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CFSAN Regulatory Codes: IA, ID, IIC
CFSAN Program Priority Codes: 1.4c
Start Date: 5/1/99    Completion Date: 5/1/02

Statement of Research Problem:
This is a multi-component project to determine factors that control microbial growth in minimally processed foods for the purpose of manipulating these factors to reduce or eliminate the pathogens. Processing of commercial produce is a rapidly expanding industry that offers convenient products with fresh-like qualities. Preservation and extension of shelf life for produce is frequently achieved through refrigeration, bactericidal rinses, modified atmosphere packaging and other technologies. It is known that produce can be contaminated with microbial pathogens but their contribution to the overall risk of microbial illness is not well defined. Understanding the rate of contamination, modeling pathogen growth and inactivation to design processes and for risk assessment, and improvement of these technologies will provide knowledge for industry and FDA to increase the safety of these foods. This research also has applications to other ready-to-eat foods.

Statement of Project Objective(s):
This project will generate data on the presence of Staphylococcus aureus and Bacillus cereus on domestic sprouted seeds. Simulation studies of environmental conditions, including modified atmosphere packaging, will determine whether they can prevent or delay the growth of these pathogens. Enumeration of Listeria monocytogenes on produce is difficult and has been one reason there is limited data on contamination rates and levels. An improved colony counting method that is rapid, easy to conduct and unaffected by the normal microflora will make surveys for L. monocytogenes contamination more feasible. Growth and survival of L. monocytogenes will be modeled to provide information for designing process systems and for risk assessments. E. coli O157:H7 can also contaminate produce and grow. Models will be developed that can predict the likely growth at different temperatures and under modified atmospheres.

Anticipated Impact on FDA Regulatory Program:


1. Better information on contamination will indicate whether additional research or regulatory effort is needed.
   
2. Understanding the potential growth or decline will also show where potential problems may lie and what might be done to reduce the risk.

Project Priority Changes During FY2000:
The scientific lead for component 2 of the FY 1999-2001 plan (phytochemicals) left FDA. This portion of the project has been inactive and the other scientists that were on that component pursued other research objectives during the year. This component has been deleted from the FY 2000-2002 plan. Component 4 was listed as a separate project in FY1999.

Note: Component 1 should be considered for moving into project 9 where these scientists are already allocating most of their time.

Administrative Liaison(s): R. C. Whiting: 202/260-0511

Research Personnel:


Name	Office/Division	FTE [00, 01, 02]	Component
R. Whiting	OPDFB/DNP	0.5, 0.5, 0.5	3
S. Mammel	OPDFB/DNP	1.0, 1.0, 1.0	3
A. deJesus	OPDFB/DNP	1.0, 1.0, 1.0	3
S. Eblen	OPDFB/DNP	1.0, 1.0, 1.0	3
G. Skinner	OPDFB/DFPP	1.0, 1.0, 1.0	4
R. Bennett	OPDFB/DMS	0.5, 0.5, 0.5	1
A. Hitchins	OPDFB/DMS	0.5, 0.5, 0.5	2
R. Duvall	OPDFB/DMS	0.8, 0.8, 0.8	2
Napier	IIT/NCFST	1.0, 0.0, 0.0	4
	Total FTE:	7.3, 6.3, 6.3

Other Personnel:


Name	Office/Division	FTE [00, 01, 02]	Component
A. McCarthy	JIFSAN student	0.2 0.2 0.2	3
Students at Moffett	IIT		4
	Total FTE:	0.2 0.2 0.2

Collaborators: none

Component 1 Objectives:
Collect data on pathogens on produce.
Component 1 FY 2000 Deliverables:


1. Conduct prevalence survey of 500 samples of produce for the four pathogens.
   
2. Initiate study on the survival of S. aureus in cilantro, lettuce and green peppers when packaged in atmoshperes of air, 2%O2-18% CO2-80% N2 and vacuum.
Component 1 FY 2000 Progress:

1. Produce survey was completed.
   
2. Initial data for the survival study was collected and is being analyzed.
   
3. Whether data for additional products or atmospheres need to be collected is being determined. S. aureus counts declined in all products and atmospheres while the indigenous flora increased.
Technical Barriers to Meeting Component 1 Objectives or Deliverables: none listed
Component 1 FY 2001 Deliverables:
Continue the studies on growth/survival in modified atmospheres.
Sprouted seeds will be tested for their ability to support growth of selected food borne pathogens.
Component 1 FY 2002 Deliverables:

Project involves the development of an enumeration method for Listeria monocytogenes contamination in food, especially fresh produce such as mixed salad vegetables and sprouted seeds. The method is based on a direct colony counts of 30° C selective enrichment cultures of samples with back calculation to yield the initial population size using predetermined typical growth rates for Listeria spp. and/or L. monocytogenes and the elapsed incubation time. The method is intended for collecting data on Listeria monocytogenes contamination concentrations while routinely monitoring regulatory samples for L. monocytogenes. Previous work established the typical growth rates in selective enrichment medium and showed that a correction factor would be necessary.

Component 2 Objectives:
Develop a rapid parametric for obtaining colony counts from produce.
Component 2 Deliverables:
Evaluation of the feasibility of using the Probelia L. monocytogenes colorimetric PCR kit to provide the parametric for colony counts.
Component 2 FY2000 Progress:
A Probelia colorimetric PCR kit for L. monocytogenes was purchased. Mr. Duvall and Ms. A. Yansaneh (U. Maryland undergraduate) familiarized themselves with the kit using pure cultures of Listeria. Mr. R. Thunberg provided them with access to a thermocycler and guidance on its use. In August, funding for a thermocycler was approved and it was ordered.
Technical Barriers to Meeting Component 2 Objectives or Deliverables:


1. Lack of dedicated thermocycler for user training and to implement the use of the Probelia colorimetric PCR kit in enumeration.
FY 2001 Deliverables:
Determine the relationship of initial spiking concentration of L. monocytogenes Scott A in selected produce matrices to the 15 h enrichment concentration determined by colony counts on selective differential agar (BCM or ALOA medium). This will show whether a correction is needed over the whole range of initial contamination concentrations likely to occur in foods. The shapes of the pre-24 h growth curves in produce containing enrichments will be determined at selected initial concentrations to find out if the correction factor is driven by the lag time or by an inhibited growth rate. Samples from the various experiments will be frozen and later assayed by the Probelia PCR, Accuprobe or other appropriate rapid methods in order to develop a colony count parametric(s). This parametric will reduce the time necessary to enumerate the enriched Listeria population.
FY 2002 Deliverables:
Complete unfinished parts of project. Assess breadth of produce matrices appropriate to the method. Present enumeration method applicable to 12-24 h selective enrichment cultures with matrix applicability and correction factors. In theory, the method could be adapted to be completed within one workday.

Component 3 Objectives:
Compare 18 strains of L. monocytogenes for their relative growth, survival and thermal death. Determine whether these characteristics correlate to serotype or Wiedmann's lineage systems to assemble information on the between strain variability.
Develop a model for the thermal inactivation of L. monocytogenes with pH, aw and prior growth with and without glucose as variables.
Component 3 Deliverables:

1. Collect data and fit models to obtain exponential growth rates, thermal inactivation D values and survival time data for the strains at selected environmental conditions. Determine variability between strains and observe any grouping by subspecies.
   
2. Collect data and fit models for D values. Generate a second level regression model to predict D values and determine quality of model.
Component 3 FY2000 Progress:
For the 18 strains, thermal death D values were collected for all strains; growth and survival data are being collected.
All data for model were collected for the thermal inactivation model and first level models (D values) were fitted to data.
Technical Barriers to Meeting Component 3 Objectives or Deliverables: none listed
FY 2001 Deliverables:
The comparative strain study will be completed. Initial data collecting to create a growth model for Listeria monocytogenes under modified atmosphere conditions will begin.
The current thermal inactivation model will be completed with additional data as determined necessary. Thermal inactivition modeling will continue and will be expanded to include other factors related to the prior history of the cells. These include growth stage and pH and temperature adaptation.
FY 2002 Deliverables:
Growth modeling under modified atmospheres will continue with additional factors added.

The mode of contamination of minimally processed fruits and vegetables is being heavily studied. Contamination with pathogenic bacteria such as E. coli O157:H7 can occur by many means. In preparing fresh produce for sale, pathogenic bacteria can easily be spread via the numerous processing steps, particularly when there is no effective reduction step involved. In addition to the spread of pathogenic bacteria, damage to the fruit or vegetable resulting from these processing steps may release extra moisture or nutrients that may create conditions where the microorganisms can grow rapidly and/or produce toxin. The number and nature of pathogenic bacteria present on finished product may reflect the bacteria present on the raw product as well as bacteria added during processing. Experimental data is necessary to determine how the various processing steps in the manufacture of fresh produce may affect the numbers of E. coli O157:H7 that may be present.

Component 4 Objectives:
Obtain experimental laboratory data for quantification of the growth of Escherichia coli O157:H7 and its behavior during the processing steps involved in the processing of minimally processed fresh produce. This data will help establish the risk associated with this pathogen and food.
Component 4 Deliverables:
Accurate scientific data for the behavior of E. coli O157:H7 during the processing of minimally processed foods can be used in risk assessments performed to determine which activities of processes represent the highest risk of propagating a food hazard. This data will allow quantification of the reduction, removal, or growth which may occur at a critical processing step. This information may lead to implementation of controls at various steps that may reduce the risks of food borne illness. These data will serve as a basis for developing guidelines for produce handling and intervention technologies.


1. Complete laboratory experiments to evaluate the behavior of E. coli 0157:H7 on model exudate or moisture that would be obtained from the chopping or shredding of lettuce, baby carrots, cabbage and radishes.
   
2. Continue and complete laboratory experiments to evaluate the behavior of E. coli O157: H7 on intact pieces of lettuce, baby carrots, cabbage and radishes packaged under air conditions.
   
3. Begin evaluation of how microbiological data obtained using real foods compared to predictions obtained using the Pathogen Modeling Program.
   
4. Evaluate need for additional experiments or model development.
   
Component 4 FY2000 Progress:
To simulate the behavior of Escherichia coli O157:H7 in liquid released during the chopping and shredding of produce, growth curves were obtained by inoculating dihydrostreptomycin resistant Escherichia coli O157:H7 into exudates obtained from baby carrots, lettuce, radishes, and cabbage and to intact produce pieces. Growth was often rapid, as for carrot exudate, where numbers increased by 3 logs in 24 hrs. All experiments were repeated 3 times under an air atmosphere. Escherichia coli O157:H7 numbers was found to grow well at 20 and 25°C in baby carrot, lettuce and raddish exudate and on whole intact pieces of produce, with growth being less or non existent at 15°C and no growth at 5°C. Escherichia coli O157:H7 grew on intact cabbage however on cabbage exudate numbers showed an initial 2-3 log decrease at 20 and 25°C after 4-6 days, then numbers showed a net 1-2 log increase by approximately 2 weeks.
Technical Barriers to Meeting Component 4 Objectives or Deliverables:
System for evaluation of growth in MAP packages will have to be developed and appropriate industry contacts will have to provide information on appropriate gas mixtures and film permeability.
(Logistics Note, Napier has accepted another position and her technician position will need to be replaced.)
FY 2001 Deliverables:

1. Initiate and complete laboratory experiments to evaluate the behavior of E. coli O157:H7 on intact pieces of lettuce, baby carrots, cabbage and radishes packaged under MAP.
   
2. Complete evaluation of how microbiological data obtained using real foods compared to predictions obtained using the Pathogen Modeling Program.
   
3. Complete laboratory experiments evaluating the behavior of E. coli during various processing steps.
   
4. Update the parameters for microbiological risk assessment models if necessary.
   
5. Complete evaluation of how microbiological data obtained using real foods compared to predictions obtained using the Pathogen Modeling Program.
   
6. Identify key parameters for minimizing risks associated with minimally processed foods, including produce and fruits.
FY 2002 Deliverables: None Listed

Whiting, R.C., Coleman, M., Narrod, C., Powell, M., Roberts, T. and W.D. Schlosser. Risk assessment in the control of VTEC. In Duffy, G. and Garvey, P. Eds. Verocytotoxigenic E. coli. Chapter 18.


Buchanan, R., Dennis, S., Miliotis, M and Whiting, R. 2000. Quantitative microbial risk assessment--FDA's perspective. NFPA.

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Hypertext updated by dav 2001-OCT-02