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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 and Antimicrobial Resistance

2006 Annual Report


1.What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? Why does it matter?
Poultry products have been clearly identified as a source of foodborne human infection with bacterial pathogens. How serious is the problem: Human pathogens such as Campylobacter spp. and Listeria monocytogenes have been associated with processed poultry and poultry products. Why does it matter: These bacteria cause disease conditions that are potentially life threatening. In the farm-to-fork continuum, the post-harvest processing represents the phase in which the greatest amount of manipulation is performed on the poultry product that could affect the microbial quality of the final product. The ultimate goal of this research is to lower bacterial contamination and incidence of foodborne pathogens on processed poultry by studying microbial interactions within the poultry processing and surrounding environments. The specific objectives to achieve this goal are: 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.

This research specifically applies to Objective 2.2 Production and Processing Ecology in the National Program 108 (Food Safety) by providing new knowledge to reduce pathogens for a safe, wholesome product and addresses the following specific goals:

2.2.1.1 Determine: sources of contamination on animals, seafood and produce and their effect on microbial load; the relationship of incoming microbial load on microbial load of the final products; and effect of feed withdrawal on microbial load. 2.2.1.2 Determine effect of microorganisms within the processing ecosystem on the microbial status of the final product. Identify which areas of the processing plant lead to a significant increase in pathogens on the product. Delineate mechanisms of pathogen transmission, and the effect of processing systems and management. 2.2.1.3 Trace sources (niches) of pathogens in processing ecosystems. Determine the physiological status of microorganisms within these niches, and determine any special characteristics required for survival. Determine role of quorum sensing and nutrient availability. Identify conditions where pathogen growth is restricted.

The presence of bacteria that cause foodborne illness needs to be lowered on processed poultry so that the final consumer will stand less of a chance of becoming ill as a consequence of handling or eating poultry products. Achievement of the project goals will promote public health and create opportunities for marketing safer poultry products domestically and abroad.


2.List by year the currently approved milestones (indicators of research progress)
Year 1 (FY ’06 – 07) 1. Determine the role that outside environmental sources of Listeria monocytogenes play in the presence of this pathogen in poultry further processing facilities: contact processing company, plan projects. 2. Develop and test intervention strategies to eliminate L. monocytogenes and Campylobacter from meat products or processing plant surfaces: test GRAS chemicals in the colon 3. Evaluate gene expression profiles of L. monocytogenes and C. jejuni in conditions relevant to poultry processing environments: establish biofilm formation, protein purification and mRNA extraction procedures. 4. Evaluate the influence of animal agriculture on Campylobacter in the environment: collect and characterize Campylobacter at the slaughter process and environmental sites.

Year 2 (FY ’07 – 08) 1. Determine the role that outside environmental sources of Listeria monocytogenes play in the presence of this pathogen in poultry further processing facilities: begin to collect isolates from plants and surrounding environments. 2. Develop and test intervention strategies to eliminate L. monocytogenes and Campylobacter from meat products or processing plant surfaces: test possible means to physically plug the vent, begin testing ultraviolet light and food grade chemicals on numbers of L. monocytogenes on raw product. 3. Evaluate gene expression profiles of L. monocytogenes and C. jejuni in conditions relevant to poultry processing environments: compare expression in biofilms versus planktonic L. monocytogenes cells. 4. Evaluate the influence of animal agriculture on Campylobacter in the environment: examine gene expression profiles of Campylobacter detected in various areas of the plant under various conditions.

Year 3 (FY ’08 – 09) 1. Develop and test intervention strategies to eliminate L. monocytogenes and Campylobacter from meat products or processing plant surfaces: measure effect on the resistance of experimental isolates to therapeutic antimicrobial drugs and meat quality. 2. Evaluate gene expression profiles of L. monocytogenes and C. jejuni in conditions relevant to poultry processing environments: study knockout mutants of genes related to biofilm development in L. monocytogenes.

Year 4 (FY ’09 – 10) 1. Determine the role that outside environmental sources of Listeria monocytogenes play in the presence of this pathogen in poultry further processing facilities: finish collection of L. monocytogenes isolates. 2. Develop and test intervention strategies to eliminate L. monocytogenes and Campylobacter from meat products or processing plant surfaces: develop method to create L. monocytogenes biofilms in drain pipes. 3. Evaluate gene expression profiles of L. monocytogenes and C. jejuni in conditions relevant to poultry processing environments: develop methods for synchronized growth of Campylobacter 4. Evaluate the influence of animal agriculture on Campylobacter in the environment: determine gene expression of Campylobacter under condition common to the poultry processing environment

Year 5 (FY ’10 – 11) 1. Determine the role that outside environmental sources of Listeria monocytogenes play in the presence of this pathogen in poultry further processing facilities: characterize isolates collected and make comparisons. 2. Develop and test intervention strategies to eliminate L. monocytogenes and Campylobacter from meat products or processing plant surfaces: test treatments to lower the numbers of L. monocytogenes associated with biofilms in drains. 3. Evaluate gene expression profiles of L. monocytogenes and C. jejuni in conditions relevant to poultry processing environments: compare expression of Campylobacter cells in different stages of cell cycle. 4. Evaluate the influence of animal agriculture on Campylobacter in the environment: collect and characterize Campylobacter isolates from environmental sites.


4a.List the single most significant research accomplishment during FY 2006.
Ultraviolet light to kill L. monocytogenes on raw poultry: In an earlier study we showed that raw poultry from a slaughter plant can act as a source of L. monocytogenes to a further processing plant. Intervening in this transfer by eliminating L. monocytogenes on raw meat prior to shipment could be a useful intervention strategy. We conceived of, planned and completed a study to measure the effectiveness of germicidal UV light to kill L. monocytogenes on raw poultry meat prior to simulated shipment. Four unique subtypes of L. monocytogenes were used to inoculate raw product prior to exposure to an UV light treatment. The UV treatment significantly lowered the numbers of all four subtypes on raw meat. Numbers were still low after simulated shipment at 4 C. These data show that UV light has potential to be used by processors to break the chain of continuous re-contamination of a poultry cooking plant with L. monocytogenes from raw meat shipments.


4b.List other significant research accomplishment(s), if any.
Controlling escape of viable Campylobacter during broiler defeathering: Campylobacter counts on broiler carcasses increase due to escape of highly contaminated gut contents. We completed a study whereby food grade organic acids (acetic, lactic, and proprionic acids) were placed in the vent of carcasses prior to feather removal. This technique was effective in killing Campylobacter in the cloaca and lessened the escape of viable cells during defeathering. This study provides poultry processors with a potential means to lower the increase in Campylobacter during feather removal and could result in lower numbers of this pathogen on fully processed broiler carcasses.

Process control in broiler slaughter plants – Campylobacter prevalence: Measuring process control in commercial broiler slaughter plants has traditionally meant counting the number of carcasses positive for Salmonella. However, legal challenges to this system have left the regulatory agency (FSIS) in need of another method to monitor processors to assure that their process is in control. We completed the sample collection phase of a large collaborative effort with FSIS and Stan Bailey (ARS) to test the use of E. coli counts as a measure of process control in broiler slaughter and processing. Our earlier findings show that E. coli counts tend to go down during processing roughly parallel to the number of Campylobacter. Four replications were conducted each with 20 processing plants; carcasses sampled at re-hang and post chill. About 3,200 Campylobacter isolates were collected and preserved for future study. Campylobacter prevalence and number data is now being analyzed. These data will provide the poultry industry and interested researchers with a very good idea of what numbers of Campylobacter are entering plants with the live birds. Furthermore, the counts on fully processed broiler carcasses will illustrate the level of reduction that is being achieved due to current processing methods. Further analyses of isolates are being performed to discover if there is selective survival of Campylobacter subtypes. (National Program Component 1, Problem Statement 1.1.2 Epidemiology and 1.1.3: Ecology, Host Pathogen and Chemical Contaminants Relationships)

Effect of subtherapeutic administration of antimicrobial (tylosin) on Campylobacter numbers on broiler carcasses during processing: Antimicrobials may be added to broiler feed in subtherapeutic doses as a growth promoter. It s not clear how this practice may affect the numbers or antimicrobial resistance profile of Campylobacter on processed carcasses. We planned and conducted a study to determine the effect of sub-therapeutic application of antimicrobial (tylosin) on the numbers of Campylobacter on broiler carcasses during processing. We also measured the antimicrobial resistance of Campylobacter isolates that remained on broiler carcasses during processing. Broiler chickens were raised with or without tylosin in the diet. All birds were intentionally colonized with C. jejuni. At 42 d, broilers were processed in the pilot plant using commercial style equipment. Campylobacter counts were measured at several points during processing. Isolates were collected and characterized relative to antimicrobial resistance. The relationship between feed and the characteristics of bacteria found on processed carcasses is new information that will help companies understand the consequences of subtherapeutic antimicrobial feeding regimens. This information will be useful to the poultry industry as they plan feed formulations.

Antimicrobial resistance of salmonellae from retail chicken: The level of antimicrobial resistance on salmonellae from US retail chicken is not readily available in the literature. We completed a survey study whereby the antimicrobial resistance profile of a collection of salmonellae isolates originally recovered from retail chicken was determined. These data are important to the industry, regulators and consumers, all of which are concerned with the possibility of antimicrobial resistant salmonellae being present in the food supply.

Elimination of L. monocytogenes from biofilms in floor drains: L. monocytogenes can form a biofilm in floor drains and thus colonize poultry processing plants. This organism is very difficult to remove from a drain once a biofilm has been established. We developed a system to create a L. monocytogenes biofilm in simulated floor drains made of PVC. We then planned and conducted a series of experiments to measure the disruption of biofilms using an ultrasonic treatment to loosen the physical structure followed by application of sanitizers. Limited success has indicated the need for a different combination of treatments and the study is being continued. Once completed these data may demonstrate a novel means to remove this deadly pathogen from an important site of poultry plant colonization.

Antimicrobial resistance of L. monocytogenes: The antimicrobial resistance of L. monocytogenes found in the environment of poultry further processing plants has not been reported. If drug resistant strains are common in this arena, the potential would exist for contamination of fully cooked ready-to-eat product with drug resistant L. monocytogenes. We conceived of, planned and completed a study to measure the antimicrobial resistance of L. monocytogenes isolates detected in a poultry further processing facility. This large group of isolates is unique because it had been collected over a year long study in a commercial plant. Most of the L. monocytogenes isolates were susceptible to all antimicrobials. However, some were resistant to ceftriaxone, oxacillin, ciprofloxacin, clindamicin, tetracycline or some combination of these. The data are useful to scientists attempting to determine the importance of L. monocytogenes in processing plants and tracking the acquisition of drug resistance in such bacteria.

Microbial community structure in the Upper Oconee River watershed: The ecological distribution of human pathogens commonly associated with agricultural products is not well known. Samples were collected from 84 sites on the Upper Oconee River watershed and the isolated bacteria were subtyped to identify the distribution of clones. Clones of Salmonella, Enterococcus, and Campylobacter were found to be clustered in areas in the rivers with a radius of about 2 kilometers, which is far larger than expected for organisms found in soil samples yet still limited enough to suggest that environmental contamination occurs as a bolus or with seepage from a point source with rapid extinction of the clone. Better descriptions of bacterial communities will help to learn ways to intervene in pathogen trafficking.


4c.List significant activities that support special target populations.
Nothing this fiscal year


4d.Progress report.
Listeria monocytogenes: Source determination in further processing plant: It is known that raw product is one source of L. monocytogenes to a poultry cooking plant; however, the importance of other sources is not certain. We planned and began an ongoing study to determine the principal sources of Listeria monocytogenes to a commercial poultry further processing plant. This required extensive interaction with and cooperation from a major integrated broiler company. Our relationship with this company made it possible to examine a new poultry cooking plant during construction, shortly after start up and monthly thereafter. Monthly samples are being drawn and analyzed in order to determine if incoming air, personnel or raw product are sources of this deadly pathogen to the poultry cooking arena. As the further processing plant (producing ready-to-eat poultry meat products) becomes colonized with L. monocytogenes, isolates are being collected, preserved, subtyped using DNA sequence methodology that we developed. Isolates found colonizing the plant will be compared with source subtypes to determine how this organism is being tracked within the plant and in the environment nearby.

Microbial ecology of major Southern river systems: The presence of a wide variety of bacteria of interest in large southern rivers that are used for commercial traffic is not widely reported. A survey study is underway of water samples from large southeastern river systems including the Mississippi, the Missouri and the Tennessee as well as contributors to the Illinois and the Alabama Rivers were collected, analyzed for presence of Campylobacter, Enterococcus, E. coli and Salmonella. Protocols for isolation of Campylobacter are being improved and plans for broadening of the geographic scope of the project are being developed. All isolates have been preserved and tested for antimicrobial resistance and subtyped. These data will be analyzed and the results will show the distribution of bacteria and also will be of interest to water quality scientists.


5.Describe the major accomplishments to date and their predicted or actual impact.
This is the first report for a new project and all accomplishments to date are shown above.


6.What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end-user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products?
Genetic sequences have been deposited in GenBank, making them available to the global research community free of charge.


7.List your most important publications in the popular press and presentations to organizations and articles written about your work. (NOTE: List your peer reviewed publications below).
Nothing to report.


Last Modified: 9/2/2014
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