1a. Objectives (from AD-416):
1: Determine the mechanisms of biofilm formation in foodborne pathogens and elucidate the role of biofilms in persistence of pathogens in food environments. 1.1 Assemble and screen a collection of Shiga toxin-containing Escherichia coli (STEC) for biofilm forming properties. 1.2 Molecular characterization of biofilm formation in non-O157 STEC. 1.3 Identification of novel factors necessary for biofilm formation in non- O157 STEC. 1.4 Mixed biofilm formation between STEC and isolates from food processing environments. 2: Examine the role of quorum sensing of microorganisms in food environments, with specific emphasis on quorum sensing in mixed biofilm formation and the role of autoinducers such as AHL in survival. 2.1 Examine the role of quorum sensing in biofilm formation by non-O157 STEC. 3: Examine the persistence and transmission of antimicrobial resistant bacteria in microbial ecosystems, with specific emphasis on mobilizable plasmids carrying antibiotic resistance genes. Specifically, conduct sequence analyses and determine phylogenetic relationships among mobilizable plasmids carrying genes encoding antibiotic resistance and investigate gene transfer in biofilms. 3.1 Examine the prevalence and persistence of the KanR ColE1-like plasmids in Salmonella serovars isolated from sick animals and their environment– a longitudinal study. 3.2 Investigate plasmid transmission and persistence in biofilms using the KanR ColE1-like mobilizable plasmids as model systems. 4: Qualitatively and quantitatively characterize microbial communities associated with food and food processing environments and examine the role of predominant species in pathogen persistence in mixed culture biofilms. 4.1 Develop a DNA-based most probable composition protocol for estimating the total number, as well as the type, of organisms in an environmental sample or biofilm. 4.2 Determine relative concentrations of various foodborne organisms. Sampling from select food or processing locales, culturable isolate plating & selection, PCR amplification, gene cloning, plating and selection.
1b. Approach (from AD-416):
Microbes rarely exist in the environment as a monoculture but are present in complex microbial communities. Microorganisms in these communities often engage in a wide range of intercellular behaviors that may affect the presence and persistence of pathogens in foods. The primary aims of this project are to gain a better understanding of some of the complex social behaviors of foodborne pathogens and to catalog bacterial communities associated with selected foods and food processing environments. These aims will be accomplished by: 1) studying the genetic factors contributing to biofilm formation in Shiga toxin-producing E. coli (STEC) and the role of cell-to-cell communication (quorum sensing) in biofilm formation in STEC, 2) studying the potential for mixed biofilm formation between STEC and non-pathogenic environmental flora, 3) examining the prevalence and persistence of antimicrobial resistance plasmids in Salmonella strains isolated from natural environments and investigating the transmission and persistence of these plasmids in model biofilms composed of Salmonella and/or STEC, and 4) developing and applying sampling and sequencing methods to qualitatively and quantitatively determine the members of microbial communities in beef products and beef processing facilities. Collaborations with ARS, corporate, and university partners have been established to ensure that all aspects of the work can be accomplished.
3. Progress Report:
Progress was made on all four objectives and several associated subobjective, all of which fall under National Program 108, Food Safety, specifically contributing to Component 1: Food Contaminants and Problem Statement(s) 1.A Populations Systems and 1.B Systems Biology of the 2011-2015 Strategic Action Plan. Microbes rarely exist in the environment as a monoculture but in complex microbial communities. Microorganisms in these communities often engage in a wide range of multicellular and intercellular behaviors such as cell-to-cell communication (quorum sensing), nutrient acquisition, biofilm formation, cellular dispersal, and the exchange of genetic material including genes encoding antimicrobial resistance. We have conducted studies to test the capacity for Shiga-toxin producing E. coli (STEC) O157:H7 and non-O157 STEC to form biofilm. While less than 5 % of STEC O157:H7 were able to form biofilm, about 20-30% of non-O157 STEC could form biofilm. We completed studies investigating the mechanisms involved in the suppression of biofilm formation and curli (a protein important for biofilm formation) expression in STEC and determined that alterations in two separate genes are important in suppressing curli expression; but both may be circumvented under certain conditions. These studies will allow us to determine the mechanisms that are used to circumvent these barriers and enhance E. coli O157:H7 survival under stress conditions and persistence in foods. We are also interested in quantitatively and qualitatively examining microbial populations as they occur in foods and food processing environments. To do so, we first developed a real-time PCR (qPCR) assay to quantify DNA from just about any bacteria. The PCR assay takes into account, and corrects, reaction efficiencies, which differ between unknown samples and standard DNA solutions. This year, we examined numerous commercially available kits to develop a DNA extraction procedure that yields nearly quantitative yields of DNA from both Gram-negative and Gram-positive bacteria. These unbiased extraction and PCR methods should yield a true quantitative assessment of mixed bacterial populations in order to better model the protection non-pathogenic species lend to the pathogens when they co-exist in food. Finally, work continued on a longitudinal study to determine the presence and persistence of small kananycin resistance plasmids in Salmonella isolates from the National Antimicrobial Resistance Monitoring System (NARMS) collections. This year, sequencing and analysis was conducted on these plasmids selected from isolates collected in 2010 and 2011, and comparison of those present in the NARMS collection from 2005, revealed that these plasmids persist in Salmonella isolated from food, animals, and humans. In addition, 4 new types of small kanamycin resistant plasmids were identified, suggesting that these plasmids continue to be acquired by and/or evolve within Salmonella. These plasmids will also be used to laboratory experiments to measure the movement of antibiotic resistance within bacterial communities.
1. Improved methods for bacterial community analysis. The examination of bacterial populations by DNA sequencing (metagenomics) has been applied to a variety of environments and the data gathered have found numerous applications in industry and medicine. Nevertheless, very few metagenomic studies have been done to measure bacterial populations on foods and in food processing environments. The metagnomic approach to examine bacteria populations involves breaking open the bacteria to extract all of the DNA, copying a specific gene from all of the bacteria, and determining the sequence of the gene copies to determine which bacteria are present. Current methods used to examine bacterial populations from environmental samples are prone to experimental biases that prevent the accurate measurements of all the different kinds of bacteria present. As a prelude to studies on metagenomic analysis of bacterial populations in food and food processing environments, ARS researchers at Wyndmoor, Pennsylvania, developed an improved method to efficiently extract DNA from all kinds of bacteria. In addition, to improve the method used to make copies of the DNA for sequencing, the scientists came-up with a way to account and correct for reaction efficiencies which differ between samples. Together, these improvements will provide a much more efficient and accurate method that will be useful to all scientists studying bacterial communities.
2. Biofilm properties of Shiga-toxin producing E. coli. Pathogenic Shiga toxin-producing E. coli (STEC) are an important cause of foodborne illness and the USDA Food Safety Inspection Service has adopted a zero tolerance policy for STEC O157:H7 and 6 other types of STEC in foods. It is not fully understood how STEC are able to persist on foods and in food processing environments. One mechanism of persistence may be through the formation of complex microbial communities called biofilms. The biofilm lifestyle helps bacteria survive harsh environmental conditions, even some sanitation procedures employed during food processing. Biofilm formation by STEC involves a complex network of regulatory genes responding to environmental signals. ARS researchers at Wyndmoor, Pennsylvania used a variety of genetic methods to better understand the molecular mechanisms necessary for STEC to form biofilm. A large collection of STEC O157:H7 and other types of STEC strains were characterized for their biofilm-forming capabilities. It was discovered that less than 5 percent of strains of STEC O157:H7 and 20-30% of other STEC strains were able to form biofilm. The genetic lesions responsible for the lack of biofilm formation in these STEC strains were identified. Some strains that were able to form biofilm carried gene mutations that would have been expected to prevent biofilm formation; thus, new mechanisms of gene regulation that allow biofilm formation must exist in these strains. This information will be of value to researchers that are developing targeted intervention strategies aimed at reducing pathogen contamination of foods and food processing environments.
Irwin, P.L., Reed, S.A., Brewster, J.D., Nguyen, L.T., He, Y. 2013. Non-stochastic sampling error in quantal analyses for Campylobacter species on poultry products. Analytical and Bioanalytical Chemistry. 405(7):2353-2369.