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:
This new Peer Review project replaces 6040-41420-004-00D, “Microbial Ecology of Human Pathogens Relative to Poultry Processing.” Continued an ongoing study to measure the flock prevalence of Campylobacter by weekly sampling of gut contents collected in a commercial broiler slaughter plant. Using data from three years of this collection and readings from a nearby weather station, we tested for a relationship between month of the year, rain fall, and temperature and the prevalence of Campylobacter in broiler flocks. Data has been collected, analyzed and manuscript prepared. Completed a study to determine if any of three different types of Campylobacter selective plating media select for specific subtypes of Campylobacter. Such a selection could skew data towards or away from one subtype and have potential to confound our understanding of Campylobacter ecology. Three types of media were used to detect and isolate Campylobacter from broiler gut contents and carcass rinses (n=100). Forty nine of 100 samples had a Campylobacter positive result on all three media (campy-cefex, campy-line and Rf-Campylobacter agars). Isolates were subjected to whole genome sequencing and the results of sequencing were used to determine MLST sequence type of each isolate. Overall, it was far more likely that all three media recovered the same subtype of Campylobacter from each sample than for any one media to result in the recovery of a different type. These data have been analyzed and a manuscript has been prepared. Antimicrobial processing aids used to kill bacteria on chicken carcasses in the processing plant can lead to a carryover of the killing effect on a carcass rinse sample. This can result in a false negative evaluation of the carcass when the rinsate is later cultured for Salmonella. Such a false negative can confound the processor or regulators in ongoing efforts to monitor Salmonella prevalence. Working with members from the Poultry Microbiological Safety and Processing Research Unit and the Quality and Safety Assessment Research Unit of Athens, Georgia, we have addressed this carry over problem. We have collaboratively defined and tested neutralizers that show promise to control carry-over killing effect of peroxyacetic acid, cetylpyridinium chloride and several other commonly used antimicrobials. More data is being collected to further replicate our results; we expect to have concrete recommendations to halt sanitizer carry-over effect within coming months. Worked with ARS and Food Safety and Inspection Service (FSIS) collaborators to formulate and test a new broiler carcass rinse medium for detection of Salmonella. This new medium is better buffered and has the ability to counter the antimicrobial activity of a wide range of processing chemicals that can be expected to possibly be present on the carcass when rinse sampling is done. Such carry over of antimicrobial activity can confound regulatory oversight relative to detection of Salmonella. The new media has been tested and shown to allow recovery of Salmonella even in presence of antimicrobial carryover. The validation stage for Salmonella is underway currently. FSIS has already begun to use the new medium for regulatory samples in broiler processing plants as of July 1, 2016. Chlorine dioxide drain decontamination: Floor drains in poultry processing plants are commonly colonized by Listeria monocytogenes, which can contaminate finished product. ARS researchers at Athens, Georgia, conceived, designed and conducted experiments to test a novel chlorine dioxide generation and delivery system as a means to sanitize floor drains. The work documented the utility of self-contained pods to significantly lower the number of total aerobic bacteria in floor drains. Further testing was conducted in model floor drains in which a Listeria monocytogenes biofilm was previously established. Conceived of a pre-chill cooking procedure to eliminate all viable bacteria from broiler carcasses prior to shipment to a further processing plant. Wrote and submitted an invention disclosure for the process. Research was conducted with ARS collaborators to test the process of cooking whole broilers before rigor to determine the effect on meat quality. Overall, meat quality as measured by tenderness was not substantially impacted by cooking before chilling. This represents a potential method to eliminate bacterial contamination on broiler meat prior to shipment to a further processing facility for deboning, dicing and other further processing. By avoiding immersion chilling, a processor may also save energy, water, plant space and equipment costs. We have shown that a 0.45 micron nitrocellulose filter placed on top of solid plating medium is useful as a means to eliminate non-Campylobacter contamination from samples with high numbers of both Campylobacter and background bacteria such as broiler feces or gut contents. This year we began a study to determine the rate of Campylobacter passage through filters and onto the growth medium below. We also began to measure the proportion of an inoculum that makes passage through the filter. We hope that these data once fully collected will allow us to use the filter method to quantitatively estimate the cellular density of Campylobacter in a complex, highly contaminated sample. Listeria (L.) monocytogenes can form biofilms and colonize floor drains in poultry processing plants causing potential to contaminate finished poultry meat products or product contact surfaces. Some protozoa, including Tetrahymena, can ingest and kill bacteria. ARS scientists in Athens, Georgia, tested Tetrahymena as a means to control L. monocytogenes numbers in model drain systems both as free swimming planktonic cells and attached to a surface as a biofilm. Preliminary work showed that co-incubation with Tetrahymena can effectively lower the number of viable Listeria monocytogenes cells both in liquid and attached to a surface. Preliminary work has also revealed that adding Tetrahymena to a pre-formed biofilm can lower the numbers of Listeria monocytogenes. Further work is planned to confirm these findings and determine the extent of Tetrahymena activity.
5. Significant Activities that Support Special Target Populations: