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
The primary aims of this research project are to understand the complex social behaviors exhibited by foodborne pathogenic bacteria and to catalog bacterial communities associated with foods and food processing environments. The knowledge gained will be useful in designing effective intervention strategies to reduce the persistence of biofilms and foodborne pathogens in food and processing environments. Accurate and quantitative microbial community analysis based on DNA sequencing is dependent upon efficient and quantitative methods for environmental sampling and DNA sample preparation yielding a consistent percentage of extractable chromosomal DNA with many different organisms. To this end we have begun thoroughly investigating various methods for semi-quantitatively extracting DNA using commercially available products. Early results reveal that the between-isolate DNA extraction in a heterogeneous sample (i.e., containing different Gram-negative and Gram-positive bacteria) was unacceptable for quantitative applications. Of all the techniques/kits tested, the most promising was mechanical cell breakage (e.g., using a French Pressure Cell) combined with various commercial extraction methods. Another aspect of the plan is the investigation of microbial social behaviors such as biofilm formation. In this regard, a large collection of strains of Shiga toxin-producing E. coli (STEC) (both O157:H7 and non-O157 STEC) have been collected. To date we have collaborated with scientists from Penn State University to screen E. coli O157:H7 isolates for the presence or absence of key genes/features involved in biofilm formation. In addition, several characteristics related to the biofilm-forming capability were analyzed, including Congo red binding on agar plates and crystal violet-binding on plastics. Additional characterization of the genetic regulation of biofilm formation will involve examining the regulation of genes involved in biofilm formation. As a prelude to these studies, novel genetic tools were constructed that will allow the facile characterization of numerous genes involved in biofilm formation by STEC.