionally human-to-human transferred pathogens, like Typhi, Vibrio and then again, we have been intermittently looking at Listeria. And as I already mentioned we look at Campylobacter in sort of a separate setting in our FoodNet sites.
We also are involved, as has been mentioned, in the Retail Food Study, and we also have now an Enterococci resistance study, which has really been converted into surveillance.
(Slide)
When we look back at NARMS, at CDC back in 1996 when it began, we had 14 states that were submitting isolates to us with two different bacteria. Salmonella and E. coli 0157. There was one epidemiologist, one laboratory and we had no dedicated office or lab space and our budget was approximately $100,000 from FDA that year.
(Slide)
Here is what we looked like in 1996. You can see that some of these were FoodNet sites, and we also had other sites back then in the beginning.
(Slide)
And now, in 2005, we are nationwide. So we receive isolates from every state in the nation. We look at nine different bacteria. Campylobacter, Enterococci, E. coli. I have already said them. I won’t keep saying them. I didn’t mention Shigella, another traditionally human-to-human transmitted enteric pathogen.
We have around six epidemiologists, nine laboratorians, we have a new office and we have a nice new lab space that CDC has given us. We are budget ed at -- I put $1.7 million. I am confused about a lot of the numbers these days, but budgeted at approximately $1.7 million. That is from both FDA and CDC. That is minus the retail food money.
That is why it looks a little lower.
(Slide)
And then, of course, for those Republicans in the room don’t get excited, but this is actually to show that we are in all 50 states.
(Slide)
This shows our submission and sampling. You can see when we began. We began with Salmonella and 0157, and we asked state sites to submit every 10th. So as they went down, what they received give us every 10th Salmonella, every fifth E. coli and 0157, and we did that for a while.
But as you see, as we started layering in more criteria, of course the burden to the lab increased and we had to do then essentially dilute out that sampling to a certain extent in 2003 where we went to every 20th. When we went nationwide in 2003 as well.
That was simply because as we were adding sites we were not changing the sampling and we were getting more and more numbers, and so -- and we are also adding pathogens. So, as was mentioned earlier, there has been tremendous growth. There was growth in funds and we were able to handle that, but we had to back off on our absolute numbers to be able to handle just the numbers of organisms coming in.
Specifically, to point out in Campylobacter we collect Campylobacter just from the 10 FoodNet sites. Again, there are lots of issues, and I will go into that in a second, with Campylobacter, because that was covered in our external review.
But just in 2005, at the recommendations of the external review and our own internal evaluations, we have now switched to a slightly different sampling for Campylobacter. You can see that we do get all of some pathogens, like Typhi, Listeria and Vibrio, and that means all that state health departments can isolate they send us.
(Slide)
Important trends. We’ve seen increase in resistance to clinically important antimicrobial agents. Again, that is one of our primary concerns and what we look at. Fluoroquinolones. You have seen data on that from the animal side; Campylobacter and Salmonella. But also, including Salmonella Typhi and Shigella.
Third generation cephalosporins. We are seeing increased resistance mainly in Salmonella, but we also now see some in 0157.
And then specifically looking at increase in multi drug resistance, like the MDR-AmpC in Newport, which now -- we now have MDRMC in over 18 different Salmonella serotypes, because it is a plasmid mediated spread, as you all know.
(Slide)
I wanted to point out some good things. This is not NARMS, but FoodNet. You guys may or may not all be aware that FoodNet has reported some nice, important declines in major foodborne diseases for 2004 compared to ‘96 when they started. O157, Campylobacter, even a little bit in Salmonella have declined between eight to 42 percent, and this was reported recently in the MMWR.
This is just a graph. We have made some tremendous success in Campylobacter. It is a really good success story. E. coli and O1057 was worrying us, but is now going down. And unfortunately, the red line, Salmonella, is not decreasing, despite a lot of effort. If you sort of look at Salmonella and get an idea and break it down by serotype, you can see sort of where the issues are.
(Slide)
Typhimurium has declined dramatically. There have been no change in Enteritidis or Heidelberg and the new kid on the block, Newport, which is a multi drug resistant Newport is up very high in Javiana because this is FoodNet and has some southern states. Javiana has particularly gone off the roof in Georgia and in some of the other southern areas.
(Slide)
Again, not so concentrating on the numbers, this is older data, but I just wanted to highlight one of the things that we do and one of the things that I think we are able to do, because we have other surveillance systems at the CDC. It is overlay data, and here is where you are seeing a FoodNet data. Again, looking at Campylobacter going down, and then we look at Ciprofloxacin, resistant Campylobacter and NARMS. Again, from these same sites.
And then we are able to actually do modeled prevalence and incidents based on those two data points and those two surveillance systems. As you can see, although Campylobacter is going down, what is going down is susceptible Campylobacter, not resistant Campylobacter.
(Slide)
So, that is surveillance. Next is epidemiological investigations. We try to pay more of a role now in outbreak investigations. Specifically, I think we have played a major role in the MDRMC and Salmonella Newport story, which was really identified in human illness through NARMS because NARMS was testing for a wide variety of antibiotics.
We get involved also in case-control studies looking at sporadic infections. One was fluoroquinolone resistant Campylobacter that was recently published, and we are currently in the throngs of doing a clinical outcome study for multi drug resistant salmonella, looking at the fact that we know there is a lot of evidence now that multi resistance Salmonella has the worst clinical outcome.
I think clearly we contributed, as was already mentioned, to the risk assessment process and to the NOH that FDC/CVM did when they were looking at fluoroquinolone resistant Campy.
(Slide)
So now, to applied research. Tim will talk a little bit more about this in the molecular characterization part. I just wanted to mention that we are obviously involved in applied research in an attempt to contribute to our understanding of the epi and maybe assist in directing specific prevention measures.
We can do this because of the surveillance framework that we have, and we conduct it upon that surveillance framework.
(Slide)
And finally, to prevention. I think one of the ways that we try to do prevention or maybe I should say education is through communication, and we have a lot of partners within NARMS. Some of them are here, and we truly enjoyed the concept of an annual scientific meeting. I think as Paula mentioned.
Unfortunately, we haven’t been able to have one this year, but this does enhance communication and collaboration around certain issues. It gives us a chance to share information with stakeholders, and we enjoy that process.
We also participate in local and state public health laboratory communication efforts. We have actually quarterly conference calls, and we have quarterly conference calls with all 50 states, which no one does at the CDC. NARMS is the only group that does that.
Just to mention, NARMS is also the only surveillance system that actually receives isolates from all 50 state health labs on a regular basis. No other surveillance system at CDC does that.
So, we take advantage of that relationship. We have a wonderful relationship with the state and local public health labs, and we use these quarterly conference calls to share information, talk about issue and problems, and this is also the forum at which are now introducing the concept of pulsing all NARMS isolates.
And then, communicating with the general public is important. We have a website, as everyone does. We have an annual report and then we do individual requests, as necessary. Obviously we get FOIA-ed quite a bit by our friends and colleagues out there, but we also with a lot of individual requests from the public.
(Slide)
We also are now involved in educational activities on prudent use. We have a program that we are involved in called Get Smart, Know When Antibiotics Work on the Farm, which is part of a larger program called Get Smart, Know when Antibiotics Work, which has been in existence for about seven years at the CDC. It is very successful.
That deals with the human issues, which we are very aware is just as important, and now we are trying to partner with groups that are working in this area as well.
(Slide)
So, after eight years we are -- NARMS, on the human side, we are clearly more robust nationwide. We collect information. Some information now about travel and other epidemiological information. We found it to be an excellent platform for studies and we are obviously conducting further studies.
We have a very good established collaboration through the retail food with the FDA Office of Research, but there is clear gaps in our surveillance and they remain. It is a challenge. It is going to be a challenge to deal with those gaps as we face level funding or probably actually reductions, as we have all been talking about.
(Slide)
I heard a question earlier about how we want to think about applying this data or how it can be analyzed, and I think one of the things that would clearly help in how we look at this data is to understand the quantity of antimicrobial agents used in specific food animals. This was a high priority action item in the Antimicrobial Resistance Action Plan.
This will give us precision to interpret surveillance on all of the arms, and obviously my goal is to integrate the report so we can actually try to interpret some of this. But this use data would help us focus prevention efforts, and there is really no surveillance on that that we can get our hands on.
(Slide)
So then, let me briefly then talk about the summary of the external review. Again, this was conducted in August of 2004. You can see the participants, two of which are here.
(Slide)
We focused on some basic issues. The first was our sampling strategy for Campylobacter, and I will go into that in a second, reporting and dissemination of data on susceptibility testing, resistance in human commensal bacterial and then molecular characterization and how to move forward in that.
And just to go over sampling on the human side, obviously sampling in laboratory based surveillance -- you have to think of this surveillance pyramid, and you have probably all seen this, but I think it is important to highlight the fact that we are catching the tip of that pyramid at CDC because a person has to become sick and has to then want to go see the doctor. The doctor hast o then want to send the sample.
The specimen then has to reach the clinical laboratory, get tested, get confirmed and that clinical lab then has to forward that to the state health department and then the state health department has to record it and tell us about it. And so, whenever we think of laboratory based surveillance, we realize we are only picking the tip of that pyramid, and CDC works very hard to try to understand what is happening at the bottom of the pyramid. In other words, to estimate burden of illness.
So one of the things in Campylobacter that we wanted to do is to see if we couldn’t move toward a sampling system that helped us delineate burden.
(Slide)
It is important to know that for Campylobacter there are no routine submission of isolates to CDC. As I mentioned, everything we do is asking the state and public health labs to submit isolates to us, which has not been the norm for any CDC surveillance system.
(Slide)
When we looked at how we sampled Campylobacter before NARMS existed, there was a sentinel county survey done. In ‘89 and ‘90, 19 U.S. counties sent the first five sporadic Campylobacters per month.
(Slide)
CDC did testing, susceptibility, they did species confirmation and they found the lessons learned from that was that the sampling scheme worked very well and that the Campylobacter isolates are very fragile and some isolates didn’t survive the shipping process.
(Slide)
That is what sort of led to the system that has been place for the last eight years, because of these challenges; the survivability of these isolates, require special shipping and then just the availability.
There are few states in this country -- I think we are at 12 now -- that require clinical laboratories to send isolates to the state public health lab. So, a lot of Campy surveillance is voluntary from the human side, and so, there are actually very few isolates often at state public health labs.
So, if we were going to get a representative sample from New Mexico, they might only have five Campylobacter. Again, I exaggerate to make the point that you just don’t know how many they have, except for the states where this is some sort of active surveillance going on or where there is mandatory submission.
(Slide)
The sampling scheme started with Campylobacter in FoodNet sites. Here are the FoodNet sites. There are 10 sites across the United States, representing about 16 percent of the population.
(Slide)
The collection for Campylobacter began in 1997. Isolates came from five sites, because that is where FoodNet was. It expanded through the years to 10 sites in 2004 and sites -- five out of the 10 sends isolates from their state public health labs, and five out of the 10 actually send isolates from a sentinel clinical lab. The sampling scheme that was chosen was the first isolate isolated each week.
(Slide)
So, we asked the reviewers, given all that history, and I really threw it at you quickly and you can read about it in a little more detail in the review, should the sampling strategy for Campylobacter be changed, and if so, how? And these are some quotes and comments from the review.
Campylobacter susceptibility testing is a higher priority and needs to be maintained. However, the limitations of the current sample need to be examined/evaluated and they emphasized that changes should be made to increase completeness of submission and representativeness; e.g., taking into account sampling by population and incidence, as well as by time of year, improving percentage of isolates received per site per week, et cetera.
(Slide)
And so, what we have done is in January of this year we started a new sampling scheme, which no longer gets this one isolate per week. Sites submit a proportion now of the total isolates their lab receives; so that now we have a proportional relationship to the number, and hopefully, that will help us them estimate burdens because now again we have a representative sample that deals with a proportion.
We are hoping to expand this now to more sites in 2006. Specifically, the second phase would be sites that mandatorily require Campylobacter submission at their state public health lab, a natural place to go. Of course, that means more testing, more isolates, more money, and again, as we are discussing today, that is one of the reasons we are sort of reevaluating everything we are doing to understand how priorities lie and where that money can be spent.
(Slide)
Again, the development and the hard work by CVM to do that broth microdilution plate is clearly helping us a lot. We normally got around 500 isolates of Campy. This year we are getting closer to 1,000, but we are able to crank them out in a minimal amount of time compared to what we were doing when we were doing E-tests. So, that is going to help. It is helping us become better efficient, but still, if we then expand to another 12 states, that is a whole bunch more isolates and we are going to have to consider the ramifications of that.
(Slide)
The other question we asked was about reporting and dissemination of data on susceptibility testing, and we said what are the advantages and disadvantages of reporting data as percent resistant versus percent non-susceptible. This is a big issue in clinical medicine. In clinical medicine I don’t care if something is resistant. I care if it is susceptible because I am treating with drug and I need to know whether my drug will work.
We wondered in reporting this surveillance data should we be changing the way we report it. Of course, then that would have to change the name from NARMS to NARSTS or something, because we would be reporting the National Antimicrobial Susceptibility Testing Service.
We asked the panel what they thought. We also asked the panel how could we improve our NARMS annual report.
(Slide)
And again, just to highlight a few of the comments, looking at percent resistance versus percent non-susceptible, a couple of the quotes: "From a clinical and molecular perspective, presenting results primarily in terms of
non-susceptible compared with susceptible is the most useful."
"However, we advocate continuing to present the basic data in tables and figures in a variety of formats."
"We recognize that this change will present challenges in terms of comparison of future reports with earlier ones."
(Slide)
What we have done as a result of these recommendations and thoughts is for the 2003 annual report which, hopefully, will be out in the next month or so -- or the next couple of months. By the summer. We will present both resistant and intermediate data, and we will have it all as resistant RIS. Essentially, SIR. So we can show that and we can see what people think and what comments are made.
We are in the process of reviewing how our international peers present their data and present their findings for enteric bacteria, because we didn’t feel comfortable to just switching to susceptible -- resistant to susceptible and non-susceptible reporting, and although the panel recommended that, they did say that we should look at a variety of formats. And so, that is what we are doing now. We really want to evaluate that change before we do it.
(Slide)
Finally, we asked that other question about how should we improve the annual report. They commented on timeliness. You already heard Lyle talk about timeliness a little bit. They talked about a summary report like DANMAP or CIPARS does a nice summary report.
Again, this is an integrated summary. But they said for each arm, if you had a summary report that was a little more streamlined, that would be very helpful.
They recommended more statistical consultation and trend analysis and to develop a web based way to query the data for those interested parties and, eventually, the general public.
(Slide)
Our actions to date. We have actually really made a tremendous amount of progress in our testing. If you saw my old isolate submission slide, we got really bogged down in 2002 and 2003 because we suddenly add a lot of pathogens, added all these states. Everything sort of happened at the same time, and it sort of -- we had to recover from that.
We also, in the middle of that, changed our database format, which has been a painful effort, but we are reaping huge rewards from that, including better collaboration with USDA because we are collaborating on less political issues. We are collaborating on data and how data is organized, and actually, that is a really good way to collaborate with people to begin to build a nice foundation for, hopefully, further integrative reporting and things like this.
We will, hopefully, have our annual reports out for both 2003 and 2004 this year and then we want to release our report every June. So, it will be a six month delay. That is our goal. That is clearly feasible for us now that our database is almost done. We also are going to change the format, as I mentioned. That format change you will see in the 2003 report this summer.
We will probably have a summary report that will be much more streamlined and much thinner than our traditional report. We found a way to collapse a lot of the data. And then, we will have a more extensive web based version that will have a lot more of the graphs and tables.
(Slide)
Again, as I mentioned, future reporting for us and for, I think, all of NARMS is that we need an integrated report. We need something integrated. As Paula mentioned, we are starting by actually just formatting our reports the same, but I want to go a step further and say can we actually put something together where we are actually looking at all the data, all the three different arms together in the same graph or the same table.
And we have all agreed that that would be a great
-- that is the appropriate step. That is why the surveillance system was established. We are going to work toward doing that in a summary -- a small summary report for the MMWR next year. We are hoping that will happen, and again, we are working toward that.
As Paula mentioned, we are making tremendous strides in the database integration, in the sense that we are designing our databases -- they are almost identical and that is huge when it comes to cranking out reports that are similar. Then our goal is to make our data web based and accessible by the year 2006, although I don’t know if that is really going to be feasible. So I put ‘06/’07.
There are a lot of hoops to jump through to make data accessible in this day and age of security. When you have a server housed within the firewall, it changes things a lot. You are going to have people accessing that server. So we have to work on a lot of issues like that, but it is going to be feasible and we are working hard toward doing that.
(Slide)
I won’t really go into resistance on human. I have talked probably too long already. Again, the review--and you can read it more extensively in the document--said it was important to continue to monitor resistance in commensals to determine the role of these bacteria as reservoirs of resistance determinants for human pathogens.
And to this end, it is important, where possible, to integrate monitoring and epidemiological data; e.g., antimicrobial use from animals, food and the environment, as well as humans.
(Slide)
NARMS should focus on the important commensals, like Enterococci and E. coli, and use susceptibility panels that, at a minimum, include those drugs important to human health, especially if members of the same class are used in animals.
(Slide)
And now, I will turn it over to Tim, and please, hold your comments or questions until Tim is done. Tim will go into a little bit of the molecular characterization issues.
DR. BARRETT: I just have a few slides. I am going to summarize the sorts of things that we were doing at the time of our review and are continuing to do now, and some of the things that we have changed as a result of our external review back in August.
As far as molecular characterization, we really are involved in three types of characterization, and I will mention something about each of them. Strain characterization. That would be subtyping, like PFGE. Identified of specific resistance determinants and the characterization of the resistance-mediating elements, being plasmids, transposons, et cetera.
(Slide)
For strain characterization, for Salmonella, as I mentioned to Lyle’s question about Typhimurium, we don’t serotype all of our Salmonella isolates. We assume that the state serotyping is correct by and large. But when something is important that we want to know that we are reporting a particular isolate serotype correctly, then we will confirm that serotype at our reference laboratory.
We do PFGE typing for special studies. That would be there in our laboratory. And we phage type Enteritidis and Typhimurium isolates for outbreaks and for special studies. For Campylobacter we are PFGE typing. We will, hopefully, finish soon the 2003 collection.
We can compare our isolates with Dave White’s retail food isolates, and after we have had a chance to see what we think that information tells us, then we will decide whether we need to do any different methods or additional methods. And again, we do PFGE on Campylobacter for special collections.
(Slide)
For Enterococcus the only strain characterization we do is to confirm the resistance. A lot of our isolates come from selective media. So we presume that they are resistant, but we confirm that. And, we do speciation.
(Slide)
For identifying specific resistance determinants one of the things we have been interested in, primarily because of the Salmonella Newport emergence, is Salmonella with decreased susceptibility to cephalosporins. We have done isoelectric focusing for beta lactamases. We routinely do PCR for the blaCMY2.
As you probably already heard or probably already know, most all of them have the blaCMY2 gene. We are also beginning to do some PCR and sequencing for other type of beta lactamases, which I will mention more about.
(Slide)
As far as resistance determinants for Salmonella with decreased susceptibility to ciprofloxacin, again we confirm the phenotypic results. We did a good bit of PCR sequencing for gyrA and parC, and we haven’t seen anything remarkable to this point. It is probably not something that we want to continue to do routinely.
We are currently collaborating with David Hooper on potential plasmid-mediated ciprofloxacin resistance, and I will mention more about that also.
(Slide)
For Campylobacter we are not routinely characterizing any genes in Campylobacter right now. At one time we sequenced a collection of QRDR genes from fluoroquinolone resistant isolates. All of the mutations that we saw had already been reported and it didn’t like anything worth continuing to do for a long time.
Likewise, we identified macrolide resistance mechanisms in a subset. They were all 23S mutations, as have already been reported, and we screened the subset with a multiplex PCR for TET determinants. They were all tet-o, as has already been reported. So we have decided that we
probably don’t want to do any of these things on a continuing basis.
(Slide)
A couple of years ago it looked like we might be seeing an emergence of E. coli 0157 with ACSSuT phenotype. Fortunately, that hasn’t happened, but we characterized the specific genes involved there by PCR. And for Enterococcus we are identifying mechanisms of resistance for quinupristin/dalfoprostin and, as Dave mentioned, we are seeing the same thing that -- probably most of our isolates don’t have one of the already described mechanisms for vancomycin and for high level gentamicin.
(Slide)
As far as characterizing the resistance mediating elements, this is something that we felt was largely outside of our area of expertise. So, we have been collaborating with others to characterize plasmids from cephalosporin resistant Salmonella and from the E. coli O157.
(Slide)
The questions that we asked our reviewers, which included Dr. Vogel and Dr. McEwen, was whether we should continue to focus on problems that would be things that we perceive to be important, such as the Salmonella Newport and on unusual isolates. Or, should we be more surveillance oriented? Should we try to find out something about everything as opposed to details about a few things?
And if that was the case, then what should we say survey. And finally, should we emphasize special collections that we have, such as FoodNet studies, for example, or outbreaks and is that what we should focus our efforts for extensive characterization?
(Slide)
Well, their response was generally that, yeah, we should continue to focus on the problems and we should continue to look for unusual isolates or unusual mechanisms. Although we may not be in a position to really define those ourselves, we are in a good position to recognize them.
So, one f the things that we have done is we have started using the SWIN Software that operates the Sensititre. It has the capability of flagging interesting isolates, and so, we have set up a number of things to get flagged so that we see this right away and don’t wait until we are looking at the data several months later.
One of those things is isolates that have reduced susceptibility to both extended spectrum cephalosporins and quinolones, and we are flagging those and -- I don’t remember who is doing it from Tom’s group, but is following up and trying to get some clinical information; travel. That sort of thing on anyone with that kind of an isolate.
We are also interested in isolates that have reduced susceptibility to ceftriaxone or ceftiofur that doesn’t look like -- phenotypically doesn’t look like it is mediated by CMY-2. That would be isolates that are not resistant to cefoxitin or to amoxicillin and clavulanic acid, and we have had a few of those.
When we see these situations, we screen for the sort of usual suspects of mechanisms by PCR, and if we don’t find them to be among our usual suspects, then we look for other people; for collaboration.
(Slide)
A good example of this is we have three isolates that apparently have a qnr, a plasmid mediated quinolone resistance gene, and we have three Salmonella. They are an ogana, abobis*, morbificans* and mendoca*. David Hooper’s lab has been doing most of this work, and we hope we are getting near publishing that information.
We are collaborating with Alesandro Carratoli in Rome to have a PCR based plasmid typing system all of the internet laboratories. Many of them are participating in a study that we are also participating in, and we hope this will prove to be a useful method for plasmid tying.
And we have worked with Dr. Fred Tinover’s lab for characterizing the bla-tem and the bla-shv genes, which I will mention in a moment here.
(Slide)
One of the things that I had in mind when I asked the question about whether we should be doing surveillance -- this is a good example. This is one that I think we needed to do.
It occurred to us that Salmonella that had the
blaCMY2 mechanism may have other beta lactamases and we wouldn’t be able to tell from the phenotype that we see, because the cmy-2 expression will mask any SBL of the phenotype. So we took all of the Salmonella -- well, in fact, all of the shigella that had reduced susceptibility to extended spectrum cephalosporins, and we did IEF and did some PCR, and we found, not surprisingly, that most of them did have the blaCMY2.
But there were 10 Salmonella that, in addition to the cmy-2, had a bla-tem gene. This is by PCR, and it is just a generic bla-tem. We didn’t identify which one. Two had only a bla-tem, one had only a bla-shv, and interestingly, that was the Mendoca isolate that had the qnr.
It looked like the presence of the bla-tem or shv was distributed among a number of serotypes that didn’t have any particular geographic relevance or anything that struck us as being -- you know, telling us a lot of information that it was worth the effort to go through to get. So we decided we probably don’t need to continue doing this.
It was worth finding out and maybe we will do it again in a few years, but we don’t need to do this every year.
(Slide)
As far as special collections, just to give you a couple of examples of what we are involved in, we are currently receiving isolates from every Salmonella outbreak in FoodNet sites, and we are receiving isolates from the study that Tom mentioned on the burden of multi drug resistant illness compared with people who have infections with susceptible strains.
(Slide)
And as far as PFGE, we are encouraging the states to do PFGE on all of their NARMS isolates. We have made this request several times. Most states actually do their Salmonella isolates anyway and many of them have agreed that they will do their NARMS isolates, if they are not already doing them.
As it turns out, most have already been done and they are submitted to PulseNet, but a lot of the time the states don’t use the same numbers for PulseNet and for NARMS. And so, we have this data already and we don’t know it. So we are making a big effort to get people to use the same number so that we don’t have to reinvent the wheel and repeat tests.
We are making further efforts to link PulseNet, FoodNet and NARMS data. I think that is all. Yes.
DR. SAHM: Yes. I guess first, Tom, I take your point early on about doing this kind of surveillance work on human isolates because these are the ones, we already know, have made it all the way to infecting the human population and what those strain characterizations are.
But in terms of part of the NARMS goal of integration that needs to happen, you showed some very interesting data about over-trending what has happened with Campylobacter and Salmonella and how Salmonella still remains relatively constant. And then amongst the Salmonella in 2004, how it was mostly -- I can’t remember the -- Javiana?
DR. CHILLER: Yes. Javiana.
DR. SAHM: Javiana. Well, back when I went to school there was only two species. But anyway, can Paula look at her data or look at any of the other two arms to see, well, geez, you know what? That is the strain or species that is most common in the retail products and is this reflective of what is happening in the animal arms of NARMS? Or is that integration just not there yet to even ask that question?
DR. CHILLER: That is a good question. When I was referring -- the majority of my referring to integration was from an annual report. So I was thinking of when we actually submit this or we present an annual report, that we present that in a yearly basis in an integrated reporting fashion.
What you are talking about is actually sort of more the detective work that we do when we see something interesting, and I think that that kind of detective work and that kind of collaboration we have definitely been doing for years. We will call Paula and we will say, have you seen this increase? She will look at her data and she will say, yeah, actually I have seen this.
I think the classic example is with Salmonella Newport when it was emerging in the late ‘90s. She was seeing it in animals. We didn’t have much of a retail food study at the time. There was just the pilot studies in D.C.
And so that kind of collaboration? Absolutely. That kind of integration we do. What I was really referring to is the kind of integration that I would like to see; is actually in a surveillance standpoint where there is ongoing reporting of these three arms looking at the data side by side in some sort of systematic fashion, and that we don’t do.
So, we will look at our data. We will see something interesting. For example, the javiana issue. Javiana we don’t think is coming from food animals. I don’t think you see it much in food animals, and we certainly don’t isolate it from any retail meats.
Well, I can give you my estimation of where we think it is coming from, but we don’t think it is coming from food animals; reptiles and things like this. It is a southern oriented issue. You don’t see it in New York for example.
So yes. We definitely call each other and ask those questions, and certainly NARMS has been a fruitful way to dig deeper into specific issues that come up.
DR. SAHM: I think the interest to the public and the scientific and medical community would be when is there and when is there not a correlation between what is being seen in food animals and what is being seen in human health to help clarify, because the other thing that points at it, interestingly, is it has to do with the question I asked Paula.
One of the recommendations from our external review is to continue, by all means, doing surveillance with commensals, such as Enterococci. But in human health the VRE rate is high and regular and there has been on the animal side. So, having that kind of information at their fingertips might give them a better perspective on what should and shouldn’t be done and going forward.
DR. CHILLER: I think you bring up a great point. I think one of the reasons we are here today to ask this question about integration. But I think, as Sue brought up earlier, you see different objectives because, honestly, we have some different objectives besides some common objectives.
With Enterococci we are finding VRE now from community isolated stools, and do we think that VRE is coming from analog use in animals? No, it is not. It is banned here. I mean, we don’t think that it is coming. But the fact that we are documenting that it is in the community and there may be now sort of a community reservoir of this within humans is important to know, but it may not necessarily be important to test for in animals, where it is important to maybe test for vancomycin in humans.
So there are some specific issues that sort of fold off of NARMS in each of arms that are specific to us. So you bring up a good point, but I think part of what we are doing here today is understanding how is the integration and how is the surveillance in these three arms to be linked together to actually inform us about food and the food supply and how resistance is moving back and forth through that system.
DR. SAHM: And I think that that is what I am a little bit fixated on today, is how the information coming from the three branches could address not only what is going on in foodborne diseases, but in terms of association. I mean, what is going on in the ecology of animal and humans.
One last question then for Tim. With all of those things that you are doing, which is a pretty impressive menu of characterizations that you are doing, is there any effort to connect with Paula and other arms so that you would have the same line listings of characteristics for strains across all three arms so that she could see whether or not there is qnr, plasmid qnr in the Salmonella that she is seeing so you could cross compare strain characteristics?
DR. BARRETT: To some extent we have different purposes. For example, the ‘tem’ and ‘shv’ issue. We looked at that. We don’t think there is really anything there. I mean, I might ask Paula have you done -- do you happen to have this information? But I wouldn’t go ask her to go and do a bunch of work when we don’t really think there is much of interest there.
Now, as far as things that we do think are of interest, like Salmonella Heidelberg, for example, we have talked with Dave and Paula about what we have seen in Heidelberg and how we could do the same things and do just what you say to try to put those together.
DR. FEDORKA-CRAY: Actually, we have a study ongoing with Jean Richard with Salmonella Heidelberg, and she has characterized --- and we are finishing the characterization of the animal one. We are going to link the two together and put out a joint publication.
DR. SAHM: I think that would be something that is very valuable. If you know these are the molecular and phenotypic characteristics of the Salmonella that you are seeing in your system or the retail food arm, versus what do the Heidelbergs look like from the CDC arm, I think it would be very valuable in terms of differentiation and molecular characterization. Thanks.
DR. KOTARSKI: Just a follow up question on that point. Are you looking for similarities between the isolates that you see in the human versus the animal isolates? And also, when you see dissimilarities between the two, are you reporting those?
I think, in all likelihood, when you find a similarity, you are going to bring it to the attention of the world. When you see a dissimilarity, how does that get a prioritization?
DR. BARRETT: That is a good question.
DR. FEDORKA-CRAY: I think I would answer that to say it doesn’t ---
DR. CHILLER: Again, one of the issues is that we may -- we on the human side may decide that this is an incredibly important resistant phenotype and we really need to look into it, and we may stomp the pavement and call our colleagues and say can you guys look into it. And it may not be a priority for Paula or Dave. It may come 10th on their list.
And so, part of that is we may think it is number one and we need to know about it now. Paula might say, yeah, it is interesting, but I have got 10 other things that I am looking at within my system now.
And so, that has led to us really focusing on probably the similarities, because for us to get to the differences then that goes way down the priority list and it becomes even more of a challenge. But I agree that both of those concepts need to be talked about.
I think that in this whole integration scheme we need to figure out some way that if we have something that is a high priority or she has something that is a high priority, each of us tries to make that a high priority for each other. I think that is what we need to figure out exactly how to do.
DR. KOTARSKI: And if that is a future goal for the integration of three arms, can you -- I think back to the population base that are being sampled. So, for example, for the animal isolates that are collected, some of those animal isolates are coming from the HACCP Program, and I think we are going to hear more about that and that sampling strategy.
In fact, in terms of what is coming from healthy animals, we are getting isolates every four years from a particular species. So, if that is the case, in terms of what is in the healthy animals that, presumably, are going into the food chain, if we are only getting isolates every four years and you start making comparisons to what is similar, then are you making similarities to what is actually on the farm or are you making similarities to something that is closer to the consumer, which might be the slaughter plant? And how do you trigger that information then?
And I am not necessarily asking for a question, because that is future based. But in terms of thinking about integration, I guess I would ask, as you make plans, is how representative are your samplings for the on-farm, for the slaughter plant and then for humans and in between?
DR. CHILLER: I think it is a great question, and hopefully, you guys will continue to battle that question after the next presentation of Neena, because it is a question that we have too. Anyone else?
DR. ALTEKRUSE: Picking up on that, some of the isolates that we -- Paula mentioned there is a lot of heterogeneity in the slaughter isolates. It is the most, perhaps, heterogeneous group of isolates and some of them really kind of fall by the wayside in terms of concern, but some other isolates are of tremendous concern to us.
A very good example is the reduced susceptibility to beta lactam Salmonella Heidelberg isolates. I think that there has been an interest in that at FSIS, and it would be helpful to have more timely feedback on that, on the status of those investigations; what are being found.
Right now most of our information relates to what we have heard from CDC, but that is just some feedback about the HACCP isolates that are going into the NARMS system. It would be tremendously useful in terms of charting agency policy direction to have a heads up on the status of that very early, and really, frequently in terms of updates, not just to wait for a publication.
(Nodding)
DR. MILLER: Thank you for the presentations. It is obvious a lot of excellent work has gone on in the year since I have been involved with NARMS. I just wanted to highlight a couple of points that I really agree with just to kind of go on the record.
I think that, Tom, you were able to really demonstrate the surveillance to the epi investigation, to the applied research and then back through prevention, and that is what I think would be ideal to be able to show for all the arms of NARMS; so that there is clear outcome use of the data; thinking and questioning that leads to other kinds of public health interventions. So, that was really nicely stated.
Obviously I agree with the integrative reporting and some of the questions that have been raised I think are good to be thinking about in terms of how to do that. Timely reporting obviously, and it sounds like you are moving towards a six-month turnaround, which would be excellent.
The other item is kind of a pet issue for me going back to working on the interagency antimicrobial resistance working group. Trying to get drug use information on the farms to link with the healthy animal data and to be able to interpret, in light of the other surveillance information.
I know it is a huge hurdle and some countries do it very elegantly, and I don’t know if anyone is really actively working on that. AVMA or not.
But that is an important piece and that will really help to make that full circle to be able to talk about outcomes and then impact use and ultimately resistance.
DR. CHILLER: Absolutely. Thanks.
DR. BARRETT: I would like to mention one more thing in terms of timeliness. Tom already alluded to this. The laboratory being behind in generating the data has been the major factor in our being behind in reports.
We got terribly behind in 2002 when we increased the number of sites without decreasing the frequency. So we got a lot more organisms, a lot more bugs in and we had problems with Sensititre. A whole lot of things went wrong that year.
As of now we are up to date now. We are up to date for 2005. So hopefully, we will stay there and we will be able to get this done faster.
DR. CHILLER: What Tim is saying basically now is that it is my fault that we are late. And that is fine. I will take the blame for that.
DR. VOGEL: This may be an unfair question, but I did send this article to you. There was a perspectives or opinion article published in Nature’s Review on microbiology in December of 2004. The title was, "Epidemiological Interpretation of Antibiotic Resistant Studies. What are we Missing?"
The abstract says that "surveillance programs examine changes in the proportion of isolates that are resistant. Although proportions are helpful for the clinician prescribing empirical therapy, proportion based analysis can be misleading to the public health professional, as they can yield biased estimates. Proportions do not adequately reflect the burden of resistance, a measure often of interest in public health."
"A more appropriate measure of this burden is the rate of isolation of resistant organisms. That is, the absolute number of resistant isolates in a population over time."
Have you had a chance to look at that article, and what is your interpretation? Could our surveillance system be changed to give us --
DR. CHILLER: Thanks. I definitely looked at that article after you sent it to us. Without going into a lot of detail, I think one of the -- for example, one of the changes that we made in Campylobacter I think will help us. I mean, we did it specifically to be able to address rates and burdens.
Clearly, when you are looking at proportions, which is what we do, because we simply can’t test every bug that comes in, we then put that in and look statistically about how that relates to a specific absolute number. And so, although we are collecting a certain number of isolates and we are reporting that as percent resistant, we are trying to determine whether we can put that into a model that allows us to determine rates.
I think that part of the issue with NARMS’ nationwide surveillance, for example, for Salmonella, is that it is difficult to put a denominator under that right now, and I think part of the advantage -- as Tim mentioned and others, of taking advantage of an active surveillance system like FoodNet where they actually go out and they count every single case in the community. I mean, they know. They know every single case that has been laboratory confirmed; to be able to put our surveillance on top of that framework.
And I think we are going to be able to get it at absolute numbers and we are going to be able to make better rate estimations. And after our external review with you guys, that is one of the things we said. You know, let’s try this now here on a platform where we think we can realistically do that.
And so, we have been involved with statistical folks at CDC now for the last six months hammering out how we might go about it. So yes. We are working on that, and I appreciate you bringing that up.
DR. ALTEKRUSE: I saw an example of that artifact. The isolates have been for --- they are really not representative of the population at large.
DR. CHILLER: FoodNet weeds that out and NARMS does now too. We are now asking about travel history because of that exact reason. But you are right. FoodNet takes that data right out of there, and we can then exclude that and we can focus on national data or we can look at just travel populations too, which is really important, because we know a lot of resistance comes in from overseas. No question about it.
DR. YOUNGMAN: Thank you very much. We need to keep pressing ahead, because we are kind of behind schedule right now. What I would like to propose is we have another talk and then, Terry, if it is okay with you, we will have a break before your talk.
I would like to introduce Dr. Neena Anandaraman, who is going to be talking about the animal isolate sampling for NARMS.
Animal Isolate Sampling
by Dr. Neena Anandaraman
DR. ANANDARAMAN: I am going to be talking about FSIS’ contributions to NARMS in just a little bit more detail than Paula went through. First, I will just give you a brief introduction to the agency and its lab and then I will talk about the Salmonella HACCP verification testing program. A
lot of you are probably already very well familiar with it, and then I am just going to briefly go over some other programs within FSIS that contribute isolates to NARMS.
(Slide)
As you all probably know, FSIS regulates nearly 6,000 meat, poultry and egg processing plants nationally. Samples from these establishments are sent for microbiological, chemical and pathological analyses. Of course, today we will be concentrating on the microbiological part.
We have got three labs. One is located in Athens, Georgia, and that is the Eastern Lab, and it is housed in the same building as Dr. Cray’s lab. Then we have the Midwestern Lab in St. Louis, Missouri, and finally, we have the Western Lab in Alameda, California.
(Slide)
In FSIS we have two categories of testing that we do. One is baseline testing, and this testing is actually weighted by production volume and it is statistically designed to estimate national prevalence levels. The other is our HACCP verification testing program, and this is a compliance testing program and it is done to verify that establishments are meeting regulatory performance standards.
(Slide)
Our HACCP verification testing program does provide the bulk of FSIS derived isolates to NARMS, and the same year that NARMS was established, in 1996, coincidentally FSIS also published its PR/HACCP Systems Final Rule. One of the provisions of that rule is that plants would meet prescribed performance standards for Salmonella and that FSIS would conduct testing to verify that these standards were being met.
The performance standards were based on prevalence estimates from baseline testing that was conducted in the early to mid 1990s, and if you are interested in more on the baseline studies, they can be found on our website.
(Slide)
So, prior to implementing our HACCP rule, which was published in 1996 and implemented in 1998, we conducted what is called pre-implementation testing. With pre-implementation testing we were trying to get an idea, both for FSIS and for the establishments, where they stood prior to implementation.
This testing was not statistically designed to estimate prevalence levels, and isolates from this program were sent to NARMS.
(Slide)
We have more information on the pre-implementation testing program in an article by Dr. Schlosser and also on our website on several documents listed.
(Slide)
So then, HACCP was implemented in stages, depending on plant size. We define large plants as having 500 or more employees. HACCP was implemented in 1998 in those. And then in 1999 in small plants, and small plants we define as having 10 to 499 employees.
For very small plants which have less than 10 employees or less than $2.5 million in sales we implemented in January of 2000. So, as of 2000 all sizes of establishments were included.
(Slide)
So, sampling for HACCP is done on an ongoing basis after the establishment’s implementation date on forward. Product class, whether carcasses or raw ground product, determines the number of samples that are collected per set and it also determines the performance standard.
Once an establishment is targeted for testing, the inspection personnel at that establishment collect a daily sample until the set is completed. Then the samples are shipped chilled the same day of collection.
(Slide)
Then there are two different categories of testing that we do. Routine testing, the ongoing testing, is what we call "A" sets, and for "A" sets establishments are scheduled approximately every six to 12 months and this is ongoing and routine. Sometimes you will see it referred to as random.
The "B", "C" and "D" sets are what we call targeted testing. These are conducted if an establishment fails a set. So, if an "A" set fails, a "B" set is done. If a "B" set fails, a "C" set is completed.
(Slide)
So, just for each of the product classes I am just going to go through how much comprise a sample set and some details of those. Fifty-one samples comprise a broiler carcass set. So, for the broilers we randomly sample after immersion chilling and we do a whole bird carcass rinse and then save 30 milliliters of the rinse fluid, which is then sent to the labs for analysis.
(Slide)
For raw ground products, for chicken, turkey and beef, we have a 53 sample set. We collect our 25 grams after final grinding and also send these on to the lab for analysis.
(Slide)
For the cattle and swine carcasses we have 82 samples for young cattle, 58 samples for mature cattle and 55 samples for swine. These are chilled carcasses which are randomly selected after 12 hours of cooling and then we swab three different sites on the carcasses. We then put the swabs in a sterile bag and send these on for further analyses.
(Slide)
This just summarizes the seven product classes that we test, the number of samples comprising a set and the maximum number of positives that are allowed. Again, this goes back to our baseline prevalence estimates.
So, for example, for young chickens they are allowed to have 12 positives out of a 51 set sample before the sample fails. If they get 13 positives, then a "B" set would be collected.
(Slide)
Our laboratory analysis can be found in our Microbiology Laboratory Guidebook that is on the web. Also, if you have more detailed questions on micro methods, Dr. Bonnie Rose is here. We confirm culturally all of our presumptive positives and then we send the isolates to APHIS’ National Veterinary Services Laboratory in Ames, Iowa. Once they have serotyped it, they send the serotype information back to the originating lab and then the isolate, along with the serotype information, is sent to Dr. Cray’s lab for antimicrobial susceptibility testing for NARMS.
(Slide)
So, this is just to give you an idea of the number of isolates that we have sent through the HACCP Compliance Testing Program over the years. Since 2000, when we have had all plants on board, we have collected approximately 40,000 to 50,000 samples a year. These are the numbers of Salmonella isolates that we then forwarded on to ARS.
(Slide)
Just some of the limitations to interpreting data derived from the program are that it is not statistically designed to estimate national prevalence levels, like our baseline studies are. We did have staggered implementation. All of the plants were not included until the year 2000.
And then, for the first several years of sampling we really heavily concentrated testing in the first half of the calendar year and we are not really taking into account any seasonal variations that may occur.
(Slide)
And then we have a couple of publications, and also, our yearly web report that talks in more detail about our testing program.
(Slide)
And just a little bit about some of the other programs that have contributed isolates to NARMS. For Salmonella we have had several egg baseline studies that we have been sending to Dr. Cray for analysis. Also, we have been sending isolates from our Ready-to-Eat Program.
(Slide)
For Campylobacter we have had a couple of programs in the past that have contributed isolates to Dr. Cray. From 1999 to October 2000 we had our young chicken microbiological baseline data collection program, and that baseline study is going to undergo review by our advisory committee, the National Advisory Committee on Microbiological Criteria of Foods.
And then since that study ended in October of 2000, in October of 2001, as Dr. Cray mentioned previously, we have been giving her spent rinse samples from our broiler rinses in the Eastern Lab, which is housed in the same building as her lab, so that she can isolate Campylobacter, generic E. coli and Enterococci.
(Slide)
Some of the descriptive information that we have provided since July 2002 with our isolates are product class information, the date of collection, region, the plant size, the set and serotype.
(Slide)
So then in summary, our verification testing program does provide the bulk of the isolates that we submit for NARMS, and though it is not a statistically designed program for looking at national prevalence levels, we feel like it provides very good information. It is quite robust.
And we do plan on forwarding any isolates from future baseline studies to NARMS for susceptibility testing. Any questions?
DR. KOTARSKI: You stated -- I would just like to compliment first on a nice presentation and quite a robust program. You said the program does not provide national prevalence data on Salmonella in meats, the different meats? It does not provide that information?
DR. ANANDARAMAN: Well, I’m saying it is not designed specifically to provide that information. It is not designed to estimate national prevalence. It is for regulatory compliance. So we are doing the program to track plant performance, rather than trying to get a national prevalence estimate.
By extension, it may be providing us with that type of information, but we just want to make sure everybody understands the limitations; that it is not specifically designed for that purpose.
DR. KOTARSKI: For clarification then, by extension it doesn’t provide us national prevalence of resistance in Salmonella, if that is the source of the samples?
DR. ANANDARAMAN: I can say that it is not designed to do that. Whether it is doing that? It may be, but I can’t say for sure.
DR. ALTEKRUSE: It is not an unbiased sample. That is the issue. On a periodic basis establishments are tested. Sometimes those tests take longer to complete because of a whole range of issues that are involved. So, you couldn’t take this percentage and say that is the percentage of resistance to this strain in these Salmonella or even that these are the percentage necessarily by serotype 100 percent. It is not an unbiased sample. It is a regulatory driven program.
One aspect of that is that we have already indicated that we are going to, in likelihood, go towards a more risk-based approach to Salmonella. So we know already that some plants have high rates of positivity and some have very low rates of positivity. And so, based on those performance standards that you saw, we are going to increase the frequency of sampling in plants that have high prevalence and decrease it in plants that have low prevalence.
So, in fact, it is going to become even more biased in its approach in the future. But one of the things that we want to try to do is to establish a parallel of sort of ongoing surveillance activity. But then, as everyone has pointed out today, money is the issue. So the design of that parallel program to obtain national representative data is going to be driven by how much funding we have.
DR. WALKER: I have two questions. Number one, on these broiler rinses that go to the Eastern Lab, are those only from the eastern United States? Do the Midwestern Labs receive isolates from -- or samples from the Midwestern and the Western states from the western states?
DR. ANANDARAMAN: Well, what happens is when a set is scheduled, it is randomly assigned to a lab. So there is a variety of states going to the Eastern Lab. It is not only eastern.
DR. WALKER: So, the eastern lab could end up with samples from Oregon or Washington?
DR. ANANDARAMAN: Sure. Absolutely.
DR. WALKER: And when you talk about shipping samples to the lab, what is the conditions and the transport time?
DR. ANANDARAMAN: Are you talking about the isolates themselves or --
DR. WALKER: The samples.
DR. ANANDARAMAN: The rinse samples?
DR. WALKER: Yes. Or the ground beef or --
DR. ANANDARAMAN: Are you talking about the spent rinse samples for Campylobacter, E. coli? Or for Salmonella isolates?
DR. WALKER: Well, for the boiler carcasses you have the rinse samples and you say shipped to the FSIS lab or the ground beef you ship to an FSIS lab.
DR. ANANDARAMAN: Okay. For the rinses that we are supplying after the Campylobacter baseline testing was completed, those aren’t getting shipped because they are only getting sent from the Eastern Lab. So the Eastern Lab -- they are walked down to Dr. Cray’s lab.
DR. WALKER: From the point of collection.
DR. ANANDARAMAN: Right. What happens with the --
DR. WALKER: Like the Oregon sample. If it were going to the Eastern Lab, how long would it take it to get there?
DR. ANANDARAMAN: Well, it is shipped overnight. It is Fed Ex’ed overnight.
DR. FEDORKA-CRAY: They are shipped overnight and processed the next morning, and as soon as they are done being processed, we get it. And they are kept refrigerated the entire time that they are --
DR. ALTEKRUSE: They are rejected -- Bonnie would know this. But they are rejected if they are above a certain temperature.
DR. WALKER: But have you ever taken like Campylobacter and ran it through it that scenario to see how well it survives?
DR. ANANDARAMAN: We are only doing that with Salmonella isolates. With the Campylobacter we only get those out of the rinse samples that are in the Eastern Lab. In the same building. We don’t ship that.
DR. WALKER: But those rinsates could come from Oregon to the Eastern Lab?
DR. ANANDARAMAN: No. No.
DR. WALKER: Just the chicken, non the rinsate.
DR. ANANDARAMAN: Oh, you’re talking about the rinsates for FSIS.
DR. WALKER: Right.
DR. ANANDARAMAN: What happens is the rinse -- once the rinse sample is collected it is refrigerated and it is supposed to be sent --
DR. WALKER: In Oregon?
DR. ANANDARAMAN: In Oregon. And it is supposed to be sent by noon that day Fed Ex’ed to our lab. So, it is Fed Ex’ed overnight to our lab so it doesn’t get there on a weekend.
DR. WALKER: So my question is have you ever done any studies to see how well Campylobacter survives that type of --
DR. ANANDARAMAN: Have we, Bonnie, that you know of? I’m not sure.
DR. ROSE: They are shipped -- all the samples, the rinses, the ground product and the sponge samples from the cattle and swine carcasses are shipped in insulated shippers with frozen gel packs. They are delivered overnight by Fed Ex to the FSIS labs.
DR. WALKER: Right. I appreciate that, and E. coli and Salmonella is probably not a problem. Or Enterococcus. They will survive, you know, standing on their heads. But Campylobacter is more fastidious and doesn’t survive as well.
DR. ALTEKRUSE: Well, one thing we do know -- we have done work occasionally with isolates or with rinses that were above the temperature that they were supposed to be brought in at, and Campylobacter doesn’t grow well in those.
We are able to isolate Campylobacter and enumerate it from rinses that are shipped overnight and under eight degrees ‘c’. Otherwise, they are rejected. What that means is that the -- an inspector in the plant is given another sample request form, and that one doesn’t count against the performance set.
DR. ANANDARAMAN: As far as specific studies done on Campylobacter survivability, we would have to check with the labs. I am not sure if we have or not.
DR. FEDORKA-CRAY: bob, we know that when we initially started to get the samples from FSIS we had a low, what we considered a low, recovery rate of Campylobacter compared to what we would have expected. We were getting about 11 percent.
What we ended up doing was we took the 10 mils of rinsate and we concentrated it by sonerfigation* step. Then we took the pellet and enriched that, and we brought our isolation up to 20, 30 percent. That is the current isolation rate that they are getting out of plants now. So we know that we are comparable to what they are seeing and it depends upon which plant you are getting these from.
Are these the ideal samples for Campylobacter? Probably not. Are they only thing that we have that would even give us a chance of getting Campylobacter? Right now? Yes.
DR. ALTEKRUSE: One additional item, if I could, and that is that keep in mind these are from birds that have just come from an online reprocessing system where they are going through some pretty harsh chemicals. So, in effect, I believe that they are not ideal samples. Under the best of circumstances, that you are dealing with injured cells as the nature of the organism. And perhaps by the time you get to retail it has some opportunity of recuperation. That is a big question right now; is what to do about damaged cells on poultry rinse carcasses.
DR. FEDORKA-CRAY: Right. And we are doing several studies with that looking at taking, in particular, those rinsates and what happens when you put them under different gas conditions or different growth conditions to see. And we have also taken a number of these rinsates and concentrated and just done PCR on them to see if we can get any Campylobacter DNA out of them at all, and all of these data are being put together and we hope to be able to have some story that we can tell from this.
The fact that we are getting any Campylobacter at all I think is, like Sean said, somewhat fairly remarkable, considering the condition that they are taken off right out of the chill tank.
DR. VOGEL: I guess to me that raises the question that if these poultry carcasses have been exposed to TSPs and other type of treatments, antimicrobial treatments, what we are getting may be preselected for resistance, --
DR. ANANDARAMAN: That is a good point.
DR. VOGEL: -- because there can be cross resistant between these antimicrobials that are used in plants as sanitizing agents and the antimicrobial drugs that we are using. So we may be biasing it towards a resistant Campylobacter.
DR. ANANDARAMAN: Unless we had some more information that we could look at those sorts of things with the isolates we wouldn’t be able to tell.
DR. ANGULO: Is every HACCP isolate sent from NVSL? Do the FSIS labs send all HACCP isolates to be serotyped to NVSL?
DR. ANANDARAMAN: Yes, they do.
DR. ANGULO: And does NVSL send every isolate to ARS for NARMS?
DR. ANANDARAMAN: Right. NVSL actually doesn’t send the isolate. They just send the serotype information back to the FSIS lab and then FSIS sends an isolate with the serotype information to ARS.
DR. ANGULO: What proportion of the HACCP isolates are "A" set isolates?
DR. ANANDARAMAN: About 90 percent.
DR. ANGULO: Have you looked to see if the other than "A" set isolates have a remarkably different resistance pattern than the "A" set isolates?
DR. ANANDARAMAN: Not yet. No.
DR. ANGULO: Because one of the easy reductions in sampling could be that just only -- in terms of NARMS. Because of this bias. It is the same plant being retested in the B and C and D sets. You can consider just submitting only the "A" set isolates into NARMS. That would reduce it by 10 percent, about 320 isolates per year, if there is an inherent bias of that.
DR. ANANDARAMAN: That is a possibility.
DR. ANGULO: Another possibility is to not sample -- not send every isolate. I mean, we do one in 20 Salmonella isolates from ill people. Has anybody done a power calculation to see what extra information is created; how much precision is created by testing 3,200 isolates this calendar year versus 1,400 or versus 1,200 isolates?
DR. ANANDARAMAN: No. I don’t think we have done that calculation.
DR. ANANDARAMAN: No.
DR. ANGULO: And the last extension of that question is how much cost savings -- how much cost saving would there be by reducing the sampling? If the shipping of every single isolate is $10.00 or something and every plate is $10.00, there could be important cost savings from reducing the number of samples tested.
DR. ANANDARAMAN: Those are costs that FSIS absorbs though. It is not coming out of the NARMS budget.
DR. ANGULO: But the plates. With the plate, NARMS purchasing the plate, and the technician time. I just question how much value is there testing. This year was a low year. That is because the positivity rates in everything except broilers is declining. I guess it was only 3,500 this year?
DR. FEDORKA-CRAY: It was only 2,500 this year, and I think part of it -- the other thing that you have to look at is this is split between -- I mean, there is chicken and then there is turkey and there is beef and there is pork in there. So it is not just 2,500 chicken isolates.
And then the other thing is that with what we are seeing now with our PFGE is a lot of diversity amongst our isolates, and that would be lost if we were cutting down. So, how would we know exactly which one to cut down? We have to run the PFGE, I would think, for at least two years to be able to take a look at that to make sure that we weren’t losing something there.
DR. ANANDARAMAN: I just wanted to add one more thing though on these numbers of isolates being different. Sometimes what happens too is that we may start a few sets at the end of one year and then a bunch the end of the next year, so it may appear that there are higher numbers of samples in the year that most of the set at the end of the year was collected.
DR. ANGULO: But this is a great discussion. It seems to me that the isolates from beef are so valuable because you get so few of them. Maybe the selection should be every one of the cattle isolates from HACCP.
But since you get so many isolates from broilers, I don’t know that -- I wonder if we -- maybe you could have a selection -- it seems there could be a relatively easily imposed sampling scheme at the FSIS labs. Tell them to send every other chicken isolate instead of every chicken isolate.
DR. ALTEKRUSE: If I could comment on that?
DR. ANANDARAMAN: Sure. Go ahead.
DR. ALTEKRUSE: Actually, we are very committed to serotyping all of our isolates. You don’t know what you have until you have serotyped it, and we want to provide that information back to the establishments.
DR. ANGULO: That makes sense.
DR. ALTEKRUSE: So, if you only take one tenth of isolates and give them information on it, you are giving them much diminished information.
DR. ANGULO: But this is a great discussion. It seems to me that the isolates from beef are so valuable because you get so few of them. Maybe the selection should be every one of the cattle isolates from HACCP.
But since you get so many isolates from broilers, I don’t know that -- I wonder if we -- maybe you could have a selection -- it seems there could be a relatively easily imposed sampling scheme at the FSIS labs. Tell them to send every other chicken isolate instead of every chicken isolate.
DR. ALTEKRUSE: If I could comment on that?
DR. ANANDARAMAN: Sure. Go ahead.
DR. ALTEKRUSE: Actually, we are very committed to serotyping all of our isolates. You don’t know what you have until you have serotyped it, and we want to provide that information back to the establishments.
DR. ANGULO: That makes sense.
DR. ALTEKRUSE: So, if you only take one tenth of isolates and give them information on it, you are giving them much diminished information.
DR. ANGULO: But I wasn’t talking about serotyping. I agree completely. Serotype all of the isolates. I was talking about the -- the burden is on the resistance testing and the cost inherent to that.
DR. ALTEKRUSE: Right. Well, there is no cost of sending them because the two laboratories are housed right there together.
DR. ANGULO: No. There is three regional FSIS sites.
DR. ALTEKRUSE: Okay. Fair enough.
DR. ANGULO: I am just raising these points about economies of scale.
DR. ALTEKRUSE: And let me follow up on it, because I am interested in Paula’s thought on this, because we know that a lot of our isolates are not the same isolates that are found to be causing human infections. And so, the question is do they -- should they have the same priority for NARMS that other isolates that are from the top 20 serotypes that cause 65 percent of the human illnesses we see?
For example, we keep on talking about Kentucky and Derby as examples of that.
DR. FEDORKA-CRAY: I think maybe we can crunch some numbers. But if you look at the -- one of the ways I look at the bang for the buck, if you will, is you look at the number of isolates that go through the animal arm of NARMS per year and you look at the Salmonella isolates, the Campy, the Enterococci and E. coli, look at the $1.5 million IAG, you take out $150,000 and that is $1.35 million that actually goes to the laboratory.
You look at about 5,000 to 6,000 Salmonella isolates, 1,000 Campylobacter, 2,000 E. coli and 1,500 to 2,000 Enterococci and we are up to 10,000 isolates. So you take those 10,000 isolates, you look at the amount of money that you are getting from it and you look at all of the other programs and they have all of the other associated costs with sending money to labs and how many isolates that you do at the end of the year, and I think that right now the savings is not going to outweigh the information that we would generate for the program.
We have 2,500 isolates that we are getting in for the slaughter samples. The other thing is that we would have to look and make sure that they are regionally representative, because in addition to just taking every 10th, you would have to make sure that you were getting an even distribution amongst regions.
I think that we talked biasing, and if you are going to have higher plants that are -- if you are going to have some plants that are going to have repeated problems and you only take an "x" number of isolates, then you are going to have the probability that you might exclude even more of the other plants that you could be testing.
I am not -- just in looking at it -- not just because I’m part of the animal arm of NARMS. But just in looking at it I don’t think that reducing the number of slaughter isolates that we do now, until we get some of these other programs up where we have the robustness going from farm to plant, is going to save us any information.
DR. ANGULO: It will save us money. That is the point. You need 10 percent less money. How are you going to cut it? You have got to cut sw. That is my point. Where would you cut? It seems to me some sampling scheme could be imposed. I am just trying to find savings somewhere.
DR. FEDORKA-CRAY: And I agree that I think we have to find savings, but I think that this would include us sitting down and looking at some of the raw numbers and asking exactly where some of that money and information is going to. We have all had a reduction over the years in the amount of monies that we have had to expend. So, it is not something that is being unequally borne by any one arm right now.
DR. KOTARSKI: As a follow up to Dr. Angulo’s and Dr. Cray’s discussion, I come to a question I asked earlier. What is the objective for the slaughter isolation monitoring component of the NARMS?
Regardless if you want to cut 10 percent arbitrarily or if you say I am going to cut the repeated trustings, I think it will help you if you go to your objective. Do you want to reflect what is actually seen at the slaughter house? Do you want to reflect your prevalence in the slaughter house? Do you want to reflect prevalence on a national basis?
What is it exactly that you are looking for and that will help guide where your cuts will come, if they need to come.
DR. ANGULO: I agree completely. The problem is though -- and I think it would be great to be explicit about that. The problem is though you have to take advantage of the isolates that are available. In this instance FSIS isolates are free. There is a system to sample them already.
So, while I agree completely with your point, we would love to have a random representative sample nationwide, but we can’t create a whole new sampling scheme. We have to use available isolates.
DR. KOTARSKI: And in no way am I trying to suggest that we recreate a sampling scheme. But since all of the samples right now, if I understand correctly, are going to Georgia, there is a subset that are actually on a randomized basis.
If you just looked at those sample sets "A", do those reflect any information about prevalence at the slaughter plant? What does that sample set represent?
DR. ANANDARAMAN: They are less bias definitely.
DR. ALTEKRUSE: But they are going to become with risk based verification, and that is the reason why there is an interest in FSIS in adding another layer, which would be a baseline, an ongoing baseline to try to get at that information, which would be nationally representative.
DR. FEDORKA-CRAY: And we are going to be involved in this upcoming baseline, as in subsequent baselines, and actually getting all of those isolates and testing them. It would be along the same lines of the NAHMS and CAHFSE program where you have nationally representative studies and sets of isolates coming in so that we can move away from bias and move to report a more national representation.
DR. ANANDARAMAN: But I agree with Paula, in that until we get those programs going, this is all we really have to look at over time. And if we start changing now, it may not be as comparable as to what we have seen in the past.
DR. YOUNGMAN: Thank you very much. We are going to be talking more about sampling after the break. What I would like to propose now is that we take a break now and continue talks after the break.
I would like us to come back at 3:30 if we could. We are also proposing to move the last talk for today to tomorrow morning. Dr. Zhao has agreed to that in the interest of time.
So, we will take a break now until 3:30, and then we will have two talks after the break. Thank you.
(Whereupon, at 3:16 p.m., a recess was taken.)
DR. YOUNGMAN: If we can start again, we are trying very hard to keep things on time. We have a few changes to the agenda as written. One is that the last talk that was planned for today we are now proposing to do tomorrow morning. Dr. Zhao will be talking about the molecular characterization tomorrow morning. That means that after this break we will only have two talks today, and hopefully, finish around 4:30, which is when we had originally planned to finish today.
What I would like to do before Terry Proescholdt comes up to talk about the retail meat sampling is to just remind the panelists in particular of the six questions that we had posed to you before this meeting. The latest version of those questions has a heading that says discussion points, and these are just a brief rundown of what those six questions are.
So if, as people are talking and as you think about things tonight and tomorrow morning, if you could be thinking about those six questions and what your individual responses would be, that would be really helpful.
Also, at the suggestion of one of the panelists, Scott McEwen, I thought what might be helpful to each of you is that tomorrow to make a bit of time at the end of the day for each of you individually to give us some of your responses to the six questions and other comments that you had for us. So, we will give you time tomorrow at the end of the day to each individually give your responses.
But just briefly, the six questions that we hoped we could get your individual responses to are: Is the current sampling process by FSIS adequate and effective for Salmonella surveillance? We have already heard some comments about that.
Are these samples adequate and effective for Campylobacter? Campy dies more quickly. So, how do we address that?
What other pathogens should or should not be isolated and susceptibility tested from the poultry rinsates? What makes sense? Again, these are brief versions of those questions that are on your pages, those six questions.
If additional sampling is suggested, please consider what we should stop doing in NARMS since no additional funding is available. It really comes down to some hard choices. If we are going to add new things, if we are going to do a better job of a certain type of testing, what do we stop doing?
Are the current sampling strategies for the retail meat arm of NARMS adequate as currently conducted? You will be hearing more about that in a minute.
Is reporting sufficient to be able to use data from the three arms effectively for public health surveillance? We have had a lot of discussions about trying to make the databases more uniform so you can more easily use data from humans, from animals, from retail meat.
How would change the reports to accomplish this so that the three arms can be used more effectively?
What should be retained and/or omitted in future reports? There is a lot in all of the reports. What are the pieces that work? What are the pieces that don’t work? How can we do a better job?
What are the top three elements of a well coordinated collaboration for the molecular characterization of isolates from all three arms? You are going to be hearing more about that tomorrow. You heard some about that today.
Molecular characterization from all three arms so we can demonstrate or refute a continuum from animal, food and human origins for specific pathogens and/or resistant phenotypes.
The fifth question: How should NARMS be involved in international efforts? We are going to hear about that tomorrow.
How do you suggest that we enhance and sustain funding for NARMS? We know that our appropriated dollars are very likely going to be going down. What can we do about that? Are there other ways that we can raise funding or other programs that we can combine somehow?
So, those are the six questions that we would really like to hear your responses.
Now, if I can turn this over to Dr. Terry Proescholdt, he is going to be talking about the retail meat sampling scheme and particularly focusing on the Iowa Pilot Study.
NARMS Retail Meat Sampling
by Dr. Terry Proescholdt
DR. PROESCHOLDT: Thank you. Actually, I am going to start with the Iowa Study, because that is where we did start, and then work up to today.
(Slide)
Our pilot in Iowa was a statewide collection. We collected the same meats as the retail arm is right now. One difference was we collected fresh or frozen product. Fresh if available. Frozen if not. The retail arm now, I think, collecting only fresh is an improvement.
In our pre-pilot we tried collecting all ground meats, and this kind of speaks to your question, Dr. Vogel. We found that all ground meats, even though they would be easier to work with in the lab, didn’t work to collect. There was no ground chicken at all. And in the smaller grocery stores, about half of them did not have ground pork. They had ground sausage, but the extra spices and whatever would just complicate the bacterial picture.
I almost expected somebody would ask about the turkey, and I tried to look it up. The closest I could find was the National Turkey Federation web pages. They said the number one product was whole turkey, which is impractical, both budgetarily and it is very difficult to put a whole turkey in a Whirl Pak bag and shake it up.
The other thing they said, but there were no numbers involved, is that the ground product is the most rapidly growing in popularity. But I cannot find whether it is number two or number 10 as far as total volume. I couldn’t find it from Ag Marketing or the Economic Research arms of USDA either. I could tell you what per capita is and this sort of thing. So, I don’t know.
Anyway, this was laid out to be a 50-week sampling, but it was not 50 consecutive weeks because we had major holidays, like Thanksgiving, Christmas and 9/11 and that sort of thing that got in the way.
(Slide)
Bacteria of interest. The first three are being done now. The fourth one we looked, instead of generic E. coli, for extended spectrum beta-lactam resistant Enterobacteriaceae.
(Slide)
We bought a list of all the chain and single store groceries in the state. From that we took a random sample of 300 grocery stores. Let me backup a half step. From that list I went through it manually to remove any obviously inappropriate stores, because the listing then was a little less exact than it is now.
I will come back to that, but there were some convenience stores, nutrition stores and that sort of thing that crept onto the list. Those were easy to remove.
We took a random sample of 300 stores. From those 300 stores we divided the state into five quadrants of 60 stores each just grouping it by latitude and longitude. Then we grouped again by latitude and longitude six stores into a route. The quadrants were sampled sequentially to help with the seasonality. The routes within the quadrants were done randomly.
We also had a list of randomly chosen backup stores, which became very useful because convenience stores still were on our list here.
The meat was shipped on ice overnight to CVM for work up. Until 9/11 it was shipped as checked luggage in a cooler on an airplane.
(Slide)
That is how the store layout looked like. You can see where the major population centers are. That is Des Moines, Waterloo, Cedar Rapids, Iowa City.
(Slide)
For the retail meat of NARMS for the first three years it was convenience sampling. The FoodNet labs were asked to sample at least one store per month, to purchase as many different brands of meat as possible and not to revisit the same store for at least two months. Again, they bought 10 samples of each meat type each month.
(Slide)
What we are doing this year instead is a stratified random sampling. The FoodNet labs were contacted. They were asked, how big an area are you willing to collect from? From that area, again, we bought store listings for everything within the zip codes or counties that they specified.
We ended up with between 30 to 300 stores per state, depending on the areas given us. The listings now are much more sophisticated, at least visually, in eliminating the convenience stores. I did again visually inspect and remove inappropriate stores. There were a couple of wine stores. One smoke shop. The Aldi chain had to go because it has nothing but frozen meat.
The stores then were sorted into four quadrants by latitude and longitude, except for California. They are sampling three full counties. So we used them as quadrants.
(Slide)
Then I randomized the order that the quadrants would be sampled and that sequence is repeated sequentially. Again, for the seasonality. The stores within the quadrants were randomized, and for each month the FoodNet people got a list of five primary stores and three backup stores.
Any stores that weren’t put on those two lists were put on an ultimate remainder list just for a second set of backups.
(Slide)
If there were more than 60 stores in a state, the primary store was only sampled once a year. If there were less than 60, obviously it had to be seen again. But I made sure it wasn’t sampled twice in the same month. When there are greater than 96 stores, the backup stores were only sampled once a year.
(Slide)
In the store the FoodNet people were asked to collect two samples of each meat type in every primary store if they could, and also, if they could, try for two different brands or lots of each meat types. And then those samples that they could not collect in the five primary stores they were asked to collect in the backup stores.
(Slide)
This isn’t perfect yet. We found out that all of our listings missed the big discount stores, like Super Target, Wal-Mart, Sam’s Club and Costco. Tennessee pointed out to us that there were a number of small mom and pop grocery stores that are also missed.
I think this came in because the chain store guide only picks up single stores that sell a $1 million or more a year and chain stores $2 million. It is probably one of their ways of eliminating convenience stores.
From a quick talk with them I think we can add the discount stores in for next year. I don’t know if we can add the small ones in, but I will follow up on that. I want to try to encourage the states with the smaller number of stores to increase their collection areas so we can have some more stores to work with.
They have been quite good working with the system. Initially there was more than hesitation. Tom did a wonderful job with selling them into agreeing to this and looking at the log sheets that have come in they appear to have done a good job of staying with the list.
And again, I will keep getting rid of the inappropriate stores so they don’t have to be bothered by going to something they can’t get their samples from. But that is it in a nutshell. I hope I helped you catch up a bit.
(Pause.)
DR. YOUNGMAN: Are there any questions for
Dr. Proescholdt?
(No response.)
DR. YOUNGMAN: Okay. Thank you, Terry.
Our next speaker is Dr. Elvira Hall-Robinson, and she is going to be talking about how the data is reported and collected.
Retail Meat Reporting
by Dr. Elvira Hall-Robinson
DR. ROBINSON: Good afternoon. For those of you who do not know me, my name is Elvira Hall-Robinson, and I am the manager and coordinator for the retail meat database activities. Today I will be talking to you about the retail meat database, the current reporting of all the NARMS Arms, and also some future plans for a more collaborative report.
(Slide)
First, I would like to start with the retail meat log sheet. After the sample is actually collected, as Terry mentioned, each meat type has a separate log sheet that is submitted from the FoodNet sites. This logsheet is sent to the Office of Research monthly or quarterly electronically. In addition, the original log sheet is Fed Ex with the isolate to the Office of Research.
Part one of the log sheet is the demographic information on the meat sample, and you have the sample I.D., the store name and city, the brand name, the lot number, whether the product was cut or ground in the store, the sell-by date, the process date and the lab process date.
Part two of the log sheet is where the isolate information is recorded from the meat sample, and it is recorded as a growth of yes or no. Once it is recorded as "yes," the sites assigned isolate I.D. number that is identical to the sample ID except a C for Campylobacter, “S” for Salmonlla, “EC” for E. coli and “E” for Enterococcus is added the sample ID number to create the isolate ID number.
We have developed protocols for the sites on guidance for completion of this log sheet, for shipping of the isolates, and a methods protocol, so that the information from site to site is consistent.
(Slide)
That was the log sheet used from 2002 to 2004. In May of 2005 a new log sheet was created so that the brand name and plant name information would remain at the site. We also added on the log sheet an organic column, and the reason we added this field which is not part of the sampling plan, but we noticed that some stores were organic that we were not a where of and they ended up on the grocery store chain guide list that Terry mention. This is column was added to distinguish this type of meat.
We also assigned a code list to 173 brand names so that we would not have misinformation. This is information is actually going to be at the site. So, prior to the site sending us the log sheet they remove brand name and establishment number, and we do not have that information anymore.
(Slide)
Now, the database that we used to mange the data for the retail meat is Access. The reason that we chose Access is it’s component of the Microsoft office suite that is loaded on all the CVM computers, It will allows easier exchange of information between Word, excel, adobe, and SAS, Access is user-friendly, we can get timely reports as the data is entered. Disadvantage is the security and the number of users using the DB at one time.
(Slide)
This is an example of the USDA’s front page. They use Access as their front page and also use Access as their back end.
(Slide)
This is an example of CDC’s front page, and CDC uses Access as their front page, but they use SQL Server as their back end. With the retail meat, we use Access as our front end and back end, but looking at using Oracle or SQL as our backend and Access as the front end. I think both USDA and FDA are looking at using another relational database as their back end because of the power.
(Slide)
This is actually an example of a model that we could use, as to how the data for the three could be brought together. One thing about relational databases is that you can bring other partners’ tables in. Okay? As you see this model, this actually was developed by John Stelling, which Paula mentioned in her talk when she talked about reporting. We have a data analysis for USDA, a data analysis for CDC and a data analysis for FDA.
There are tables in the back end of this that actually links to those particular tables from the three arms. You don’t have to integrate the actual data, but you can have their tables in it to create reports that are very similar.
(Slide)
Now, I would like to talk about how the data for the retail meat is actually managed. I am going to through our database and then talk about reporting, as far as what is the current reporting with all three arms, and then go into what is in the future. Actually, that Paula and Tom has already mentioned.
(Slide)
Again, this is our front page and we have three buttons. We have the data entry button, search data and the reports button. As far as the data entry, the fields are very similar to the monthly log sheet that I went over prior to this.
The information from the log sheet is just extracted as it comes in electronically. So, once it comes in, it actually gets entered into the database, which means we can get prevalence data out very quickly.
Again the brand names are going to be changed from a name to a code. We will not be recording any information on the lot number field.
(Slide)
Now, the second part of the log sheet, as I mentioned, was part two, which is the isolate information, and that is here. What happens is the isolate I.D. is actually extracted down here into the database, and once the site recovers the bacterium, then they actually put in the yes or no and an Isolate ID is entered.
Once it becomes a yes, the sites actually assign an isolate I.D. number. This isolate I.D. number is very similar identical to the sample ID except if Salmonella is isolated we put an "s" on the end of it, for Campylobacter we put a "c" and for E. coli we put an "ec", and for Enterococcus we put an "e" on the end of the isolate ID number.
Once the isolate comes into the Office of Research, it is assigned a CVM number, which is going to be changed to what we call a NARMS number. We are calling it a NARMS number instead of a CVM number. What that number is will link the sample data and isolate data to the MIC data.
(Slide)
Here is the actual form for the MIC data. Currently the data that we are collecting is the susceptibility method, the drug, the MIC sign and also the MIC number goes here and then we determine whether the isolate is Susceptible, Intermediate or Resistant.
(Slide)
Now, the second part of the database is the search data, and we can search the data by state, sample I.D. and we can also search by CVM number. If someone called up and said can you actually find this isolate in the database, what is going on with the resistance, we can actually find it very quickly.
(Slide)
The last part of the database, the last button, is the reports button, this button is linked to our reports page, and there are different types of reports on this reports page. We have formal reports, there are bar graphs for MIC distributions, there is a percent positive sample tables, pivot tables and then there is percent resistant pivot tables that we can convert to a pivot chart.
The main buttons that we really use a lot is the percent positive samples that we provide on the quarterly conference call with the FoodNet sites that go through CDC. The other button we use a lot is the percent resistant button.
(Slide)
Here is an example of the percent positive samples by state and meat product for Campylobacter and Enterococcus for 2002 and 2003. This is an example of that, and that is in your book.
(Slide)
The next button that we use I mentioned before is the percent resistance, and this is the pivot table for the percent resistance. We can convert the pivot table to the pivot chart from that pivot table, and we can just hit a button and it actually we can get that chart.
So, the blue bars are your percent susceptible, the burgundy bar is your intermediate susceptible and the yellow bar is your percent resistance.
(Slide)
As far as the current reporting published on the web, both CDC and USDA published reports in 1997, as they mentioned. USDA has published a different variety of formats for their reports and they have some data for 2003. CDC published similar reports from year to year, from 1997 to 2002, and they were published, as Tom mentioned, this summer, their 2003 report.
As far as retail meat, we have published the 2002 annual report last year, and it looks like we are going to be publishing the 2003 report this summer and then hopefully, at the end of the year publishing the 2004 data.
(Slide)
As far as all of the reports, most of the reports cover all or most of this; the interpretive criteria, the frequency positive samples, the frequency of the percent resistance for antimicrobials, also the MIC bar graphs, MIC distribution, multi drug resistant tables and multi drug resistant patterns. In addition, the retail meat for the 2002 report we published PFGE patterns for Salmonella and Campylobacter.
(Slide)
As far as future reporting, as Paula and Tom mentioned, FDA, USDA, CDC and CIPARS met in March of this year to discuss comparing reports of all the three U.S. agencies. During the meeting, we discussed an overview of our databases. We actually share databases; so that we could actually share queries, we can share coding and all of that.
We also discussed developing a new report and, as Tom mentioned, the level of reporting. Should we be looking at drug versus drug class or both? How do we define multi drug resistance, dichotomization? As Tom mentioned, looking at susceptible and non-susceptible resistance.
Also, putting confidence intervals into the percent resistance tables and modeling the Danish and the CIPARS reports. In the meeting we discussed strategies for presenting and coordinating the reports. We talked about executive summaries, web based tables, graphs and reports, as Tom mentioned, and paper-based reports.
One deliverable we hope to have is a collaborative report that is going to be published in the MMWR in 2006 for the 2004 data.
(Slide)
After the meeting we the USDA and CDC programmers John Stelling, Terrell Miller, Tom Chiller and Lauren Stancik came up to the Office of Research and we actually decided to put together a report layout of what a collaborative report will look like. The NARMS working group meeting that occurred in May we decided to present this and this is the percent positive resistance for Campylobacter with the three agencies: CDC, FDA and USDA.
I only actually put this out to Tetracycline because the chart was too large, it would not fit.
(Slide)
And that is all I have. Are there any questions?
DR. KOTARSKI: I have a question as a point of clarification. You had mentioned early on, in terms of the data entry, there was a column or an entry regarding whether or not the meat was cut in the store or not or cut or ground in the store, and they would answer yes or no. What is the significance of that entry?
DR. HALL-ROBINSON: That is a good question. Before I came the log sheet was actually created. But I guess the importance is to know how a product is handled as far as the process of the product itself, if there a more contamination rate based on how the product is handled and processed. Your pork chop is cut in the store, and usually the hamburger is ground in the store. That is routinely done.
DR. KOTARSKI: Okay. And that is reported out?
DR. HALL-ROBINSON: We have not actually published any data on that at this time.
DR. KOTARSKI: Okay. Is there any reason why you have decided not to report whether it was cut in the store or not?
DR. HALL-ROBINSON: We haven’t even talked about it. There is no reason why we don’t publish it or not publish it. We haven’t discussed it.
DR. KOTARSKI: Thank you.
DR. McEWEN: Maybe Terry is the one for this question or maybe you can take a stab at it, Elvira. When it comes to integrating the information that you get from the human side of things and retail and also slaughter I guess, if you -- the tendency will be, I guess, to see if we have something pop in an area. In people, let’s look at the retail and see if we have a comparable sort of phenomenon there.
The tendency would be to treat them as kind of catchments areas I guess and the assumption being perhaps that what is in the retail stores will reflect human exposure, peoples’ purchasing practices in those areas and exposure. If you sort of go further I guess, another assumption might be that what goes in through the slaughter houses through that region will turn up in retail.
I wondered to what extent you have given the thought to investigating the sort of food distribution system to see how it reflects that sort of catchment area?
DR. HALL-ROBINSON: I don’t think we have actually looked at the food distribution, but I can say that as far as when we were collecting the information on the plant that some product for example is process in Indiana may be distributed in Michigan, from the same processing plant. So we haven’t, looked at that information at this time.
DR. WALKER: I think an example of that is when we had that first BSE positive cow in, I think, it was Washington or Oregon. How many states had the meat from that animal been distributed to?
It is a very good question, but I think it would be extremely difficult in this country because of the marketing practices.
DR. HALL-ROBINSON: Products can get to California very quickly, within hours or days.
DR. KOTARSKI: Another question or point of clarification. So the sampling strategy that you have currently for 10 states now, that reflects human populations in terms of exposure? Or does it reflect the number of stores? What is the population base you are representing by the selection of the stores?
DR. HALL-ROBINSON: Terry, I don’t know if you want to explain that as far as when we came up with the -- how we can up with the randomized sampling, because there were some limitations with the sites.
DR. PROESCHOLDT: Are you asking why the 10 FoodNet states or why the areas within each state?
DR. MILLER: What does it represent I would like to know?
DR. KOTARSKI: Yes.
DR. PROESCHOLDT: What does FoodNet represent?
DR. MILLER: No. What does your sampling --- represent?
DR. PROESCHOLDT: It represents Tom; correct me if I put my foot in my mouth. It represents as big an area as we could force the FoodNet labs to cover.
DR. CHILLER: It basically we have 10 FoodNet sites. Some of those FoodNet sites are actually statewide. Other FoodNet sites are very localized. For example, in California it is the three bay area counties. And again, we are not here to talk about FoodNet. That is a whole two and a half day lecture in itself.
But essentially, FoodNet is active surveillance for defined regions within each of these states. So FoodNet goes out. They know every single clinical lab in those regions and they ascertain every month the number of lab confirmed cases of these pathogens, which is why -- when I showed you the model data and E. coli O157 going down and Campylobacter going down, that is all based on FoodNet active surveillance so they can make statements about incidents, unlike when we are just doing lab based surveillance or passive surveillance.
It is harder to make statements about that. They can then estimate burdens because they know the populations living in these counties and they know every single lab based organism coming in.
So what we do in the retail food study is we tried to go after those exact areas, because that would be nice. Again, you guys are getting at the exact point. It would be nice to relate that back to a population where we are actually studying disease, and ultimately, that is what we would like to be able to do.
Some sites we are not willing to drive all over the same catchment areas that they are calling clinical labs to get. In most states we actually are in that same catchment area. So we have taken that chain store list from that catchment area, randomized all the stores within that catchment area based on the chain store list and they are buying meat essentially from stores where they are also collecting active surveillance data on disease.
DR. MILLER: And what proportion of the population does that represent?
DR. CHILLER: FoodNet represents about 15 percent of the U.S. population, and so, it is going to vary in each state, depending how big the catchment area is in each site.
DR. MILLER: Have you done any work in comparing the outcome from the retail meats and looking at human disease in that same area?
DR. CHILLER: Not really. We have begun to look at some interesting issues. I think Dave mentioned one where the prevalence of Campylobacter seems very geographical distinct. So they might isolate a ton in Maryland little in California or vice-versa, and we wondered if that related to prevalence in humans.
And we looked at that, and it didn’t seem to relate to prevalence in human disease. So then we wondered -- I mean, it is -- yes. It is never that simple. You would like it to be a simple association. Of course, it never is.
So then we wondered well, maybe they happen to be buying more organic or maybe they are grinding differently. I think as Bob mentioned and as Scott eluded to, it would be nice to sort of trace, to trace distributors, but distribution in the United States ends up being a 15-legged spider very quickly and things just tend to go out.
So we have -- but we are interested in these regional differences. We are trying to understand why can one state have half as much Campylobacter prevalence on chicken than another and why do some states have a lot more Campylobacter illness than others. So we wondered is there a relationship to the food that we eat or is there something else.
These questions have been generated by this data, but unfortunately, we are not answering them yet. But we are trying to methodologically work out answers to those things. The nice thing is having this retail food study within FoodNet and having the FoodNet investigators who are very committed to understanding these issues and questions provides a wonderful platform for pretty intensive research questions into these various issues.
DR. WHITE: If I could add too? I was invited down to the FoodNet meeting this year to talk about NARMS retail for the first time. So, we were able to talk to them about the data we had and to start coordinating using that data with their active disease surveillance. So, we are starting to go ahead with that.
DR. YOUNGMAN: One thing that I was going to add though is -- I mean, it is not just that we are doing this work and we don’t see any correlations that make sense. For example, the most prevalent serotypes that you find in retail meats aren’t the same most prevalent serotypes that you find in humans and so forth. So, there are associations that we are seeing that make sense.
DR. CHILLER: Yes. I definitely didn’t want to give the impression -- you’re absolutely right. It has been very interesting. As Dave mentioned, we just now are coming along with three years of data from a convenience sampling.
In any surveillance system, as you all know, it is hard to really know how to evaluate the first year. You are working out a lot of methods and testing, especially in complex lab based surveillance systems where you are getting labs and sites involved. Now that we have three years of data and now that we are more comfortable with that, we are now making a lot of effort to look at things together.
I think, as Linda mentioned, one of the first things you can see right away is there are some top serotypes that we are finding in human food. And in Paula’s. I mean, we look at Paula’s as well there. So there are going to be some commonalities and there is going to be some major differences.
Kentucky is one serotype that comes to mind that they are finding a lot of and we don’t find much human illness. So, there are a lot of questions being asked essentially by the data and by comparing data, which is great.
DR. VOGEL: I think this question is either for Dave or Terry. I don’t know that I wasn’t listening. What is happening with the sample size on the retail over the three years and in the future?
In 2005 I understand you are going to randomized. But is the sample size, the number of meats selected, changing or increasing?
DR. WHITE: Yes. I can answer that. The numbers have increased over the number of states that have been involved in the program. We started off with six states in ‘02, and remember, they are doing 40 meats per month. So that is 240 meats per month in ‘02. Now we have 10 states doing 40. So, we are 400 per month. So, we are up to 4,800 meats.
That is plateaued. We have no more states to add. So for the current time that is what we have.
DR. VOGEL: So, what are you getting for serotype numbers in 2004? For example, all you have is available is the 2002. You had eight isolates of Newport that year. Well, you can’t tell a whole lot about resistance trends or prevalence if you have only got eight isolates.
DR. WHITE: I agree completely. But in a way that is good that we are not seeing that much Salmonella in the retail foods. 2004 serotyping is undergoing right now. So we don’t know yet. But hopefully, we will have three years of data to compare.
Unfortunately, as we talked about earlier, the majority of our Salmonella is coming from poultry and we rarely have any Salmonella from a ground beef product or a pork product. So those numbers, from year to year, are only in the single digits.
DR. McEWEN: (Microphone not turned on.) -- according to classification of drugs --- human importance may have come up?
DR. HALL-ROBINSON: Yes, it did.
DR. McEWEN: I just wondered what you folks thought about that, whether you considered doing that at all.
DR. HALL-ROBINSON: Well, I know that FDA -- and, Dave, you can step in at any time -- actually had important drugs with the Guidance 152. So the question is do we use that or do we use another classification of important drugs that CDC used.
I think Tom mentioned at the meeting we talked about that. I think it was WHO. We were getting together and coming up with a list of drug classes.
DR. CHILLER: Yes. It is a great question, Scott. CIPARS, as you know, does do that and we like that. We started talking about that concept at this joint meeting when all of us were together. Lucille and others were there from CIPARS.
We were less -- we thought that maybe the best way to approach that, if we were to present drugs, is in classes of critical importance. So what this is, for those of you who don’t know, is that there are -- in the Guidance 152, for example, FDA has ranked drugs as critically important, highly important and important.
So the way to report on resistance would be then to start with the highly important ones and show resistance and then go to critically important and then go to important.
WHO is about to come out -- they drafted a similar list that will be a WHO drafted list where they have got representatives from all over the world, all the different regions, to come together and draft that same list. When we talked there, we thought that maybe the best thing to do was for CIPARS and the U.S. to use the WHO list.
It wasn’t seen as something we developed in our own countries and then sort of imposed that. The idea is maybe we could all be using that critically important list to rank our drugs and then we would have comparative rankings and comparative lists, including even European colleagues or other people that moved into this sort of reporting. So, that was discussed as an idea.
DR. WHITE: The good thing too is I think our two most critically important drugs in surveillance for Salmonella would be ciprofloxacin and ceftriaxone. If you look at those numbers over the years, they are extremely low. I think resistance is less than one percent to both of those drugs.
DR. McEWEN: (Microphone not turned on.) to comment --- talking about integration --- the surveillance on policy. I think the CIPARS group in Canada --- an example for us to --- this past year --- Lucy talked about it. When they rank them, the resistance results according to human importance, --- and there was one province where there was a lot of Salmonella resistance --- so that begged the question why I guess.
And also, there was a -- there appeared to be a relationship between -- I think it was in the retail isolates. It was a high prevalence and samples from poultry from this one province --- it raised a lot of flags and questions --- and public health ministry got interested and it appears that the poultry industry had made a switch in the --- use of --- may explain it and then decided to switch back and make the corrective action, which I understand has been reflected in this year’s data. So, a good example of integration.
DR. YOUNGMAN: Are there any other questions? Maybe not for Elvira, but questions that you wanted to ask of some of the other speakers from this morning?
(No response.)
DR. YOUNGMAN: Okay. Well, that takes us about to 4:20, close to 4:30 when we were planning to close for today. I just wanted to remind you, those of you who have parked in the garage, if you punch in #1470 that will allow you to leave the garage without paying. Your cost for parking today has been covered by this conference.
DR. McEWEN: Is there any chance we could do the other talk today? If we are going to have --- my calculations each person takes 15 minutes ---
DR. YOUNGMAN: That is fine with me. Are there objections from people in the audience to that, people with kids to pick up or whatever? Because our speaker is here.
(No response.)
DR. YOUNGMAN: An alternative that has been proposed is that we could started earlier tomorrow. We are slated to start at 8:30 tomorrow. Are there objections if we started -- sorry?
DR. ZHAO: I am willing to do it however you decide.
DR. YOUNGMAN: Would you like to give the talk today so we can carry on and then have more time tomorrow for discussion?
(Multiple discussion.)
DR. YOUNGMAN: If everybody is okay with that, that sounds like a great suggestion.
(Multiple discussion.)
DR. YOUNGMAN: Well, then our next speaker is
Dr. Shaohua Zhao, who is going to talk about molecular characterization of isolates from the retail meat arm.
Okay. Thank you.
Molecular Characterization
by Dr. Shaohua Zhao
DR. ZHAO: Good afternoon. I know this is the last talk for today. I will keep it short and speak as fast as I can.
I will talk about the molecular characterization of --- isolates. I think previously our team and Dr. Paula Cray has talked about the --- isolates on the human side, as well as the animal side. There are many aspects --- as the interest area.
(Slide)
We are focusing on the three areas. On molecular subtyping, detection and identified of antimicrobial resistance genes and study the mechanisms of antibiotic resistance and resistance gene transfer.
I would like to give you even more about the molecular subtyping work at CVM, but before I do that I will just briefly talk about the history --- identification of antimicrobial resistance and mechanisms of resistance.
(Slide)
We have used PCR and followed it by DNA sequence analysis to detect and identify the antimicrobial resistant gene. We are particularly interested in the --- integron
--- resistant mechanism. Integron is a mobile --- plays a significant role to spread the antimicrobial resistant gene among the gram lactam bacteria.
So we use PCR to detect the integron, then we use a DNA sequence analysis to identify what kind of specific gene is in the gene cassette. So we have identified many resistance genes that are associated with resistance to
beta-lactam, aminoglycosides, trimethoprin, chloramphenicol, and sulphonamides.
We also identified the many beta lactam resistance in genes including the members of the blaCMY gene. As the previous talker has mentioned, the blaCMY gene is so important to public interest because of the cause --- third generation --- drug of choice to treat --- Paula talked about a microarray.
Right now at CVM we are studying the development of microarray to detect the antimicrobial resistance gene. Microarray has a greater capacity. They can simultaneously detect 100s of 1000s of genes in a single DNA chip. We have selected more than 100 resistance genes and virulence genes and selected 75 --- on each gene put on the array.
So, once this project that --- increase all capacity to detect the antimicrobial resistance gene.
(Slide)
And the studies of mechanisms of antibiotic resistance. For certain resistance profile we like to find out what is the mechanism of that. We are particularly interested to identify the resistance. You know, we are they located at. Is it a plasmid or chromosome? If it is a plasmid, can it transfer to a new different strain or species by conjugation or transformation?
So we have identified many plasmid-borne integron medicated resistance genes transferred by conjugation and transformation. We also clone and express the blaCMY gene into the new host to see if they are resistant to capacity. We find out that the blaCMY gene can resist to a 1st, 2nd and 3rd generation of cephalosporins.
Not just one particular antibiotics. Actually, there is a whole bunch of antibiotics that they can cause resistance.
We are also interested in -- you know, for fluoroquinolone resistance mechanism. As you know, fluoroquinolone resistance is caused by the --- gene, the --- mutation. So we are the PCR and follow the DNA sequence analysis to identify where the mutation occur. If it was a single mutation or a double mutation or the factors there for fluoroquinolone.
Dr. White and McDonough has a collaboration with the University of --- by gene knock-out mutagenesis. They have identified several efflux pumps, such as acrA, acrB and tolC. If highly expressing those efflux pumps, they can cause resistance to tetracycline and chloramphenicol.
Also, expressing high levels of those efflux pumps will --- mutation that can cause highly resistance to ciprofloxacin -- dramatically increased.
(Slide)
Now I would like to talk a little bit about molecular subtyping. The goal of molecular subtyping is to determine genetic relationships among Salmonella and Campylobacter obtained from NARMS isolates, identify emerging multi drug resistance serotypes or strain that is associated with human diseases and share the DNA fingerprinting data with other public health agencies through PulseNet.
(Slide)
The technique we have been using at CVM include Pulsed Field Gel Electrophoresis, multi local sequence, plasmid profile and repetitive PCR and ribotyping. We compare those methods in terms of the discriminatory power, reproducibility, cost and PFGE comes out as still the best method.
(Slide)
These methods is standardized by PulseNet. As previously talked this morning about the best methods, I would to just briefly talk about PulseNet. PulseNet is the National Molecular Subtyping Network for Foodborne Disease Surveillance. It is a collaborative between CDC, the Public Health Laboratory and USDA and FDA.
(Slide)
The goal of this program is to reduce the burden of foodborne illness by assisting investigations and improving outbreak detection through the rapid linking of cases by DNA fingerprinting patterns comparison and leading to faster intervention and establishment of control measure.
(Slide)
Since PulseNet was established, according to statistic models, the size of the outbreak has significantly reduced. Two thirds of cases could have been prevented because of the PulseNet, because when you find out the source you can recall the product or the meat from the market.
(Slide)
Right now PulseNet in the USA have over 17 laboratories participating. It includes all the 50 state public health laboratories. Some of the bigger states, like California or Texas, they have county or the city laboratories participating. We have two USDA and eight FDA laboratories, including six --- and CVM.
(Slide)
This is a database from CVM. We have over 4,000 database entries. Salmonella is the biggest database. We have over 2,700 data entry. Out of that about 1016 isolates are from NARMS isolates. That includes 1999, and in 2000 Paula sent us about 600 --- for us to type.
And then we have Campylobacter, 1,3000 isolates and 761 is from NARMS retail meat. All of this data was submitted to the PulseNet. We have E. coli and Vibrio. Those are mostly from internal responsibility activity.
(Slide)
Now, I would like to share with you some PFGE profiles for multi drug --- Typhimurium. This is actually the PFGE DNA fingerprint pattern. This is a denogram generated by software --- based on the DNA fingerprinting pattern.
If there is a streak line here, that means they have a distinguishing pattern. That is 100 percent pattern similarity. Here is an antimicrobial resistance profile. Each black square shows the resistance to particular antimicrobials.
This is the CVM number, from different state and the source and the isolation date. We do not have -- put the FDA’s and CVM’S PFGE pattern because we compare the database with PulseNet. So this pattern number is from CDC.
This comparison here is not to say this particular -- this isolate is associated with outbreak. It is just to say in particular that this pattern is not associated with human disease because there is no epidemiology information to link it together. We just want to say particular --- cause human disease.
Okay. Look at this. This is a chicken isolate, isolates from New York in 2003. We also have two human isolates from New York that show identical pattern. Again, because of state labs -- some state labs do not provide the antimicrobial data here. So we just leave it empty, but that does not mean --- particular sensitive strain.
There is another cluster here. This is another identical pattern from the ground turkey. We also have human isolates. This cluster here is a typical DT104 pattern here because they are resistant to ampicillin, chloramphenicol, cipromycin --- and tetracycline. We have this isolates isolated from chicken breast, pork, chicken breast again and pork. They all have human isolates and show identical patterns here.
(Slide)
Okay. Some random Newport -- you know, everybody has some mentioned it in the previous talks. It is emerging in multi drug resistance, the serotype. In our retail meat isolates 42 percent of them are resistant to nine or more antimicrobial. So it is quite a resistance compared to Typhimurium.
Again, we have pork isolates, ground beef isolates, ground turkey isolates. In each of these patterns we have human isolates which match this pattern.
(Slide)
This is a PFGE profile of Campylobacter jejuni from Iowa. I think Terry has talked about the Iowa Retail Meat Study. So we did it by --- because what happened with Campylobacter is sometimes it was -- when inside the discriminatory power is limited. So we used the second --- to confirm the commonality. So you can say we have --- there is chicken isolates, we have human isolates that show the same pattern and also turkey isolates and chicken isolates shows the PFGE pattern. All it is from the Human Iowa Retail Meat.
(Slide)
So, in summary of our study, molecular studies on mechanisms of resistance and resistance transfer have provided valuable information for better understanding the emerging and dissemination of antimicrobial resistance in bacterial pathogens. Our data shows that certain multi drug resistance to Salmonella and Campylobacter were frequently present in the retail foods and also recovered from human patients.
(Slide)
For the success of the NARMS program to collaborate on molecular characterization I think there are three important elements. First of all, isolates and data sharing is so critical to determine the emergence of antimicrobial resistance of foodborne pathogens. We really hope eventually we all do the PFGE and we somehow link this data together.
Like Tom this morning mentioned, hopefully the three sites and the data can be put on the front page to say, okay, this is the emerging serotype. What is the commonality on the three. Hopefully, we can some day be able to do that comparison.
Of course, we have to use standard methods to make it comparable our subtyping a DNA fingerprinting pattern.
Lastly is linking the molecular subtyping to other epidemiological data to provide valuable information on origin and transmission of multi drug resistant pathogens. Thank you.
DR. ALTEKRUSE: My understanding is that all of the isolates of the three arms are going into the PulseNet now? Is that correct?
DR. ZHAO: No. According to this morning, Tom or Tim said that --- isolates or maybe it is already in the PulseNet, but that is not identified because the number was not matching. Is that correct?
DR. CHILLER: Yes. Basically, from -- traditionally, NARMS has not requested surveillance isolates to be pulsed, because remember we are getting one out of every 20 isolates. The majority of the isolates that are in our public health lab are sporadic. So maybe 20 percent outbreak, eight percent sporadic.
We are getting majority sporadic cases and those come into us and no one has ever asked state labs to pulse those specific isolates before.
As Tim mentioned, what probably has been happening is a lot of times states pulse their isolates anyway. I mean, Minnesota pulses everything. So the data is actually in there, but there have been discrepancies of the I.D. number that was sent NARMS versus what sent to PulseNet.
We are sort of moving forward on two fronts. The first front is we have sort of a national campaign to standardize I.D. numbers from states. Any isolate that comes to CDC for any reason, if we could use one I.D. number, whether it is for susceptibility testing, pulsed field, MLST, whatever, if we could use one I.D. number, then we could link to all that data.
DR. ZHAO: But CVM isolates --- every single Salmonella and the Campylobacter --- PulseNet.
DR. ALTEKRUSE: And then my question -- the other part of it is the FSIS slaughter HACCP isolates. Those are going into VetNet. Correct? Which is a component of PulseNet. So, is it now possible to do a query?
DR. FEDORKA-CRAY: Not yet. But there will be when we get our database.
DR. ALTEKRUSE: Things are -- it started?
DR. FEDORKA-CRAY: Yes. We don’t have a database up yet. I mean, we have a hard time even querying our own stuff right now in that database. When it is up, then we will be able to use -- there will be cross talks between the systems.
DR. ALTEKRUSE: I just want to agree with something you said, and that is that the real value of NARMS will increase tremendously when that data sharing of molecular information is possible.
DR. ZHAO: Absolutely.
DR. FEDORKA-CRAY: Right. We discussed with Swami -- we have been discussing this for three years now. The idea to keep it separately was based mostly on a personnel and a funding issue. Swami doesn’t have the personnel, nor does he have any monies to deal with animal isolates, per se.
So this was a way to get it up and going and have somebody else worry about a database and keeping it going, and they are going back and redoing parts of their database now and trying to change these numbers over and renaming some old patterns because they were done in a different way, where our database gets to start clean.
Again, it is like you want to improve the wheel instead of reinvent the wheel, and that is where the VetNet system comes in.
DR. ZHAO: You know, Paula, my understanding is that eventually your VetNet will connect with the PulseNet. Right?
DR. FEDORKA-CRAY: I’m sorry? What?
DR. ZHAO: Will connect with PulseNet.
DR. FEDORKA-CRAY: Yes.
DR. ZHAO: And we have access to compare -- to access your database in other words?
DR. FEDORKA-CRAY: Sure.
DR. ZHAO: So we can --- right now we can just easily compare our data to PulseNet, but the --- we can compare all data to your data and you can compare it,
vice-versa.
DR. FEDORKA-CRAY: Yes. That is the idea. We would all be able to look at each other’s databases. Right now we don’t have access to CDC’s database. At some point in time we will have access to their database to be able to query theirs, just like a PulseNet site. They will be able to query ours. Hopefully we will be able to query yours, you will be able to query ours and PulseNet’s too. So that would be the idea. An integrated system.
DR. ZHAO: I think that is very important. When you have the capability to compare the three arms, you can make sense what is commonality, what is human, what is animal and what is retail food and that would make it meaningful.
DR. McEWEN: (Microphone not turned on.) I wonder how useful it is to know that. My understanding is that PulseNet is good for helping you identify --- local or geographically disbursed. The further you get from Atlanta it seems people are less enamored with PFGE and ask questions about how discriminatory it is I guess or how peaceful --- genetics.
I am wondering how well all of the efforts going into --- we have talked a bit about looking at some similarities between animals and people and that sort of thing, but I wondered if ---
DR. BARRETT: I think the critical issue in understanding how this will be helpful is to think in terms of clustering instead of matching. When we are talking about an outbreak, you know, is this strain from this person the same as this one, we want them to match. But I don’t think we are going to see a lot of that in animal and meat and human isolates.
But I think we will see clustering where you will see very clearly -- the Kentucky is a good example. The chicken Salmonella Kentucky isolates cluster separately from the human isolates. I would say that is a pretty strong indication that those human isolates are probably not coming from chickens.
I think we will be able to make those sorts of observations by seeing these cluster more tightly; this group clusters more tightly than that group. You know, they are most closely related. There is probably a link here.
As far as matching, there will be some, like you have seen, but I don’t think that is the strength of it. I don’t think that is what we want to look at.
DR. McEWEN: (Microphone not turned on.) -- how you define the clusters? I don’t know --- distribution and --
DR. BARRETT: Yes. I would say that is true. I think that it is sort of an early area. That is what we will do. You know, at two percent, boy, I don’t see anything here. They are just randomly scattered. Let’s go to five percent. What do we see?
To some extent the only way you can do that is to have the information already that you would like to have to validate it, and I am not sure, with NARMS, where that will come from. I think in other avenues of PulseNet we are able to do that and perhaps we can transfer that to NARMS.
DR. ZHAO: I think a lot of good example is Salmonella Newport. In most of the multi drug resistance to Newport, compared with the CDC database, most of them were cattle. But if you look at the susceptible strain for the human, --- isolates from the turkey. You know what I’m saying here?
So you can say it is a multi drug resistance from animal through the food and to the human. You know, comes from which animal? The PFGE will provide the information of this linkage together.
DR. McEWEN: (Microphone not turned on.) So, you think it may be helpful in --- of exposure to different species ---
DR. ZHAO: Yes. Not for all, but for certain serotypes. Not for all, but for certain serotypes. Also, I think that the PFGE --- has a very important --- to say certain pattern only from certain store or from certain brand of the type of meat. So maybe if you put those information together maybe you can provide us to say that this MDR is due to the retail store cross contamination or from slaughter or originally from animal.
So, if you put the PFGE with the --- pattern, plus this other information, maybe we can find out where the MDR come from there.
DR. KOTARSKI: I wondered. When you find matches between humans and a certain retail store or if it is an animal species or whatever, how many matches become compelling evidence that, for example, all of the Newport was in humans that was susceptible is coming actually from turkey?
How many matches do you see to come forward with that hypothesis or come forward even with a conclusion?
DR. ZHAO: Well, we just see a phenomena --- how many of them are --- turkey isolates are most susceptible compared with the cattle isolates. When you compare the -- look at the one particular pattern and compare with the --- database, then you have a match. --- pattern.
But this has to be maybe to compile more data ---
ground beef we will --- phenomenon.
DR. KOTARSKI: So, it allows hypothesis generation conclusions?
DR. ZHAO: Yeah.
DR. WHITE: And I think too, Sue, there is no magic number. There is not like five or three. I mean, the more data we generate, the larger our associations become or not become too.
DR. ALTEKRUSE: What becomes compelling? Is it finding it in food from a patient? It seems like you are saying the denograms are exploratory, but it is not about causation. It is not even really associations. It is just looking at a pattern.
DR. ZHAO: Yeah. For this comparison, not necessarily this particular isolate are associated with --- epidemiology of information is much more critical to say this outbreak is caused by a particular strain. You have to have where the human --- they have a time, a place or have that information linked together to make that --- linkage or calculation.
DR. WHITE: I think the best case scenario would be if we could be real time with pulsed field and from the FoodNet sites, active disease surveillance, human disease and the retail from those same sites, and we could do pulsed field immediately and submit those patterns. Then we would have a much shorter time frame for comparison.
Right now we are submitting patterns a year to two years later. I mean, you can’t do any type of analysis, except it looks like it came from this.
DR. ALTEKRUSE: So, when will you all have that in place?
DR. WHITE: Remember, we have a budget of so much money.
DR. FEDORKA-CRAY: We are giving FSIS our in two weeks.
DR. ZHAO: --- for our --- and we are not participating --- because we have a web --- communication every day. So if we have a match mostly we feel guilty --- they are asking for last month’s --- pattern. We have the same pattern, but it is last year.
DR. WHITE: I think the last thing, as Tim mentioned as well, the more data we generate, the more we start to see maybe particular associations with particular patterns with particular foods or particular animals over time. So, the more we generate that and see these differences -- say like between like the Kentuckys with chicken breast.
They are very different from the Kentuckys you see from ground beef or swine, and you start to see these outbreaks of human disease, we will be able to say, well, we can steer the public health officials in that direction possibly. We are not going to come out and say it is chicken.
DR. ZHAO: Yeah. That is a really good example. We have a high frequency isolate from the animal arm and the retail meats, but they have very low incidence on the human side. But if you look at the PFGE profile, they are very different.
DR. ALTEKRUSE: Shaohua, with the case of Campylobacter, for example, it seems like there are so many different laboratories that have different preferences for how they subtype their Campylobacter. How do you arrive at a preferred method?
DR. ZHAO: Well, CDC’s --- already. But our experience with Campylobacter, the second --- is very --- because the discriminatory pattern for Campy. One enzyme is not good enough. So we find out that you use two --- for Campy.
DR. ALTEKRUSE: But, for example, in Europe I believe they use amplified polymorphism.
DR. ZHAO: Yeah. You know, but those methods I think -- first of all, it is not standardized and it --- problems. So I think the PulseNet --- so we can make the data comparison, otherwise you compare apples to oranges ---
DR. ALTEKRUSE: But even in the United States, for example, there are some people who have been very strong advocates of FLA sequencing. I don’t know. That is my question. How do you arrive at a pathogen specific method?
DR. ZHAO: Well, CDC -- you know, PulseNet has
a --- R&D group where they --- methodology and look at more distinct power methods.
For example, even PFGE’s golden standard for --- is not powerful enough so that --- additional methods are --- a certain serotype or other methods it is better than PFGE, and therefore, it can be --- they are also looking for other methods.
But for the time being, I think the PFGE is the gold standard, you know, --- for Campy.
DR. ALTEKRUSE: Thank you.
DR. FEDORKA-CRAY: I think that is one of the things that I wanted to point out. If you talk to Swami, he sits there and doubts that PFGE will be used by PulseNet in five years, but there will be an evolution of methods and there will be other methods.
Tim is working on other methods. Our labs are working on other methods and there will be others. I think microarray technology might be something that comes on or comes up faster, and there will be more standardized methods over time.
So, even Swami doesn’t think that PFGE will stay as the gold standard over time and that there will be other things developed, other techniques refined and made more standard.
DR. ZHAO: Yeah. This is a concern because the PulseNet --- deal with over --- state labs --- in terms of the costs, that is another important concern besides the methods --- for the time being --- it is one of the best methods I have seen.
DR. BARRETT: Over the years I have been accused many times of being PFGE-centric. I really don’t like it, PFGE. The bottom line is that it has worked out the best for what it has been used for, and it continues to.
DR. ZHAO: Yeah. I just went to the Canada --- Study. Actually, Bob --- talk about the molecular subtyping and compare --- PFGE is their number one. You know, there is no question about it. You know, --- and the other countries and whatever, and everybody comes to the conclusion, for the time being, that PFGE is their number one.
DR. YOUNGMAN: Okay. Thank you very much, Shaohua.
I guess this concludes things for today. I am sure some of you will want to talk afterward, but I am aware of the time and feel that we need to close for today.
We would like to reconvene tomorrow morning at 8:30, and we will start by talking about a recap and then we will also be talking about international issues related to NARMS.
Thank you very much to all of you for coming today, and we will see you tomorrow morning at 8:30.
(Whereupon, at 4:54 p.m., the hearing was recessed, to reconvene Friday, June 24, 2005, at 8:30 a.m.)
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