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ARS Home » Southeast Area » Athens, Georgia » U.S. National Poultry Research Center » Bacterial Epidemiology & Antimicrobial Resistance Research » Research » Research Project #430181

Research Project: Control Strategies and Evaluation of the Microbial Ecology Associated with Foodborne Pathogens and Poultry Processing

Location: Bacterial Epidemiology & Antimicrobial Resistance Research

2017 Annual Report

1a. Objectives (from AD-416):
Objective 1: Develop, evaluate and optimize processing treatments to reduce, control and potentially eliminate foodborne pathogens in poultry processing. Objective 2: Evaluate and define the potential role of protozoa in shaping the ecology of bacterial pathogens in controlling foodborne pathogens in poultry processing environments. Objective 3: Develop algorithms for interpreting and handling sequencing data to aid in epidemiological tracking, defining differences in isolates of foodborne pathogens, including antibiotic resistance patterns, and predicting and determining the source of the isolate.

1b. Approach (from AD-416):
The goals of this project fit into two major approaches: 1) development of alternative methods for processing poultry products, and 2) development of methods that accurately monitor the microbial quality of poultry products processed by alternative methods. The alternative methods include testing several novel chemical and physical decontamination procedures. The approach for most of this work is to apply the intervention strategy and compare the microbial quality of the treated poultry product with control product treated by standard methods. A long term objective is to develop systems of using protozoa as natural controllers of foodborne pathogens. This will involve studying the ecology of protozoa that feed on the pathogens and determining methods to enrich the processing environment with effective protozoa. Approaches for monitoring microbial quality will include enhancing the sensitivity and specificity of microbial detection. The project will also use genetic typing methods including whole genome sequencing and metagenomic sequencing to track specific clones of pathogens in and around poultry processing environments.

3. Progress Report:
Objective 1: Control of Campylobacter relies on sensitive and accurate detection of the organism. A study was completed to characterize the passage of Campylobacter through 0.45 and 0.65 µm filters. These filters can be used to separate Campylobacter from other organism present in complex samples. This provides critical information relative to how many Campylobacter cells must be present per mL in order for the filter method to be a useful detection method. The data may also be used to estimate the actual number of motile Campylobacter cells per mL of original sample based on number found by the filter method. The data have been analyzed, a paper prepared and submitted for publication. A survey for Campylobacter in a commercial broiler processing plant was continued. As part of this survey we have collected ceca from more than 300 flocks and now have data from four continuous years. These data are being continually collated and will be used to further examine flock prevalence and seasonality of Campylobacter. Gut contents are also being archived and analysis is underway to compare the microbiome of Campylobacter positive and negative flocks to determine if there is a previously undescribed relationship between other bacteria and the presence or absence of Campylobacter. We conducted a study to test the use of chlorine dioxide as an antimicrobial litter treatment for killing bacteria in broiler chicken bedding material. A two-part litter amendment that generates chlorine dioxide was tested for lessening the number of bacteria in litter, thus decreasing the effect of previous flocks on the establishment of flora in a new flock. This work is being done in collaboration with a colleague at Western University of Health Sciences, College of Veterinary Medicine, who has developed a microbiological population that when fed to young chicks may be helpful to both improve growth and exclude human pathogens from the gut. More work is planned for optimization of litter treatment to kill background bacteria before adding the helpful inoculum. To control the contamination of poultry carcasses during processing, a novel method was designed and developed for plugging the cloaca of killed broiler carcasses prior scalding. Multiple experimental replications have been conducted at the U.S. National Poultry Research Center, Athens, Georgia, pilot processing plant. Preliminary data indicates Campylobacter contamination is well controlled. More work is ongoing to further optimize the process. A new culture medium was developed for sampling processed broiler carcasses to detect Salmonella in the presence of residual antimicrobial carcass washing chemicals. This new medium, neutralizing buffered peptone water (n-BPW) has been accepted by regulators and is in use nationwide. However, the medium had not been validated with Campylobacter. An experiment was designed and multiple replications have been conducted to determine the efficacy of the mew medium relative to the traditional carcass sampling medium. Data have been collected, and analyzed. A manuscript is under preparation. There have been several outbreaks of Campylobacteriosis associated with broiler chicken livers. We have undertaken a study to determine the prevalence of Campylobacter on the outside and inside surfaces of chicken liver both in a commercial processing plant and at retail. Objective 2: Protozoa can be bacterivorous, ingesting and killing bacteria as a food source. Natural streams were tested and some were found to harbor Listeria monocytogenes. Wild protozoa were collected from these streams and used in experiments along with Tetrahymena and Euglena for the ability to kill L. monocytogenes as both free-swimming planktonic and biofilm cells. We found that Tetrahymena out performs the other kinds of protozoa tested for this purpose. Next, we will test the efficacy of Tetrahymena against L. monocytogenes when it is present as one member of a complex biofilm and free-swimming community. A microfluidic device was developed for evaluating the motility and trapping of protozoa. Euglena was tested in the device and it was shown that the protozoa were attracted to an extract from Listeria monocytogenes, a food-borne pathogen. The device may be used to select protozoa that are preferentially attracted to pathogens that are then destroyed by the protozoa. Objective 3: Whole-genome multi-locus sequence typing (wg-MLST) is a data-intensive method for evaluating populations of bacteria. Transforming raw DNA sequence data into a matrix that can be readily analyzed is a key step that needs more automation. We have developed a script that can use reference databases for any desired organism to create the analysis matrix. Work is continuing to make the algorithm more rapid for near-real time analyses. A gene that confers resistance to colistin (polymyxin E) has frequently been found on a type of plasmid known as IncI2. A multi-locus sequence typing (MLST) scheme was developed for IncI2 plasmids and has been applied to 110 known IncI2 plasmids. Preliminary analyses have shown that the plasmid is more stable than expected, meaning that the core of the plasmid does not readily change even though it can readily shift to other host bacteria. Control of this plasmid needs to start while it is still relatively rare.

4. Accomplishments
1. Pre-chill cooking of poultry carcasses. Cooking is an effective method for destroying food-borne pathogens. ARS researchers at Athens, Georgia, hypothesized that cooking poultry carcasses before chilling would help control pathogens if the quality of the meat was not adversely affected. Carcass portions were cooked before or after chilling and the tenderness and yields were compared. It was found that the tenderness was actually improved when intact non-deboned breast fillets were cooked before chilling and the yields were not significantly different. For poultry products that are intended for the fully-cooked market, cooking before chilling will control food-borne pathogens and result in a tender product and substantial energy savings for chilling.

2. Neutralizing-buffered peptone water carcass wash. Broiler carcasses are tested for presence of human pathogens by a whole carcass rinse method using buffered peptone water (BPW). During the course of commercial broiler processing, carcasses are washed with various antimicrobial processing aid chemicals to lower bacterial contamination. ARS scientists at Athens, Georgia, showed that enough of the antimicrobial chemical is carried with a broiler carcass into a carcass rinse sample to confound detection of Salmonella by continued antimicrobial action in the rinsate during simulated shipment to an offsite lab. The scientists developed a new medium, neutralizing – BPW (n-BPW) that was effective in counteracting four of the most common antimicrobial treatments currently used in broiler processing and allow detection of Salmonella. This is very important as it makes a false negative result far less likely. The new medium has been adopted by the Food Safety and Inspection Service for regulatory sampling of broiler carcasses.

Review Publications
Berrang, M.E., Ladely, S.R., Meinersmann, R.J., Line, J.E., Oakley, B., Cox Jr, N.A. 2016. Variation in Campylobacter multilocus sequence subtypes from chickens as detected on three plating media. Journal of Food Protection. 79(11):1986-1989.
Gamble, G.R., Berrang, M.E., Buhr, R.J., Hinton Jr, A., Bourassa, D.V., Johnston, J.J., Ingram, K.D., Adams, E.S., Feldner, P.W. 2017. Neutralization of bactericidal activity related to antimicrobial carry-over in broiler carcass rinse samples. Journal of Food Protection. 80(4):685-691. doi:10.4315/0362-028x. JFP-16-412.
Bourassa, D.V., Wilson, K.M., Bartenfeld, L.N., Harris, C.E., Howard, A.K., Ingram, K.D., Hinton Jr, A., Adams, E.S., Berrang, M.E., Feldner, P.W., Gamble, G.R., Frye, J.G., Jackson, C.R., Johnston, J.J., Buhr, R.J. 2017. Surface water accumulation and subsquent drip loss for processed broiler carcasses subjected to a post-chill water dip or spray. Poultry Science. 96(1):241-245.
Hudson, L., Harrison, M., Berrang, M.E., Jones, D.R. 2016. Alternative antimicrobial commercial egg washing procedures. Journal of Food Protection. 79:1216-1220.
Landrum, M.A., Cox Jr, N.A., Cosby, D.E., Berrang, M.E., Russell, S.M. 2016. Treatment with a low pH processing aid to reduce campylobacter counts on broiler parts. Poultry Science. 96:1028-1031.
Cox Jr, N.A., Richardson, L., Cosby, D.E., Berrang, M.E., Wilson, J.L., Harrison, M.A. 2016. A four-quadrant sequential streak technique to evaluate Campylobacter selective broths for suppressing background flora in broiler carcass rinses. Journal of Food Safety. doi:10.1111/jsf.12311.