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United States Department of Agriculture

Agricultural Research Service

2011 Annual Report

1a.Objectives (from AD-416)
The purpose of the Poultry Microbiological Safety Research Unit is to further reduce or eliminate bacterial pathogen contamination in poultry operations and the following objectives are to:.
1)Assess the effectiveness and further development of bacteriocins (anti-bacterial peptides) and bacteriophage by in vitro bacterial growth inhibition in culture and in vivo experimentation via challenge in chickens..
2)Reduce bacterial populations in chicken litter by monitoring poultry houses for bacterial pathogens carried by chickens during use of intervention technologies. Decontamination techniques, such as in-house foam applied disinfectants will be examined for reduction of Campylobacter spp., Salmonella spp., Clostridium perfringens and Listeria monocytogenes..
3)Improve cultural methods for Campylobacter spp. in poultry in further support of FSIS needs, including the improvement of recovery. Use microarray expression analysis under various cultural conditions to identify nutrients necessary for optimal growth, colonization and culture of Campylobacter spp.

1b.Approach (from AD-416)
New antimicrobial agents will be developed that are usable by the poultry farmer on a large scale to reduce Campylobacter spp. and salmonellae by applying in feed at the appropriate times before slaughter. Also, new means for the drug-free production sector to control clostridial disease without relying on antibiotics are the major expected approaches of Objective 1. Providing a science based source of data to better dispose of spent poultry litter without major energy or financial inputs is the approach for Objective 2. A more reliable and quantitative method to routinely culture Campylobacter spp. from food and environmental samples is the approach of Objective 3.

3.Progress Report
Bacteriocin/phage discovery and applications. Bacteriophages are viruses that infect and can potentially kill their bacterial hosts. Because biotechnological uses of bacteriophage gene products as alternatives to conventional antibiotics will require a thorough understanding of their genomic context, we sequenced and analyzed the genomes of four closely related bacteriophages isolated from Clostridium perfringens, an important agricultural and human pathogen. Comparative analyses of the evolutionary history and genomic context of common phage proteins revealed two important results:.
1)strongly significant host-specific sequence variation within the endolysin; and.
2)protein domain architecture apparently unique to our phage genomes in which the endolysin is located upstream of its associated holin. Endolysins in our phage genomes may be subject to different selective pressures than the rest of the genome. These findings may have important implications for potential biotechnological applications of phage gene products.

Improved cultural methods for pathogen recovery. Phenotype microarrays were utilized to evaluate the ability of Campylobacter coli strain 49941 to oxidize 190 different substrates as sole carbon sources at 37oC and 42oC. Our improved understanding of the metabolic pathways and nutritional requirements of C. coli is being used to develop enhanced media for detection and isolation of the pathogen.

This inhouse research project has been replaced by project #6612-32000-057-00D.

1. Antimicrobial activities of bacteriocins against pathogenic bacteria. Countries that have complied with the ban on antimicrobial growth promoters (AGP) in feeds have had increased incidences of Clostridium perfringens-associated necrotic enteritis in poultry and an increased potential to cause human food-borne bacterial diseases. To address this issue, new antimicrobial agents, putative bacteriophage lysins from the genomes of bacteriophages were identified and expressed as recombinant proteins. The proteins were identical in the C-terminal putative cell-wall binding domain, but only 55% identical to each other in the presumptive N-terminal catalytic domain. Both recombinant lysins were capable of lysing both parental phage host strains of C. perfringens as well as other strains of the bacterium in spot and turbidity reduction assays but other bacteria were resistant to the lysins. Consequently, it is possible to develop species-specific interventions for food-borne pathogens while not reducing the numbers of probiotic helpful bacteria during food-animal production. Also, a new bacteriocin L-1077 appears to hold promise in controlling C. jejuni/S. Enteritidis among commercial broiler chickens during production.

2. Understanding metabolic pathways and nutritional requirements of Campylobacter coli strain 49941. Campylobacters are small, asaccharolytic bacteria having unique nutritional and environmental requirements. These bacteria are commensal organisms in chickens, but remain the leading cause of bacterial gastroenteritis in humans. C. jejuni is most often associated with poultry, while C. coli are more frequently associated with swine. Temperature may trigger potential colonization or virulence factors in C. jejuni and temperature-dependent colonization factors are responsible for species-specific colonization by C. coli. ARS researchers in Athens, Georgia, determined utilization of 190 carbon substrates by C. coli at 37oC and 42oC using a phenotype microarray (PM). L-asparagine and L-serine allowed significantly more respiration by C. coli at the lower temperature of 37oC as compared to 42oC. Conversely, L-glutamine was utilized to a significantly greater extent at the higher temperature of 42oC. Other organic substrates exhibited temperature dependent utilization including succinate, D, L-malate and propionate which all supported active respiration by C. coli to a greater extent at 42oC. A better understanding of the metabolic pathways and nutritional requirements of campylobacters will provide improvements in culture media for detection and isolation of the pathogen leading to intervention methods to reduce human exposure.

Review Publications
Seal, B.S., Siragusa, G.R., Simmons, I.M., Fouts, D.E., Garrish, J.K., Kuntz, R.L., Woolsey, R., Schegg, K.M., Kropinski, A., Ackermann, H.W. 2011. Clostridium perfringens bacteriophages FCP39O and FCP26F: genomic organization and proteomic analysis of the virions. Archives of Virology. 156(1):25-35.

Line, J.E., Hiett, K.L., Guard, J.Y., Seal, B.S. 2010. Temperature affects sole carbon utilization patterns of Campylobacter coli 49941. Current Microbiology. 62:821-825.

Last Modified: 11/24/2015
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