2012 Annual Report
1a.Objectives (from AD-416):
1. Develop, evaluate, and validate alternatives to current antimicrobials and sanitizers for processing poultry carcasses that meet national and international food safety standards. Specifically, alternatives to chlorine and chlorine dioxide, such as GRAS surfactants, ozone, acidified sodium chloride, and peroxyacids should be examined.
2. Characterize and evaluate poultry processing conditions associated with processed broiler carcasses that are heavily contaminated by Salmonella. This objective will include determining microbial recovery/extraction rates for poultry rinse sampling and examining survival characteristics for Salmonella serotypes that survive processing operations. Sampling should include whole carcasses that are high shedders, and mechanically-separated poultry parts.
3. Conduct studies on extrinsic factors that influence the survival and attachment of pathogenic, spoilage, and indicator microorganisms recovered from commercial poultry processing operations. These include water activity, pH, ozone, metal chelators, and organic acids.
4. Collaborate with the QSARU at Athens to develop emerging technologies for process control and correlate molecular and cultural analysis of microbial communities in poultry with process control and pathogen contamination.
1b.Approach (from AD-416):
Technologies will be developed that can be utilized by commercial processors to reduce microbial contamination associated with poultry processing. Formulations for novel sanitizers will be developed, and improved techniques utilizing sanitizers already approved for commercial use in processing will be designed. Surfactant based sanitizers used alone or in combination with non-chlorine based sanitizers will be examined as alternatives to chlorine and chlorine dioxide for decreasing microbial contamination of poultry. Additionally, poultry processing conditions associated with broiler carcasses heavily contaminated by Salmonella will be evaluated and characterized. Cross contamination during processing will be examined by studying the role of these heavily contaminated carcasses in the spread of Salmonella during processing. Furthermore, factors that influence survival and attachment of pathogenic, spoilage, and indicator microorganisms on poultry skin will be examined. Microorganisms on poultry skin will be examined utilizing Benchtop scanning electron microscopy (SEM) with SEM software and with standard microbiological methods. Finally, in collaboration with the Quality Assessment Research Unit (QARU), correlations between molecular and cultural analysis of microbial communities on poultry skin will be examined and emerging technologies for process control will be developed. Technology will be designed for use in the rapid detection and identification of pathogenic bacteria associated with poultry processing and for the characterization of microbial biofilms on broiler skin. All research goals will be achieved through an interdisciplinary team approach utilizing knowledge and skills of Unit scientists and other scientists who possess unique skills and resources that will aid in the successful completion of this project.
Extensive progress was made on 3 of the objectives. Under Objective 1, experiments that studied the effect of power of hydrogen (pH) on the ability of alkaline salts of three fatty acids (FA) to inhibit growth of bacteria associated with poultry processing were conducted. Findings from these studies demonstrated that the pH of solutions of alkaline salts of FA may influence the antibacterial activity of the surfactants towards bacteria associated with poultry processing. Under Objective 3, research was conducted that demonstrated that Campylobacter bacteria can be grown aerobically in media supplemented with organic acids that are metabolized by these pathogenic bacteria. The development of this bacteriological media will simplify procedures for growing Campylobacter since utilization of the media will allow scientists to grow the bacterium without the use of specialized gaseous atmospheres and expensive incubation equipment. Studies were also conducted that examined changes in the microbial quality of scalder and chiller water during poultry processing operations in commercial poultry processing facilities. Results from these studies showed that there was a significant increase in the number of pathogens recovered from scalder water; however, there was a smaller increase in the number of pathogens recovered from water taken from immersion chiller tanks. These experiments will provide researchers and commercial processors with additional data that can be used to understand cross contamination and pathogen survival in poultry processing water. Experiments were also conducted to develop a new technique for rapidly identifying Salmonella bacteria. The new technique identifies the bacteria based on the structure of the bacterial Deoxyribo Nucleic Acid (DNA), and it will provide researchers with a tool for rapidly identifying Salmonella. Additional studies developed a new method for Campylobacter detection using specific antibodies that were developed for the bacterium. This biosensor-based detection technology will provide the poultry industry with a novel, rapid method for detecting Campylobacter species (spp.) in poultry production and processing. Under Objective 4, studies on the development of rapid methods for the detection and identification for foodborne bacterial pathogens associated with poultry processing were conducted. A procedure called the Fourier Transform Infrared Spectroscopy (FT-IR) method was investigated for its ability to detect and differentiate different species of Salmonella. Experiments showed that the FT-IR can rapidly detect and differentiate between Salmonella Typhimurium and Salmonella Enteritidis species, and it can also distinguish between live and dead cells of these bacteria with 100% accuracy.
Temporal study of pathogen ecology in poultry processing. Agricultural Research Service (ARS) scientists in Athens, Georgia, demonstrated changes in the bacterial quality of scalder tank water during commercial processing of poultry resulted in the recovery of higher levels food safety-related pathogens from the water within those tanks. Chiller tank water quality did not change as significantly, however, and no pathogens were recovered from chiller water. Results of this study will provide data to researchers and commercial processors on the importance of understanding the role of microbial ecology of processing water in controlling poultry food safety-related pathogens in the processing operations.
Transmission of Salmonella and Campylobacter among caged and cage-free laying hens. ARS scientists in Athens, Georgia, conducted a series of experiments to evaluate the potential for the transmission of the foodborne pathogens, Salmonella and Campylobacter, among table egg producing hens in different housing systems. Among Salmonella-challenged hens, the percentage of cecum and reproductive tract samples positive for Salmonella were similar, and infection from the cloaca likely resulted in contamination of the reproductive tract. The horizontal transmission of Campylobacter among non-challenged hens was significantly greater on shavings than in cages. The litter in the shavings pen most likely contributed to the survival of the Campylobacter that was shed through the feces of challenged hens. Findings of this study will assist commercial poultry growers in determining best growing methods to reduce the spread of human foodborne pathogens among laying hens.
Classification and structural analysis of live and dead salmonella cells using Fourier Transform Infrared (FT-IR) Spectroscopy and Principle Component Analysis (PCA). ARS scientists in Athens, Georgia, conducted experiments on the rapid detection, identification and differentiation of different bacteria. In recent years, research on the development of rapid methods for identifying and characterizing bacteria has increased. In these experiments, the Fourier Transform Infrared spectroscopy (FT-IR) method was used to detect and differentiate Salmonella. Live and dead cells of Salmonella Typhimurium and Salmonella Enteritidis were used in this study. Live cells of both Salmonella Typhimurium and Salmonella Enteritidis were classified with 100% accuracy and the technology was able to differentiate between live and dead cells. This technology may be used to develop methods that can be used for the rapid detection and identification of Salmonella.
Effect of power of hydrogen (pH) of alkaline salts of fatty acids on the inhibition of bacteria associated with poultry processing. ARS scientists in Athens, Georgia, conducted experiments using the agar diffusion assay to examine the effect of pH on the ability of alkaline salts of three fatty acids (FA) to inhibit growth of bacteria associated with poultry processing. FA solutions were prepared by dissolving fatty acids in potassium hydroxide (KOH), and citric acid was added to the mixtures to adjust the pH. Results indicated that reducing the pH of some of the fatty acid mixtures caused decreases in the size of zones of inhibition of some of the bacteria. Findings demonstrated that the pH of solutions of alkaline salts of FA may play an important role in the antibacterial activity of these surfactants towards bacteria associated with poultry processing. Research will be used in the development of novel, non-chlorine based sanitizers that can be used to reduce microbial contamination during poultry processing.
Development of a Salmonella enterica identification assay based on Deoxyribo Nucleic Acid (DNA) structure of the bacteria. ARS scientists in Athens, Georgia, conducted laboratory experiments to develop genetic primers and probes that specifically target portions of Salmonella DNA. The newly developed assay will allow scientists to accurately distinguish between any combinations of the DNA from strains of Salmonella enterica. Research is being expanded to test environmental Salmonella enterica isolates. Results of this assay will provide researchers with a rapid serotyping tool for major poultry food safety-related pathogenic Salmonella enterica strains, and a method to understand the survival dynamics of these strains in environments along the entire poultry production spectrum.
Colonization of a marker and field strains of Salmonella Enteritidis and Salmonella Typhimurium in vancomycin pretreated and non-pretreated laying hens. ARS scientists in Athens, Georgia, conducted a study to evaluate the effects of pre-treating laying hens with the antibiotic vancomycin on the hen’s susceptibility to colonization by Salmonella. The ability of Salmonella strains to colonize the intestinal and reproductive tracts the hens were compared. Hens were orally challenged with the Salmonella and housed for 72 weeks. Periodically, hens from the study were euthanized, organ samples were collected, and the presence of Salmonella in the organs was determined. The ability of the Salmonella to colonize the hens was based on the ability of the bacteria to successfully compete for nutrients and to become resistant to vancomycin. Findings of this study confirm the importance of bacterial antibiotic resistance in the colonization of poultry by human pathogenic bacteria.
Aerobic growth of Campylobacter in media supplemented with a-ketoglutaric, lactic, and/or fumaric acids. ARS scientists in Athens, GA conducted a series of experiments to determine if cultures of Campylobacter bacteria can be grown under aerobic conditions. Campylobacter is a major human, foodborne pathogens, and presently microbiologists must grow these bacteria in closed jars under microaerobic conditions. Producing microaerophilic conditions requires the use of expensive equipment and technical training. Recent experiments have indicated that growth of Campylobacter is stimulated in media supplemented with selected organic acids; therefore, experiments were conducted to determine if Campylobacter that are provided some of these acids can also grow under aerobic conditions. Findings indicated that aerobic growth of all Campylobacter isolates was produced in media containing a mixture of organic acids. This study may lead to the development of media that will simplify and reduce the cost of growing cultures of a major human foodborne bacterial pathogen.
Dunkley, C.S., Cunningham, D.L., Ritz, C.W., Dunkley, K.D., Hinton Jr, A. 2011. Using mortality compost in vegetable production: A comparison between summer and winter composting and its use in cabbage production. Agriculture, Food and Analytical Bacteriology. 1:6-14.
Hannah, J.F., Wilson, J.L., Cox Jr, N.A., Richardson, L.J., Cason Jr, J.A., Buhr, R.J. 2011. Horizontal transmission of Salmonella and Campylobacter among caged and cage-free laying hens. Avian Diseases. 55:580-587.
Hannah, J.F., Wilson, J.L., Cox Jr, N.A., Richardson, L.J., Cason Jr, J.A., Buhr, R.J. 2011. Colonization of a marker and field strain of Salmonella Enteritidis and a marker strain of Salmonella Typhimurium in vancomycin pretreated and non-pretreated laying hens. Avian Diseases. 55:588-592.
Hinton Jr, A., Ingram, K.D. 2011. Influence of ethylenediaminetetraacetic acid (EDTA) on the bactericidal activity of fatty acids. International Journal of Poultry Science. 10:500-504.
Hinton Jr, A., Ingram, K.D. 2011. Influence of ethylenediamine-n,n’-disuccinic acid (EDDS) concentration on the bactericidal activity of fatty acids in vitro. Journal of Food Safety. DOI: 10.1111/j.1745-4565.2011.00307.x.
Rothrock Jr, M.J. 2011. Comparison of microvolume DNA quantification methods for use with volume-sensitive environmental DNA extracts. Journal of Natural and Environmental Science. 2(1):34-38.
Rothrock Jr, M.J., Frantz, J., Burnett, S.E. 2012. Effect of volumetric water content and clover (Trifolium incarnatum) on the survival of Escherichia coli O157:H7 in a soil matrix. Current Microbiology. 65(3):272-283.
Rothrock Jr, M.J., Vanotti, M.B., Szogi, A.A., Gonzalez, M.G., Fujii, T. 2011. Long-term preservation of Anammox bacteria. Applied Microbiology and Biotechnology. 92:147-157.
Sundaram, J., Park, B., Yoon, S.C., Hinton Jr, A., Windham, W.R., Lawrence, K.C. 2012. Classification and structural analysis of live and dead salmonella cells using fourier transform infrared (FT-IR) spectroscopy and principle component analysis (PCA). Journal of Agricultural and Food Chemistry. 60(4):991-1004.