2012 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.
Progress was made on all three objectives and their subobjectives, all of which fall under National Program 108. Progress was made under Objective 1.1 by mapping sequencing reads to reference genomes to identify genetic variation in strains of Shiga toxingenic Escherichia coli (STEC) serotypes O26 and O111. These single nucleotide polymorphisms (SNPs) will be validated and used to develop an evolutionary model for the emergence of these foodborne pathogens. DNA was extracted and sequencing libraries made from STEC serotype O103 strains. Under Objective 1.2, progress was made by sequencing and assembling 11 Salmonella strains. Additional strains will be determined once a current experiment is finished that will show the relatedness of strains and allow the most diverse strains to be sequenced. Under Objective 2.1, we have made significant progress towards a functional metagenomic system that will utilize the DNA from bovine intestinal microbiota to screen for inhibitors of E. coli O157:H7. DNA isolation methods, utilizing bead beating and enzymatic cell lysis, have been developed to obtain high quality DNA from bovine fecal samples. This DNA is sheared to an optimal length of 40kb and purified using a BluePippin DNA size selection system. We have begun sampling cattle to identify those individuals that do not harbor E. coli O157:H7 as they may be colonized with microbial antagonists that prevent the growth of the pathogen. Under Objective 2.2, we have made significant progress demonstrating that biofilm formation and the effectiveness of food grade sanitizers to remove biofilm is quite variable in STEC and Salmonella strains. The sequence of bacterial colonization and extracellular polymeric substances significantly affect biofilm formation and resistance to sanitization. Under Objective 3.1, substantial progress has been made in understanding plasmid maintenance and stability in Salmonella. Progress was made by sequencing and comparing 11 plasmids from Salmonella isolates before examining their adaptive changes in the batch fermenter. Under Objective 3.2, we have made significant progress culturing and identifying antibiotic resistant E. coli. Progress was made by completing the survey of antibiotic resistant E. coli from a cattle feedlot and characterizing a majority of the isolates by PFGE and molecular methods.
Genomic markers for identifying specific pathogenic Escherichia coli strains. The Food Safety Inspection Service recently declared E. coli strains O26, 045, O103, O111, O121, and O145 adulterants in beef trim and recently started screening for these foodborne pathogens. The current method for detecting these specific strains (serotypes) takes several days because there is not a specific genomic marker for each serotype. ARS scientists at Clay Center, Nebraska identified strain specific DNA markers for each serotype by comparing portions of the DNA from each serotype. The DNA markers were licensed to a company that makes diagnostic kits for foodborne pathogens and are being used as part of a commercially available assay.
Bono, J.L., Smith, T.P., Keen, J.E., Harhay, G.P., McDaneld, T.G., Mandrell, R.E., Jung, W., Besser, T.E., Gerner-Smidt, P., Bielaszewska, M., Karch, H., Clawson, M.L. 2012. Phylogeny of Shiga toxin-producing Escherichia coli O157 isolated from cattle and clinically ill humans. Molecular Biology and Evolution. 29(8):2047-2062. doi: 10.1093/molbev/mss072.