2011 Annual Report
1a.Objectives (from AD-416)
1. Determine particular ecological niches/reservoirs for pathogenic/antimicrobial-resistant bacteria, identifying nutritional/biological/environmental factors affecting ability to colonize/survive/persist within gut of food-producing animals & their production environment. a) Determine effect of feeding distillers grains & other diets & organic acids on gut microbial ecology; b) Determine effects of feeding diets high in rumen undegradable intake protein & of feeding monensin on colonization/carriage/shedding of Campylobacter in cattle; c) Evaluate mechanistic effects of short chain nitrocompounds & free fatty acids on hydrogen balance/fitness of foodborne pathogens; d) Determine if specific or shared reservoirs exist for Clostridium difficile in populations of swine & humans in integrated agri-business operations; phenotypically/genotypically characterize Cl. difficile isolates; e) Determine effects of lactoferrin & whey-protein concentrate on fecal shedding & gut populations of Salmonella Typhimurium in weaned piglets; f) Determine influence of vitamin D supplement on fecal prevalence/concentrations of E. coli O157:H7 in cattle. 2. Evaluate ways to better characterize/control Salmonella, in particular multi-drug-resistant Salmonella, for use on commercial dairy farms to enhance food safety. a) Develop Salmonella serotype-specific real-time PCR method; b) Determine if Salmonella serotype changes due to changing environment or acquisition of antimicrobial resistance; c) Determine if feeding sodium chlorate, with/without nitroethane, is effective in reducing populations of Salmonella as well as E. coli O157:H7 & generic E. coli in milk-replacer fed calves & cull dairy cattle. 3. Develop interventions that prevent/mitigate colonization of gut of food-producing animals (particularly lower GI tract before slaughter) or that reduce pathogenic/antimicrobial-resistant bacteria in production environment. a) Determine if porcine-derived competitive exclusion culture can stimulate innate immune responses in neonatal gnotobiotic piglets; b) Develop/characterize bactericidal effectiveness of thymol & diphenyliodonium chloride products bound to clay-based adsorbents; c) Establish feasibility of fresh/dried citrus peel & purified essential oils as feed additives to reduce pathogen populations in ruminants; d) Develop targeted management interventions to facilitate exclusion of antimicrobial-resistant bacteria from gut of treated animals. 4. Develop understanding of microbial adaption to intrinsic/extrinsic stressors on acquisition/exchange/expression of incompatibility plasmids & antimicrobial resistance elements in foodborne pathogens in production/processing environments. a) Determine if number/size of plasmids possessed by different wildtype E. coli affects growth rate in nonselective & mixed culture environments, and if these plasmids are lost during long-term maintenance without selection pressure; b) Determine if IncN/Incl1 can mobilize IncA/C in tri-parental conjugations; c) Determine if occurrence of bacterial resistance to disinfectants is important in disinfectant control or impacts antibiotic resistance in microorganisms of animal origin.
1b.Approach (from AD-416)
Basic and applied research will be conducted to achieve project objectives. Ecological studies utilizing metagenomic analysis will elucidate niches or reservoirs where pathogens may exist and when combined with traditional epidemiological and microbiological cultural methods these studies will help reveal environmental, nutritional, and biological factors affecting fitness characteristics related to the persistent colonization, survival, and growth of these pathogens in food animals and their production environment. Research involving both in vitro and in vivo experimentation will be used to measure and characterize adaptive responses microbes may exhibit to intrinsic and extrinsic stressors, such as those exerted by disinfectants and antimicrobials, as well as to determine the role these stressors may play in pathogenicity, virulence, and resistance. Animal studies conducted under clinical and field situations will be used to develop and evaluate interventions, thereby revealing specific metabolic endpoints, cellular mechanisms, and sites of action of cellular processes that may ultimately be exploited to decrease carriage and shedding of pathogens during production and at slaughter. Opportunities for mitigation will be validated in the field. In some cases, Cooperative Research and Development Agreements will be implemented with industry partners to aid in technology transfer.
This is a new project that is continuing and expanding upon the work of the precursor project 6202-32000-020-00D. Work under this project during FY 2011 resulted in significant progress in evaluating and developing new strategies and best-use applications of existing strategies for the control of foodborne pathogens in cattle and swine. Project work in FY 2011 provided new knowledge on the application of a number of novel feed additives to reduce pathogen colonization in swine and cattle, establishing that feeding appropriate amounts of organic acids can reduce fecal shedding of Salmonella by pigs; and showing that a monoester of the 12-carbon fatty acid, lauric acid, significantly inhibits mastitis-causing pathogens in dairy cattle. Project work also generated new, important information on factors influencing the carriage and potential dissemination of disinfectant and antimicrobial resistance among bacterial populations, establishing that environmentally ubiquitous organisms such as Pseudomonas aeruginosa may be important reservoirs of disinfectant and antimicrobial resistance in agricultural environments. Project work in FY 2011 expanded on research initiated under the previous project to develop practical, cost-effective interventions and/or management practices to reduce the carriage and environmental dissemination of pathogenic and antimicrobial-resistant microbes by food-producing animals. New technology and protocols developed by this work will help U.S. farmers and ranchers produce safer and more wholesome meat products, at less cost, for the consumer.
Disinfectant and antimicrobial resistance in Pseudomonas aeruginosa. Pseudomonas aeruginosa is a pathogenic bacterium that can cause disease in both humans and animals; it occurs throughout the world in many environments. The specific characteristics and traits of P. aeruginosa make it well suited as a potential reservoir of antimicrobial resistance in certain food processing environments. ARS researchers at College Station, TX, established the disinfectant and antimicrobial resistance patterns of more than 300 P. aeruginosa isolates and found in many of these isolates high levels of antibiotic and disinfectant resistance. Of specific importance was the identification of isolates highly resistant to the commonly used disinfectants cetyl ammonium, cetyl pyridinium halides, and chlorhexidine. This work has important food safety/human health implications because it has identified a potential reservoir of disinfectant and antimicrobial resistance in agricultural and food processing environments.
Organic acids reduce Salmonella in swine and poultry. Salmonella bacteria are human pathogens that can reside in the gut of food animals such as swine, cattle, and poultry; these bacteria can contaminate meat products reaching the consumer and thus cause illness or even death. Organic acids are a dietary additive that can improve animal growth efficiency and change the microbial population of the intestinal tract. ARS researchers at College Station, TX, demonstrated that including specific organic acids in the diets of pigs and chickens could reduce populations of Salmonella from 10 to 100 fold in the live animals. This work has important food safety implications because it identifies another tool to help producers reduce the carriage of foodborne pathogens in meat-producing animals. Reduced pathogen loads in animals at slaughter will result in microbiologically safer meat products reaching the consumer.
Xu, Z., Wang, H., Shen, Y., Nichkova, M., Lei, H., Beier, R.C., Zheng, W., Yang, J., She, Z., Sun, Y. 2011. Conformational changes of hapten-protein conjugates resulting in improved broad-specificity and sensitivity of an ELISA for organophosphorus pesticides. Analyst. 136:2512–2520.
Li, X., Lui, P., Tang, S., Beier, R.C., Wu, X., Yang, L., Li, Y., Xiao, X. 2011. Development of an immunochromatographic strip test for rapid detection of melamine in raw milk, milk products, and animal feed. Journal of Agricultural and Food Chemistry. 59:6064-6070.