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United States Department of Agriculture

Agricultural Research Service

Research Project: Microbial Ecology of Human Pathogens Relative to Poultry Processing

Location: Bacterial Epidemiology & Antimicrobial Resistance Research

2012 Annual Report

1a. Objectives (from AD-416):
1. Using population genetics, track bacterial migration and adaptation of foodborne pathogens through poultry processing and the associated environment. Evaluate the variations and influence of genetic and strain diversity from animal through the processing plant. 2. Examine the role of protozoa and other potential biological populations in the microbial ecology of foodborne pathogens through poultry processing. 3. Evaluate the potential for protozoa and other biological controls to be used as intervention or mitigation strategies for human pathogens in poultry processing and processing facilities. 4. Based on objectives 1-3, develop and evaluate physical and chemical intervention strategies to reduce contamination by foodborne pathogens of poultry products.

1b. Approach (from AD-416):
The focus of this research would be called the “transmission phase” by epidemiologists or the “migration phase” by ecologists. Processing of poultry products creates many severe barriers to transmission such that most of the pathogens are lost. However, it is clear that the barriers are incomplete and enough pathogens survive and pass to human consumers to cause foodborne disease. It is reasonable to assume that bacteria have adaptive strategies that improve the chances that some clones will survive processing making transmission to humans possible. The objectives of this project are designed to determine the relative ability of genetically different clones of foodborne pathogens to survive barriers that are encountered in the poultry processing plant. This will be followed by studying specific biological barriers that are common to ecosystems and are often responsible for limiting migration of bacteria. It is also likely that protozoa will be found in the processing environment that are not only ineffective in killing pathogens but may even be protective. Therefore, we plan to study the mechanisms of destruction or protection as they are uncovered. The knowledge that is gained from these studies will be used to design enhanced barriers in an attempt to improve the microbiological benefits of poultry processing.

3. Progress Report:
We have previously reported that floor drains in a poultry further-processing plant can harbor strains of Listeria monocytogenes, a bacterial species that can contaminate food and cause disease in humans. Some floor drains continually harbor the organism and others are continually negative. It is known that the wet surfaces in a floor drain have a complex community of microorganisms, but the influence of these organisms on the pathogens is not understood. We performed assays to compare the species that are present in drains that were shown to harbor Listeria with drains that were negative. We identified 83 species of fungi, 70 species of bacteria and 28 species of protozoa. Differences between Listeria-negative and Listeria-positive drains were seen for all three classes, most often exhibited as an increase in the numbers of some species in the Listeria-positive drains. The significance of these differences is being analyzed. Completed a follow up study to measure the contamination of raw meat by airborne drain Listeria and the fate of such contamination on meat during cold storage. This data has been collected and is currently being analyzed. Carried out preliminary work and started data collection on an intervention strategy to kill Listeria on raw poultry meat exposed to airborne cells. Using a previously developed germicidal ultra-violet light method, we treated breast fillets with the low numbers expected to be present due to cross contamination from drain spray. Working with scientists from the Poultry Microbiological Safety Research Unit and an outside company, we tested electroporation technology as a means to sanitize poultry carcasses during slaughter. The method promises limited utility in the current state. The cooperators are working to fine tune the equipment towards best effect. Collected carcass drip samples from commercial processing plants representing approximately 1,500 broiler carcasses per replication. The drip samples are being used to detect Campylobacter, compare a panel of Campylobacter specific growth media to determine the best performer and working with PMS to develop a microbiological census of all bacteria likely to be present on broiler carcasses during slaughter and first processing. Continued to study a proprietary broiler chill water additive designed to maintain the efficacy of chlorine as an antimicrobial even in the presence of organic matter (such as broiler carcasses). This compound was found to limit the number of total aerobic bacteria on carcasses and lessen the amount go cross contamination with human pathogens: Salmonella and Campylobacter. This data is currently being analyzed prior to manuscript production.

4. Accomplishments
1. Dissemination of Campylobacter in a Georgia river. The greater part of the life cycle of Campylobacter jejuni is not defined – the sources of chicken infection are unknown and passage of the organism outside of the chicken to human is uncharacterized. This study was designed to see if open waterways are part of the life cycle for the organism involved in human disease. Over the course of four years 560 water samples were taken from the Upper Oconee River Watershed from which 47 strains of Campylobacter were obtained. The strains were genetically typed in order to classify them by family lineages and this data was compared to types known to be found in chickens and humans. Of the recovered strains, some have previously been found in cattle and very few that were ever found in humans whereas most of the strains have only been associated with environmental water. This means that open water plays only a minor role if at all in the transmission of human associated Campylobacter and need not be a focus of intervention.

2. Investigation of Listeria monocytogenes isolates related to a large cantaloupe-borne outbreak. Listeria monocytogenes is a deadly human pathogen that has been associated with a variety of foods including cantaloupe in a 2011 outbreak in the U.S. We gathered L. monocytogenes isolates from the cantaloupe outbreak, isolates from other food-related outbreaks, clinical collections, defined epidemic clones and isolates from chicken processing plants. All isolates were characterized and compared to one another using sophisticated molecular subtyping methods. It was discovered that the cantaloupe outbreak isolates matched isolates from food-related outbreaks in Canada, other worldwide clinical collections and chicken processing plants in the U.S. None of the cantaloupe isolates fit into previously described epidemic clones but two novel epidemic clones were created to include these strains. Thus, a new type of highly virulent Listeria has been recognized and needs to be monitored.

3. Airborne transfer of Listeria monocytogenes from floor drains during wash. Listeria monocytogenes, a human pathogen, can be found contaminating the environment inside poultry processing plants especially the floor drains. It is unclear if or how Listeria can travel from a floor drain to product. The objective of this study was to determine if an accidental discharge of a water hose into a contaminated floor drain could result in airborne transfer of live Listeria cells to other surfaces. Using a two second spray into an experimental model drain systems, we were able to detect airborne Listeria within the experimental rooms. Listeria was detected settling out of the air as far away as 4.0 m (13 ft) on the floors and even 2.4 m (8 ft) high on the walls. Poultry processors will use this information to guide sanitation standard operating procedures relative to avoiding inadvertent hose spray into floor drains. Researchers will find this information critical as they design and test intervention strategies to prevent the escape of live Listeria from contaminated sites during poultry plant wash down.

4. Validation of sequence-based typing for Campylobacter jejuni/coli. Campylobacter is the genus of bacteria that is responsible for the greatest number of human diarrheal disease. Up to now, type profiling of these organisms has relied on the use of a group of genes from the organism that were selected with the intent that they would be representative of the entire genome, an assumption that can now be tested using total genome sequences. A method was invented to simultaneously compare the variation in all of the 1029 genes we identified as being contained in a panel of genomes. The method involved measuring the differences in each of these genes for each of the 25 genomes to create a data file that was then subjected to specialized statistical analysis for determining clusters. We were able to define clusters that correlated with specific evolutionary influences, such as selective pressure for frequent changes (‘mutations’) or participation in cross-bacterial exchange events (‘recombination’ also known as ‘lateral gene transfer’). These are factors that affect interpretation of population genetic data for analysis of migration, so knowing which genes have these influences will be useful in future interpretations.

Review Publications
Berrang, M.E., Smith, D., Meinersmann, R.J. 2011. Variations on standard broiler processing in an effort to reduce Campylobacter numbers on postpick carcasses. Journal of Applied Poultry Research. 20(2):197-202.

Berrang, M.E., Meinersmann, R.J., Hofacre, C.L. 2011. Spray washing, absorbent cornstarch powder, and dry time to reduce bacterial numbers on soiled transport cage flooring. Journal of Applied Poultry Research. 20(3):378-382.

First, M.R., Park, N., Berrang, M.E., Meinersmann, R.J., Bernhard, J.M., Gast, R.J., Hollibaugh, J.T. 2012. Ciliate ingestion and digestion: flow cytometric measurements and regrowth of a digestion-resistant campylobacter jejuni. Journal of Eukaryotic Microbiology. 59(1):12-19.

Berrang, M.E., Meinersmann, R.J., Cox Jr, N.A., Cray, P.J. 2011. Application of chlorine dioxide to lessen bacterial contamination during broiler defeathering. Journal of Applied Poultry Research. 20(1):33-39.

Berrang, M.E., Windham, W.R., Meinersmann, R.J. 2011. Campylobacter, Salmonella and Escherichia coli on broiler carcasses subject to a high pH scald and low pH postpick chlorine dip. Poultry Science. 90(4):896-900.

Berrang, M.E., Meinersmann, R.J., Hofacre, C.L. 2011. Forced Hot Air to Dry Feces and Kill Bacteria on Transport Cage Flooring. Journal of Applied Poultry Research. 20(4):567-572.

Hunter, S.M., Berrang, M.E., Meinersmann, R.J., Harrison, M. 2009. Genetic diversity of campylobacter on broiler carcasses collected preevisceration and postchill in 17 U.S. poultry processing plants. Journal of Food Protection. 72(1):49-54.

Oakley, B., Line, J.E., Berrang, M.E., Johnson, J., Buhr, R.J., Cox Jr, N.A., Hiett, K.L., Seal, B.S. 2012. Pyrosequencing-based validation of a simple cell-suspension polymerase chain reaction assay for Campylobacter with application of high-processivity polymerase with novel internal amplification controls for rapid and specific detection. Diagnostic Microbiology and Infectious Disease. 72(2):131-138.

Last Modified: 06/21/2017
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