Progress 02/10/11 to 01/03/16
Outputs
Progress Report 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. 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. Progress made falls 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 surface associated microbial communities called biofilms. The ability to form biofilm is considered an important mechanism by which foodborne pathogens persist in foods and food processing environments; ultimately leading to human illness. Numerous studies have revealed molecular mechanisms of biofilm formation in non-pathogenic E. coli, but considerably less is understood of biofilm formation in pathogenic foodborne Shiga toxin producing E. coli (STEC). Studies conducted as part of this research project revealed that only about 5 % of STEC O157:H7 and 20-30% of non- O157 STEC are able to form biofilm. Thorough phenotypic and genetic characterization of a collection of STEC O157:H7 and non-O157 STEC strains revealed that, in almost all STEC strains, the inability to form biofilm could be explained by just a few genetic defects: 1) the presence of a stx1 prophage or other prophage in the mlrA gene required for expression of biofilm formation genes; 2) mutations in the rpoS gene that encodes another important regulator required for biofilm formation; and 3) lack of cellular motility. Despite the disruption in these genes required for biofilm formation, some strains of STEC retain, or have regained, the capacity for form biofilm. Whole genome sequencing and transcriptomic gene expression analysis studies of these strains revealed new mechanisms of biofilm formation and pathogenesis as well as several new regulatory mechanisms (e.g., gene excision, gene duplication, functional substitutions, gene expression from disrupted/truncated genes) that allow STEC to form biofilm despite the previously identified mutations. Additional studies were conducted to further demonstrate how E. coli responds to stress, such as the presence of sub-lethal levels of antibiotic, to induce biofilm formation. One such study revealed that sub- lethal concentrations of antibiotics induce the excision of the loss of stx1 prophage, resulting in an intact mlrA and restoration the biofilm phenotype and increased expression of virulence-associated genes. Studies of the regulation of biofilm formation by STEC are providing a better understanding of the regulatory barriers to biofilm formation in STEC and the mechanisms used by STEC to circumvent these barriers enhancing STEC survival under stress conditions and their persistence in foods. In an effort to better understand STEC persistence in foods and food processing environments, effort were undertaken to study the microbial communities with which STEC may associate. Initial studies were undertaken to quantitatively and qualitatively determining 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. Using the most efficient DNA extraction methods determined in this study, we have evaluated next generation sequencing of microbial communities by targeted 16S rDNA analysis using experimentally developed artificial mixtures of bacteria and have initiated studies of the microbial communities on commercial beef products. These studies will be continued in an effort to gain a better understanding of these microbial communities, the biofilm forming members of these communities, the roll these communities play in STEC persistence in beef processing facilities, and, ultimately, STEC persistence and presence in beef products. Antibiotic resistant pathogenic bacteria pose serious public health concerns and the use of antibiotics as veterinary drugs and growth promoters in food animals has the potential to increase the incidence of antibiotic resistant pathogens in foods. Furthermore, biofilms provide a physical barrier that can protect bacteria from a variety of stresses, including antibiotics, and may provide conditions that promote for the spread of genes encoding antibiotic resistance. To better understand the contribution of gene transfer in biofilms to antibiotic resistance in foodborne pathogens the mobility of plasmid-associated antibiotic resistance genes between pathogens in biofilms was studied. Several small mobilizable plasmids isolated from strains from the National Antimicrobial Resistance Monitoring System for Enteric Bacteria (NARMS) collection were sequenced and characterized. Many of the sequenced plasmids were tested and were found to be capable of transferring antibiotic resistance to other bacteria within biofilms at equal or greater frequencies compared to experimental controls on agar plates. This research enhances our understanding of resistance plasmid transfer within biofilms and may ultimately lead to better control against the spread of antibiotic resistant bacteria and antibiotic resistance genes between bacteria. The biofilm lifestyle confers protection from a number of stresses, including oxidative stress. The molecular mechanisms and regulation of genes related to oxidative stress were studied in biofilms formed by pathogenic E. coli. This follows a previous study that focused on non- biofilm, free swimming cells and allows, by comparison, the identification of regulatory changes in resistance mechanisms during and following the transition from swimming cells to biofilm cells. This is the first study that examined the gene expression changes in all four major peroxide resistance genes in biofilm cells. Some important findings in this study include: 1) peroxide resistance was greater in biofilm cells than in swimming cells; 2) the major protective peroxidases for biofilm cells were identified; 3) regulatory genes required for full resistance were identified; and 4) complex regulatory systems for peroxidase gene regulation were determined. These studies provide important information on the protection against oxidative stress in E. coli biofilms. Determination of the regulatory mechanisms involved can allow a rational approach to developing targeted interventions to decrease the persistence of E. coli biofilms in food processing facilities. 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. While the development of new antimicrobial compounds by conventional methods can be costly and time-consuming, the chemical synthesis of compounds that mimic the structures of the natural host defense peptides is facile and economical. As part of a collaborative project with a local technology company [CRADA #58-3K95-0-1448-MTA] several of the company�s antimicrobial peptide mimics were tested to determine their efficacy in killing an important deadly foodborne bacterium. These compounds appeared to kill the cells by causing them to break open, thus are less likely to result in the development of resistant strains than traditional antibiotic compounds. The amount of the antimicrobials necessary to kill the pathogenic bacterium was determined and more extensive analyses (survival/kill curves and time-to-inactivation) were conducted on the top candidate compounds. Several lead compounds were identified. These antimicrobial peptides have the potential to be used to improve human and animal health. In addition, these compounds might be used to sanitize food contact surfaces or be incorporation into materials such as conveyer belts to reduce the bacterial and pathogen load in food processing facilities and foods, ultimately reducing the incidence of foodborne illness. Harmful E. coli bacteria are major foodborne pathogens, causing an estimated 250,000 illnesses in the US each year. The United State Department of Agriculture Food Safety Inspection Service (FSIS) regularly tests beef products for 7 different types of pathogenic E. coli and has a zero-tolerance policy for samples that test positive. During pathogen testing, it is typical to include a positive control sample (i.e., a sample inoculated with a strain of pathogenic E. coli) along with the test samples to ensure that the procedure is working properly. When this is done, there is always a slight risk that the pathogenic E. coli used to inoculate the positive control sample might cross-contaminate the test sample, leading to a false-positive result. At the request of FSIS a collection of pathogenic E. coli strains, one each of the 7 regulated types, were genetically manipulated to contain a unique segment of DNA that allows them to be distinguished from any naturally occurring E. coli. Thus, a positive test sample can be distinguished from the E. coli in the positive control sample. In addition to being used by regulatory agencies and food testing laboratories as positive control strains for detection of pathogenic E. coli, the E. coli strains constructed in this study could also be used in developing new methods of detection and to model the growth of pathogenic E. coli in foods. Accomplishments 01 Transmission of antibiotic resistance within biofilm. Antibiotic resistant pathogenic bacteria pose serious public health concerns and increase the burden of disease treatment and the use of antibiotics as veterinary drugs and growth promoters in food animals has the potential to increase the incidence of antibiotic resistant pathogens in foods. Furthermore, the complex microbial communities (biofilms) found in foods and food processing environments provide a physical barrier that can protect bacteria from a variety of stresses, including antibiotics, and may provide conditions that promote for the spread of genes encoding antibiotic resistance. To better understand the contribution of gene transfer in biofilms to antibiotic resistance in foodborne pathogens, ARS researchers in Wyndmoor, Pennsylvania conducted laboratory experiments on the mobility of plasmid-associated antibiotic resistance genes between pathogens in biofilms. The researchers found that many of the plasmids tested were capable of transferring antibiotic resistance to other bacteria within biofilms at equal or greater frequency compared to experimental controls on agar plates. This research enhances our understanding of resistance plasmid transfer within biofilms and may ultimately lead to better control against the spread of antibiotic resistant bacteria and antibiotic resistance genes between bacteria. 02 Naturally occurring variants of E. coli O157:H7 form strong biofilms. The formation of complex bacterial communities (biofilms) is important for the persistence of pathogens in foods and food processing environment. Early events in biofilm formation involve stable binding of bacteria to surfaces and similar binding events are known to contribute bacterial persistence during the establishment of an infection. Nevertheless, most strains of the major foodborne pathogen E. coli O157:H7 are generally weak biofilm-formers under environmental conditions. ARS researchers in Wyndmoor, Pennsylvania have previously described several genetic defects that contribute to reduce biofilm formation by pathogenic E. coli. Here they report additional genetic and environmental factors necessary for biofilm formation by these deadly foodborne pathogens ascertained by careful physiological and genetic characterization of naturally-occurring biofilm-forming variants of E. coli O157:H7. The regulatory mechanisms reveal elements that are shared among cellular processes related to biofilm formation, responses to environmental stress, and pathogen virulence. The mechanisms remain unknown in approximately 25% of the variants and await further analyses by whole genome sequencing. Understanding bacterial stress response, biofilm formation, and pathogenesis will no doubt lead to better design of the intervention strategies to minimize impact caused by the foodborne pathogens.
Impacts
(N/A)
Publications
- Zhou, X., Zhang, L., Shi, C., Fratamico, P.M., Liu, B., Paoli, G., Dan, X., Zhuang, X., Cui, Y., Wang, D., Shi, X. 2016. Genome-scale screening and validation of targets for identification of Salmonella enterica and serovar prediction. International Journal of Food Microbiology. 79:376-383.
- Paoli, G., Sommers, C.H., Scullen, O.J., Wijey, C. 2014. Inactivation of avirulent pgm+ and delta pgm Yersinia pestis by ultraviolet light (UV-C). Food Microbiology. DOI:10.1016/j.fm.2014.06.002.
- Irwin, P.L., Nguyen, L.T., He, Y., Paoli, G., Gehring, A.G., Chen, C. 2014. Near-quantitative extraction of genomic DNA from various food-borne eubacteria. BMC Microbiology. DOI: 10.1186/s12866-014-0326-z.
- Irwin, P.L., Nguyen, L.T., Chen, C., He, Y. 2014. Variability in DNA polymerase efficiency: effects of random error, DNA extraction method, and isolate type. JSM Mathematics & Statistics. 1(1):1003.
- Chen, C., Nguyen, L.T., Cottrell, B.J., Irwin, P.L., Uhlich, G.A. 2015. Multiple mechanisms responsible for strong Congo red-binding variants of Escherichia coli O157:H7 strains. Pathogens and Disease. 74:123.
- Bai, Y., Cui, Y., Paoli, G., Shi, C., Wang, D., Shi, X. 2016. Synthesis of amino-rich silica coated magnetic nanoparticles and their application in the capture of DNA for PCR. Colloids and Surfaces B: Biointerfaces. 145:257-266.
- Uhlich, G.A., Chen, C., Cottrell, B.J., Yan, X., Hofmann, C.S., Nguyen, L. T. 2016. Stx1 prophage excision in Escherichia coli strain PA20 confers strong curli and biofilm formation by restoring native mlrA. FEMS Microbiology Letters. 10.1093/femsle/fnw123.
- He, Y., Ingudam, S., Reed, S.A., Gehring, A.G., Strobaugh Jr, T.P., Irwin, P.L. 2016. Study on the mechanism of antibacterial action of magnesium oxide nanoparticles against foodborne pathogens. Journal of Nanobiotechnology (Biomed Central Open Access). DOI:10.1186/s12951-016- 0202-0.
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Progress 10/01/14 to 09/30/15
Outputs
Progress Report 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. 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. Microbes rarely exist in the environment as a monoculture but usually exist in complex microbial communities often growing on surfaces. Microorganisms in these communities often engage in a wide range of multicellular and intercellular behaviors, nutrient acquisition, biofilm formation, cellular dispersal, and the exchange of genetic material including genes encoding antimicrobial resistance. As part of this project, we previously discovered that only about 5 % of Shiga toxin producing E. coli (STEC) O157:H7 were able to form biofilm, while about 20-30% of non-O157 STEC could form biofilm and the genetic mechanisms responsible for disrupting biofilm formation in STEC were identified. We also found that, despite the disruption in some genes previously thought to be required for biofilm formation, some strains of STEC retain, or have regained, the capacity to form biofilm. This year whole genome sequencing and transcriptomic gene expression analysis studies have revealed new genes involved in biofilm formation and pathogenesis as well as several new regulatory mechanisms (e.g., gene excision, gene duplication, functional substitutions, gene expression from disrupted/ truncated genes) that allow STEC to form biofilm despite the previously identified mutations. Additional studies were conducted to further demonstrate how E. coli responds to stress, such as the presence of sub- lethal levels of antibiotic, to induce biofilm formation. The results of these studies are expected to allow a more science-based rational approach to the development of intervention technologies to reduce the incidence of foodborne illness. We also are interested in studying plasmid transfer within microbial communities. As a prelude to these studies, the plasmids were characterized, including determining the complete nucleotide. This year, studies were completed to select appropriate strains and helper plasmids that will allow the examination of plasmid transfer in mixed populations. Accomplishments 01 Understanding the mechanisms contributing to persistence of pathogenic E. coli in foods. Shiga toxin-producing E. coli (STEC) is an important group of foodborne pathogens, leading to an estimate of over 175,000 illnesses, over 2000 hospitalizations, and 20 deaths in the U.S. each year, resulting in an economic burden of almost $300 million. STEC persists in food processing environments by adhering to foods and food processing surfaces. ARS researchers at Wyndmoor, Pennsylvania, have identified several new mechanisms by which pathogenic E. coli regulate the genetic and physiological processes required for binding of the organisms to solid surfaces the potential for biofilm formation and persistence. A thorough understanding of the mechanism of STEC biofilm formation is critical to understanding their persistence in foods and identification of new mechanism for regulation of biofilm formation in STEC will be helpful in identifying targeted intervention strategies to reduce foodborne illness caused by STEC. 02 Mechanism for spread of antibiotic resistance between bacteria demonstrated. Antibiotic resistance in foodborne pathogens poses serious public health concerns and can be a confounding factor in the treatment of foodborne illnesses. Antibiotic resistance genes can reside on the bacterial chromosome or on small self-replicating plasmids antibiotic resistance can spread by movement of these antibiotic resistance-encoding plasmids between bacteria. ARS researchers at Wyndmoor, Pennsylvania, have demonstrated that some large plasmids that carry resistance to several antibiotics can assist in the movement of some small plasmids between bacteria. The smaller plasmids typically carry resistance to only one or two antibiotics but cannot move on their own. These studies demonstrate a mechanism for the movement of these small plasmids between bacteria and for the spread of antibiotic resistance. Understanding the mechanisms and preferences by which the antibiotic resistance plasmids are transferred will enhance our knowledge on the spread and transmission of antibiotic resistance genes and may help devise intervention technologies to interfere with or prevent the spread.
Impacts
(N/A)
Publications
- Paoli, G., Uhlich, G.A., Wijey, C. 2015. Genetically marked strains of Shiga toxin-producing O157:H7 and non-O157 Escherichia coli: Tools for detection and modelling. Journal of Food Protection. 78(5):888-901.
- He, Y., Reed, S.A., Bhunia, A.K., Gehring, A.G., Nguyen, L.T., Irwin, P.L. 2015. Rapid identification and classification of Campylobacter spp. using laser optical scattering technology. Food Microbiology. 47:28-35.
- Yan, X., Fratamico, P.M., Bono, J.L., Baranzoni, G., Chen, C. 2015. Genome sequencing and comparative genomics provides insights on the evolutionary dynamics and pathogenic potential of different H-Types of Shiga toxin- producing Escherichia coli O104. BMC Microbiology. DOI: 10.1186/s12866-015- 0413-9.
- Gunther, N.W., Liu, Y., Nunez, A., Paul, M., Uhlich, G.A. 2014. Non- labeled quantitative proteomic comparison identifies differences in acid resistance between Escherichia coli O157:H7 curli production variants. Foodborne Pathogens and Disease. 11:30-37.
- Chen, C., Yan, X., Wang, S., Jackson, C.R. 2015. Application of metagenomics technologies for antimicrobial resistance and food safety research and beyond. In: Chen, C.Y., Yan, X., and Jackson, C.R., editors. Antimicrobial Resistance and Food Safety-Methods and Techniques. New York, NY: Elsevier B.V. p. 401-422.
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Progress 10/01/13 to 09/30/14
Outputs
Progress Report 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. 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. 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, nutrient acquisition, biofilm formation, cellular dispersal, and the exchange of genetic material including genes encoding antimicrobial resistance. As part of this project, we previously discovered that only about 5 % of Shiga toxin producing E. coli (STEC) O157:H7 were able to form biofilm, while about 20-30% of non-O157 STEC could form biofilm. Genetic mechanisms responsible for disrupting biofilm formation in STEC were identified. This year additional progress was made on elucidating the mechanisms of biofilm formation and evaluating the effect of environmental factors on the biofilm forming capability in STEC. The results of these studies are expected to allow a more science-based rational approach to the development of intervention technologies to reduce the incidence of foodborne illness. We are also interested in quantitatively and qualitatively examining microbial populations in foods and food processing environments. To do so, we developed a quantitative real-time PCR (qPCR) assay that corrects for differences in reaction efficiencies between unknown samples and standard DNA solutions, and examined developed an efficient DNA extraction procedure. This year progress was made to further address issues related to qPCR quantitation of microbial communities and the developed methods were applied to a model community system. The tested methods performed well and are expected to yield a true quantitative assessment of foodborne bacterial populations. Progress was made on characterizing a predominant food spoilage organism isolated during a survey of the microbial population associated with commercial chicken products. The isolate is a well-known food spoilage organism, Brochothrix thermosphacta, but has not been well characterized to date. It grows very rapidly at refrigeration temperatures (less than 5 hour doubling time) and displays a unique morphology, forming visibly large roughly spherical aggregates of cells of about 1000 cells/CFU and tightly woven strands of cells when observed by electron microscopy. We are interested in determining if these morphological properties contribute to biofilm formation and the incorporation of pathogens into preformed biofilm. As no B. thermospacta genomic sequences are currently available, we sequenced the genomes of two isolates, one that expressed the unique morphology and one that does not, in order to do comparative genomic analyses to determine the molecular mechanisms that lead to this unique growth pattern. Finally, we will use a collection of small kanamycin resistance plasmids from Salmonella from the NARMS collections to study plasmid transfer within microbial communities. As part of this project, these plasmids have been characterized, including determining the complete nucleotide. This year, as a prelude the studies on plasmid transfer within microbial communities, progress was made on examining plasmid stability, plasmid transfer, and identifying appropriate bacterial strains. Accomplishments 01 Environmental factors affect biofilm formation in pathogenic E. coli. 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. Contamination of food products by STEC may be in part due to the formation of complex microbial communities called biofilms that lead to persistence of the pathogens in food and food processing environments. 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. The scientists previously determined that STEC O157:H7 are generally poor at forming biofilms (only 5% of O157:H7 strains formed biofilm experimentally), while 20-30% of other STEC strains (non -O157 STEC) were able to form biofilm. More recently the genetic mechanisms by which biofilm formation is disrupted in these STEC strains were determined. In addition, environmental factors (e.g., temperature and growth media) that affected biofilm formation in STEC were identified and the mechanism of biofilm regulation by these environmental factors was determined. 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. 02 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. While the development of new antimicrobial compounds by conventional methods can be costly and time-consuming, the chemical synthesis of compounds that mimic the structures of the natural host defense peptides is facile and economical. In collaboration with a CRADA partner, an ARS researcher at Wyndmoor, Pennsylvania, tested several of the company�s antimicrobial peptide mimics to determine their efficacy in killing an important deadly foodborne bacterium. These compounds appeared to kill the cells by causing them to break open, thus are less likely to result in the development of resistant strains than traditional antibiotic compounds. The amount of the antimicrobials necessary to kill the pathogenic bacterium was determined and more extensive analyses (survival/kill curves and time-to-inactivation) were conducted on the top candidate compounds. Several lead compounds were identified. These antimicrobial peptides have the potential to be used to improve human and animal health. In addition, these compounds might be used to sanitize food contact surfaces or be incorporation into materials such as conveyer belts to reduce the bacterial and pathogen load in food processing facilities and foods, ultimately reducing the incidence of foodborne illness.
Impacts
(N/A)
Publications
- Chen, J., Shi, X., Gehring, A.G., Paoli, G. 2014. Automated immunomagnetic separation of Escherichia coli O157:H7 from spinach. Food Control. 179:33- 37.
- Chen, C., Hofmann, C.S., Cottrell, B.J., Strobaugh Jr, T.P., Paoli, G., Nguyen, L.T., Yan, X., Uhlich, G.A. 2013. Phenotypic and genotypic characterization of biofilm forming capability in non-O157 Shiga toxin- producing Escherichia coli strains. PLoS One. 8(12):e84863.
- Xu, J., Song, M., Yang, P., Shi, C., Paoli, G., Shi, X. 2014. Phenotypic and genotypic antimicrobial resistance traits of foodborne Staphylococcus aureus isolates from Shanghai. Journal of Food Science. 79:635-642.
- Uhlich, G.A., Chen, C., Cottrell, B.J., Nguyen, L.T. 2014. Growth media and temperature effects on biofilm formation by serotype O157:H7 and non- O157 Shiga toxin-producing Escherichia coli. FEMS Microbiology Letters. 354:133-141.
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Progress 10/01/12 to 09/30/13
Outputs
Progress Report 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. 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. 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. Accomplishments 01 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. 02 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.
Impacts
(N/A)
Publications
- 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.
- Uhlich, G.A., Chen, C., Cottrell, B.J., Hofmann, C.S., Dudley, E., Strobaugh Jr, T.P., Nguyen, L.T. 2013. Phage insertion in mlrA and variations in rpoS limit curli expression and biofilm formation in Escherichia coli serotype O157:H7. Microbiology. 159:1586-1596.
- Bai, Y., Song, M., Cui, Y., Shi, C., Wang, D., Paoli, G., Shi, X. 2013. A rapid method for the detection of foodborne pathogens by extraction of a trace amount of DNA from raw milk based on label-free amino-modified silica-coated magnetic nanoparticles and polymerase chain reaction. Analytica Chimica Acta. 787:93-101.
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Progress 10/01/11 to 09/30/12
Outputs
Progress Report 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. 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. 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. Accomplishments 01 Gene regulation in E. coli biofilms. Pathogenic E. coli is able to form biofilms that increase the bacteria�s resistance to environmental assaul This also increases their persistence in food processing facilities. 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 ca allow a rational approach to developing targeted interventions to decrea the persistence of E. coli biofilms in food processing facilities. 02 New antimicrobial compounds kill foodborne pathogens. The incidence of antibiotic resistant pathogens is on the rise, reducing the effectivenes of antibiotic treatment of human infections and necessitating the development of new antibiotics. ARS researchers at Wyndmoor, Pennsylvan in collaboration with scientists at PolyMedix, Inc., Radnor, PA, tested several of the company�s antimicrobial peptide mimics and determined tha 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.
Impacts
(N/A)
Publications
- 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.
- Davis, R., Paoli, G., Mauer, L.J. 2012. Evaluation of fourier transform infrared (FT-IR) spectroscopy and chemometrics as a rapid approach for subtyping E. coli O157:H7 isolates. Food Microbiology. 31:181-190.
- Uhlich, G.A., Chen, C., Cottrell, B.J., Irwin, P.L., Phillips, J.G. 2012. Peroxide resistance in Escherichia coli serotype O157:H7 biofilms is regulated by both RpoS dependent and independent mechanisms. Microbiology. 158:2225-2234.
- Uhlich, G.A., Chen, C. 2012. A cloning vector for creation of Escherichia coli lacZ translational fusions and generation of linear template for chromosomal integrations. Plasmid Journal. 67(3):259-263.
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Progress 10/01/10 to 09/30/11
Outputs
Progress Report 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. 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. 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.
Impacts
(N/A)
Publications
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