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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

2013 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:
ARS scientists completed a study 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. Data show that treatment at 800 micro-Watts/cm2 for times as short as 5 seconds is adequate to significantly reduce numbers of L. monocytogenes on raw chicken breasts. This type of intervention could be applied to cut up parts in the slaughter plant and has potential to break the cycle of Listeria contamination from slaughter plant to cooking plant with the transfer of raw product. This data has been collected, analyzed and the resultant paper is currently under review at an international journal. ARS scientists completed a study on 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 of cross contamination with Salmonella and Campylobacter. Work was conducted both on a bench scale level and in a pilot plant with whole broiler carcasses. These data have been collected and analyzed, the resultant manuscripts are currently in preparation. ARS scientists completed a study to determine the best means to sample broiler skin for the recovery of food-borne pathogens, Salmonella and Campylobacter. Carcasses were inoculated with known numbers of both pathogens and breast skin was sampled by a non-destructive sponge method and by a skin excision method. These data have been collected and are currently being analyzed prior to preparation of a manuscript. In collaboration with ARS scientists from Clay Center, NE, the genome of a Urease Positive Campylobacter lari was fully sequenced. Preliminary annotation of the sequence shows a unique gene set but analyses has not been completed to show if there is sharing of genes between this population of bacteria and Campylobacter that commonly contaminate poultry products. In collaboration with a scientist in Denmark, total genome sequences for 42 isolates of Listeria monocytogenes have been analyzed to find which genes are evolving jointly. Preliminary analysis indicates that the genes are not as linked as expected; that is to say, there appears to be extensive trading of genetic material between different lineages. Such trading of genes means that the organism is more adaptive than expected and more information is needed to fully trace lineages of the organism.

4. Accomplishments
1. 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. The objective of this study by ARS scientists at Athens, GA, 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 raw poultry meat placed on a work surface 8 feet distant from the drain. Using a two second spray into an experimental model drain system, Listeria became airborne within the experimental rooms and was detected settling out of the air on raw breast meat and the organism was found on breast halves exposed to the airborne cells and on breasts later exposed to the contaminated work surface. Poultry processors will use this information to guide sanitation standard operating procedures relative to avoiding inadvertent hose spray into floor drains and 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.

2. Evolution of IncA/C plasmids. Plasmids are important pieces of DNA because they are often exchanged between different families of bacteria and because they usually carry genes that give the bacteria a survival or growth advantage, such as antimicrobial resistance genes. How plasmids change and what regulates the changes is not known but it will help control the spread of injurious genes if we better understand this phenomenon. To develop a model of how the class of IncA/C plasmids evolved ARS scientists at Athens, GA, determined the DNA sequence for 39 recently isolated plasmids and compared these with each other and 26 more sequences that are publicly available. The pattern of changes made it clear that the major situation that leads to change is during the transfer of the plasmid from one strain to another strain of bacteria. This study showed how IncA/C plasmids are related to each other so that tracking the plasmids should be easier and the ultimate goal of controlling the plasmids will benefit from interrupting bacterial transfer between different strains of bacteria.

3. Multiple copy genes. Multiple copies of a gene are inherently unstable that results in loss of the copies, but there are some genes that are beneficial to have in multiple copies, such as the ribosomes that are needed to put together proteins in the cell. It turns out that the multiple copy genes are more similar to each other within a single cell than would be predicted based on the amount of differences seen between cells through a process known as “concerted evolution” that uses each copy to edit the other copies. Three sets of multiple copy genes have been studied by ARS scientists at Athens, GA, in Campylobacter to date: the 23S ribosomal gene, the flagellin genes, and the ribosomal intergenic sequence. This is important to interpret in the foodborne pathogens Campylobacter jejuni and C. coli because resistance to some antimicrobial drugs is based on changes to the ribosomal genes.

Review Publications
Meinersmann, R.J., Berrang, M.E., Little, E. 2013. Campylobacter spp. recovered from the Upper Oconee River Watershed, Georgia, in a four-year study. Microbial Ecology. 65(1):22-27.

Berrang, M.E., Frank, J.F., Meinersmann, R.J. 2013. Contamination of raw poultry meat by airborne Listeria originating from a floor drain. Journal of Applied Poultry Research. 22(1):132-136.

Berrang, M.E., Bailey, J.S., Altekruse, S.F., Shaw, Jr., W.K. 2008. Presence and numbers of Campylobacter, Escherichia coli and Salmonella determined in boiler carcass rinses from United States processing plants in the hazard analysis and critical control point-based inspection models project. Journal of Applied Poultry Research. 17(3):354-360.

Lloyd, T., Alvarado, C.Z., Berrang, M.E. 2012. Organic acid formulation and dip to control listeria monocytogenes in hot dogs. International Journal of Poultry Science. 11(7):469-473.

Lloyd, T., Alvarado, C.Z., Mckee, S.R., Berrang, M.E. 2010. Control of Listeria monocytogenes in Ham Deli Loaves using Organic Acids as Formulation Ingredients. Journal of Food Safety. 30(4):793-803.

Berrang, M.E., Frank, J.F. 2012. Generation of airborne listeria from floor drains. Journal of Food Protection. 75(7):1328-1331.

Cox Jr, N.A., Richardson, L.J., Maurer, J.J., Berrang, M.E., Cray, P.J., Buhr, R.J., Byrd Ii, J.A., Lee, M.D., Hofacre, C.L., O'Kane, P.M., Lammerding, A.M., Clark, A.G., Thayer, S.G., Doyle, M.P. 2012. The evidence for horizontal and vertical transmission in Campylobacter passage from hen to her progeny. Journal of Food Protection. 75(10):1896-1902.

Oakley, B., Morales, C., Line, J.E., Berrang, M.E., Meinersmann, R.J., Tillman, G.E., Wise, M.G., Siragusa, G.R., Hiett, K.L., Seal, B.S. 2013. The poultry-associated microbiome: network analysis and characterization along the farm-to-fork continuum. PLoS One. 8(2):e57190.

Trimble, L.M., Alali, W.Q., Gibson, K., Ricke, S.C., Crandall, P., Jaroni, D., Berrang, M.E. 2013. Salmonella and Campylobacter prevalence and concentration on pasture-raised broilers processed on-farm, in a Mobile Processing Unit, and at small USDA-inspected facilities. Food Control. 34(1):177-182.

Last Modified: 05/21/2017
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