2007 Annual Report 1a.Objectives (from AD-416) 1)Determine the role that outside environmental sources of Listeria monocytogenes play in the presence of this pathogen in poultry further processing facilities. 2)Develop and test intervention strategies to eliminate L. monocytogenes and Campylobacter from meat products or processing plant surfaces. 3)Evaluate gene expression profiles of L. monocytogenes and C. jejuni in conditions relevant to poultry processing environments. 4)Evaluate the influence of animal agriculture on Campylobacter in the environment. 1b.Approach (from AD-416) Poultry products are commonly associated with the human pathogens Campylobacter and Listeria. The processing plant is a post-harvest site in which numerous manipulations are made to the poultry product, many of which may impact the microbial quality of the product. This research project is designed to study the distribution and dispersion of bacterial pathogens in poultry processing plants and poultry products. The goal is to develop information so that knowledge-based methods can be developed to improve the microbiological quality of poultry products. Sub-types of organisms will be studied to determine if they are specific for the poultry processing ecosystems from which they are derived. Gene expression will be examined in order to find genes that are regulated in response to environmental conditions or are growth phase specific. As work progresses, the results of these efforts may uncover possibilities for definition of critical control points or potential intervention strategies. 3.Progress Report Ribosomal differences in individual Campylobacter: Campylobacter have multiple copies of the ribosomal genes. Mutations of the ribosomal genes are involved in resistance to some antibiotics. In excess of 10% of Campylobacter isolates were found defective in their ability to coordinate changes, which results in strains that have substantial differences between the copies. Research is being done to determine how this effects acquisition of antimicrobial resistance. Antimicrobial resistance of biofilm forming Listeria: L. monocytogenes is known to have increased resistance to antimicrobial agents when a biofilm has been formed. This research, in conjunction with Project Number 6612-41420-015-05S, is to study differences in gene expression when exposed to antimicrobial agents in a biofilm compared to in a free-living state. Listeria have been grown in the required conditions and messenger RNA has been prepared. The samples are awaiting application to hybridization arrays. Sources of Listeria in a poultry plant: We have studied the colonization of a new poultry further processing plant by monitoring floor drains for L. monocytogenes. We have collected isolates from the drains as well as from incoming raw product, smaller numbers of isolates have been detected in other potential sources such as the surrounding environment. We have isolated, identified and stored cultures and have begun the process of typing using DNA fingerprinting techniques. The types of isolates detected in incoming sources will be compared to those detected within the plant and on condemned fully cooked product. These data will allow us to determine the most important sources of L. monocytogenes. Tracking Campylobacter types through poultry processing: We examined a large collection of Campylobacter previously collected from broiler carcasses in 20 US broiler processing plants. Isolates were identified to the species level and typed by DNA sequence methodology. The data has been collected and analysis is under way. This will show the diversity of Campylobacter on broiler carcasses, the geographical diversity of Campylobacter, the possibility of carcass contamination with new subtypes during processing, and the effect of broiler processing on Campylobacter diversity. Interventions for improving microbial quality of poultry: Completed a study to examine the effect of a series of in-plant intervention procedures on the numbers of Campylobacter, E. coli and presence of Salmonella on broiler carcasses. Carcass samples were collected before and after 5 different wash and/or brush steps in a commercial broiler processing plant. Carcass rinses were examined for presence and numbers of the bacteria. The information will be useful to poultry processors as they evaluate potential intervention strategies to control human pathogens on processed carcasses. 4.Accomplishments Selection of Macrolide resistant Campylobacter: Macrolides are antibiotics that are commonly used for the treatment of Campylobacter infections. It has been hypothesized that poultry production methods encourage the selection of resistance to macrolides. A study was completed in which simulated treatment of poultry with therapeutic and sub-therapeutic levels of tylosin was performed. The birds were then challenged with either Campylobacter jejuni or C. coli and the emergence of resistant isolates was monitored. It was found that both species generated resistant isolates, but C. coli were more likely to become resistant, especially with sub-therapeutic levels of tylosin. It was also found that the most resistant isolates had changes in all three ribosomal genes; a few had changes in two copies of the gene; and none of the resistant isolates had changes in only one copy of the gene. This information is important for developing strategies of using antimicrobials in a manner that does not encourage development of resistance. (National Program Component 1, Problem Statement 1.1.2 Epidemiology and 1.1.3: Ecology, Host Pathogen and Chemical Contaminants Relationships) Cold-air versus ice-water chilling of poultry carcasses: Planned, conducted and completed a study to examine the use of cold air rather than ice water to cool broiler chicken carcasses during processing. In the US most commercial plants use a water immersion method to chill carcasses, while cold air is more common in Europe. We hypothesized that a cold air method may cause skin drying and thereby kill Campylobacter that is on broiler carcass skin. We designed an air chill method to simulate what may be done in commercial conditions and compared Campylobacter from carcass halves cooled in air to other carcass halves cooled in ice water. We determined that air chilling does not result in lower numbers of Campylobacter. Further, when isolates were characterized from chilled carcass halves, we found that air chilling did not select for different species, subtypes or levels of resistance to antibiotic drugs. Thus, producers do not need to select current chilling methods based on microbial quality of carcasses. (National Program Component 1, Problem Statement 1.1.2 Epidemiology and 1.1.3: Ecology, Host Pathogen and Chemical Contaminants Relationships) Evaluation of FSIS HACCP inspection model program: Planned, conducted and completed a collaborative study with FSIS and Stan Bailey to examine the microbiological quality of broiler carcasses processed in plants under the HACCP inspection model program (HIMP) of FSIS inspection. HIMP plants are part of a test program to test a new inspection system. FSIS collected carcass rinse samples from every HIMP plant in the US and we used those rinses to measure the numbers of Campylobacter, E. coli and coliforms as well as the presence of Salmonella. Samples were drawn early and late in the processing continuum to monitor the effectiveness of processing under the model inspection system. These data will be useful in evaluating HIMP plants and may help regulators in future decisions relative to inspection system implementation. (National Program Component 1, Problem Statement 1.1.2 Epidemiology and 1.1.3: Ecology, Host Pathogen and Chemical Contaminants Relationships) 5.Significant Activities that Support Special Target Populations None. 6.Technology Transfer Number of non-peer reviewed presentations and proceedings 9 Review Publications KAPCZYNSKI, D.R., MEINERSMANN, R.J., LEE,, M.D. ADHERENCE OF LACTOBACILLUS TO INTESTINAL 407 CELLS IN CULTURE CORRELATES WITH FIBRONECTIN BINDING. CURRENT MICROBIOLOGY. 2000. Berrang, M.E., Bailey, J.S., Alterkruse, S., Patel, B., Shaw, W., Meinersmann, R.J., Cray, P.J. 2007. Prevalence and Numbers of Campylobacter on Broiler Carcasses Collected at Rehang and Postchill in 20 US Processing Plants. Journal of Food Protection. 70(7):1556-1560. Berrang, M.E., Ladely, S.R., Meinersmann, R.J., Cray, P.J. 2007. Sub-therapeutic Tylosin Phosphate in Broiler Feed Affects Campylobacter on Carcasses During Processing. Poultry Science. 86(6):1229-1233. Northcutt, J.K., Berrang, M.E. 2006. Influence of chicken transportation cage washing system on wastewater characteristics and bacteria recovery from cage flooring. Journal of Applied Poultry Research. 15:457-463. Lyon, S.A., Fletcher, D.L., Berrang, M.E. 2007. Germicidal Ultraviolet Light to Lower Numbers of Listeria Monocytogenes on Broiler Breast Fillets. Poultry Science. 86(5):964-967.