2013 Annual Report
1a.Objectives (from AD-416):
1. Molecular characterization of the genomic and transcriptomic differences present in foodborne pathogens (particularly Shiga-toxigenic Escherichia coli(STEC) and Salmonella spp.) to provide an understanding of genetic variation and how this variation is associated with the ability to cause disease in humans.
2. Survey ecological niches and reservoirs using a systems approach to identify sites for potential interventions to reduce foodborne pathogens.
3. Identify how foodborne pathogens acquire, maintain and transmit genes for antimicrobial resistance and virulence within cattle from production to processing.
1b.Approach (from AD-416):
Prevention and control of foodborne pathogens entering the food chain remain elusive goals, despite intensive research efforts. Information is lacking regarding the genetic variation among these pathogens in terms of the virulence and metabolic genes present, nucleotide polymorphisms, and differences in the transcriptional response and control mechanisms employed when they are exposed to adverse environmental stimuli. The advent of novel, high throughput DNA sequencing methods has revolutionized the fields of microbial genomics and microbial transcriptomics. Herein, we propose to make use of these methods and a systems approach in experiments designed to address three key knowledge gaps:
1. How are foodborne pathogens gaining entry into the food chain?
2. What are the genetic elements that facilitate a foodborne pathogen’s ability to cause disease and how are they acquired and maintained?
3. What are the novel DNA targets that can be exploited for detection, traceback and intervention development of more virulent serotypes?
The successful completion of this project will result in the development of methods and techniques to detect, characterize and target foodborne pathogens’ ability to survive in their different environments, cause disease in humans and gain entry into the food supply--which ultimately will provide a microbiologically safer food supply.
Under Objective 1, the effort continues to develop molecular epidemiological tools and evolutionary models for the classification and emergence of Shiga toxin-containing Escherichia coli (STEC) and Salmonella using single nucleotide polymorphisms (SNPs). Initial results suggest that the different pathogenic STEC serotypes have a group of strains that are more pathogenic for humans than the rest of the strains. Using a Salmonella Newport strain sequenced last year as a reference, we simulated the gene expression response of a multi-drug resistant (MDR) Salmonella Newport strain to multiple hurdle beef carcass processing interventions using high-throughput DNA sequencing methods. The data collected show that intervention exposure results in the increased expression of ~16% of Newport genes, including those involved in the heat shock response, acid stress response, DNA repair, and a number of regulatory systems that govern the Salmonella virulence response. Under Objective 2, we continue to make significant progress towards identifying potential interventions to reduce foodborne pathogens. Recto-anal junction swabs from cattle were taken to determine those animals that don’t shed STEC O157:H7. Swabs samples will be used to screen for inhibitors of E. coli O157:H7 from bovine intestinal microbiota. One potential source of foodborne contamination in the slaughter facilities is from biofilm formation. We showed that solid surface materials could significantly affect STEC biofilm formation on solid surfaces made of materials commonly used in meat industry. Also this biofilm formation made the bacteria more resistant to disinfecting agents. In nature, bacteria are able to form single – species biofilms, but more frequently coexist in multispecies communities and form mixed biofilms. We showed that mixed biofilm development was highly dependent upon companion strain properties in terms of the expression of bacterial extracellular polymeric substance, including curli fimbriae and exopolysaccharide cellulose. We proposed a new beef contamination model, in which biofilm formation and sanitizer resistance play critical roles that lead to days with high E. coli O157:H7 beef contamination in commercial meat plants. Under Objective 3, we participated in a joint project with the FDA-USDA National Antimicrobial Resistance Monitoring System pilot feedlot survey of antimicrobial resistant Enterobacteriaceae and collected 900 E. coli isolates from various feedlot settings that will be used in future conjugation and continuous culture experiments.
Theraputic treatment for disease does not increase antimicrobial resistance in cattle at harvest. Some classes of antibiotics are critically important to human medicine and are prescribed for the treatment of serious E. coli and Salmonella infections. Concerns have been raised that therapeutic treatment of feedlot cattle with antibiotics in the same classes as those used for humans increases the prevalence of resistant E. coli. ARS scientists at Clay Center, Nebraska detected a baseline, low level of antibiotic resistant E. coli in cattle upon arrival at the feedlot, temporarily increased resistance after antibiotic treatment, and a return to baseline levels after several weeks. Genetic analysis of 312 resistant E. coli isolates demonstrated that the baseline level of resistant E. coli in this cattle herd was more likely due to the persistence of a few feedlot-adapted resistant E. coli strains rather than the transfer of the genes conferring resistance between E. coli strains. These results indicate that antibiotic treatment of disease in cattle feedlots does not increase the prevalence of antibiotic-resistant E. coli in those cattle when they are harvested.
Gragg, S.E., Loneragan, G.H., Brashears, M.M., Arthur, T.M., Bosilevac, J.M., Kalchayanand, N., Wang, R., Schmidt, J.W., Brooks, J., Shackelford, S.D., Wheeler, T.L., Brown, T.R., Edrington, T.S., Harhay, D.M. 2013. Cross-sectional study examining Salmonella enterica carriage in subiliac lymph nodes of cull and feedlot cattle at harvest. Foodborne Pathogens and Disease. 10(4):368-374.
Schmidt, J.W., Griffin, D., Kuehn, L.A., Harhay, D.M. 2013. Influence of therapeutic ceftiofur treatments of feedlot cattle on fecal and hide prevalences of commensal Escherichia coli resistant to expanded-spectrum cephalosporins, and molecular characterization of resistant isolates. Applied and Environmental Microbiology. 79(7):2273-2283.
Beier, R.C., Poole, T.L., Brichta-Harhay, D.M., Anderson, R.C., Bischoff, K.M., Hernandez, C.A., Bono, J.L., Arthur, T.M., Nagaraja, T.G., Crippen, T.L., Sheffield, C.L., Nisbet, D.J. 2013. Disinfectant and antibiotic susceptibility profiles of Escherichia coli O157:H7 strains from cattle carcasses, feces, and hides and ground beef from the United States. Journal of Food Protection. 76:6-17.
Edrington, T.S., Loneragan, G.H., Hill, J.E., Genovese, K.J., He, L.H., Callaway, T.R., Anderson, R.C., Brichta-Harhay, D.M., Nisbet, D.J. 2013. Development of a transdermal Salmonella challenge model in calves. Journal of Food Protection. 76:1255-1258.
Harhay, G.P., Koren, S., Phillippy, A.M., McVey, D.S., Kuszak, J., Clawson, M.L., Harhay, D.M., Heaton, M.P., Chitko-McKown, C.G., Smith, T.P.L. 2013. Complete closed genome sequences of Mannheimia haemolytica serotypes A1 and A6 isolated from cattle. Genome Announcements. 1(3):e00188-13. DOI: 10.1128/GENOMEA.00188-13.