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:
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 initiated a number of studies to determine the factors associated with a number of social behaviors exhibited by Shiga-toxin producing E. coli (STEC) O157:H7 and non-O157 STEC. We have assembled a collection STEC strains and completed studies investigating the mechanisms involved in the suppression of biofilm formation and curli (a protein important for biofilm formation) expression in E. coli O157:H7. We have determined that alterations in two separate genes are important in suppressing curli expression but that 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. Similar studies have been undertaken to characterize these phenomena in non-O157 STEC. In addition, we constructed novel genetic tools to aid in the construction of strains that will allow us to study the expression of genes important for biofilm formation in STEC. We are also interested in quantitatively and qualitatively determining 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. We used this assay to determine the efficacy of about a dozen well-known methods for quantitatively extracting DNA from various bacteria. We found that two commercial kits, when operated in tandem, give nearly quantitative results for the most bacteria. If successful true quantitative assessment of mixed bacterial populations can be achieved in order to better model the protection non-pathogenic species lend to the pathogens when they co-exist in food. Finally, preliminary studies to examine the transmission and persistence of antibiotic resistance in foodborne pathogens within environmental communities were undertaken. By examining numerous Salmonella isolates from National Antimicrobial Resistance Monitoring System collections from three different years a group of small plasmid DNAs with the potential to be transferred between bacteria and encoding resistance to the antibiotic kanamycin was identified. The incidence of these plasmids was consistent between years. The DNA sequence of these plasmids will be determined to see if the same plasmids persist over time or if new plasmids and antibiotic resistance genes were acquired. These plasmids will also be used to laboratory experiments to measure the movement of antibiotic resistance within bacterial communities.
1. Gene regulation in E. coli biofilms. Pathogenic E. coli is able to form biofilms that increase the bacteria’s resistance to environmental assaults. This also increases their persistence in food processing facilities. A better understanding of how these biofilm-associated bacteria respond to stresses is needed in order to reduce the incidence of foodborne illness. ARS researchers at Wyndmoor, Pennsylvania investigated the molecular mechanisms and regulation of genes related to oxidative stress in pathogenic E. coli biofilms. This is the first study that examined the gene expression changes in all four major peroxide resistance genes in biofilm cells, and determined the regulatory mechanisms involved that can allow a rational approach to developing targeted interventions to decrease the persistence of E. coli biofilms in food processing facilities.
2. New antimicrobial compounds kill foodborne pathogens. The incidence of antibiotic resistant pathogens is on the rise, reducing the effectiveness of antibiotic treatment of human infections and necessitating the development of new antibiotics. ARS researchers at Wyndmoor, Pennsylvania, in collaboration with scientists at PolyMedix, Inc., Radnor, PA, tested several of the company’s antimicrobial peptide mimics and determined that these compounds appeared to kill the cells by causing them to break open, and so are less likely to develop resistant strains than traditional antibiotic compounds.
Irwin, P.L., Nguyen, L.T., Chen, C., Uhlich, G.A., Paoli, G. 2012. A method for correcting standard-based real-time PCR DNA quantitation when the standard's polymerase reaction efficiency is significantly different from that of the unknown's. Analytical and Bioanalytical Chemistry. 402:2713-2725.