1a. Objectives (from AD-416):
1: Molecular identification and characterization of the genetic factors that influence biofilm formation by Shiga toxin-producing Escherichia coli (STECs). 1.1 Genomic, transcriptomic, and molecular analyses to identify novel genetic factors and regulatory mechanisms for biofilm formation in STEC. 2: Examination of the influence of extrinsic (biotic and abiotic) and intrinsic factors on biofilm formation by STECs. 2.1 Microbiological properties and comparative transcriptomic analyses of serotype O157:H7 biofilms on abiotic and biotic surfaces, and in various environmental conditions. 2.2 Evaluation of the roles and interactions of various plasmids (conjugative or mobilizable) carried by mixed-biofilm partners (intrinsic factors). 2.3 Mixed culture biofilm of STEC with beef-associated biofilm-forming flora. 3: Qualitative and quantitative characterization of microbial communities associated with beef, and how the various populations influence the presence of STECs. 3.1 16S rDNA-targeted metagenomic studies of microbiomes on ground and intact beef. 3.2 16S rDNA-targeted metagonomic studies of microbiomes associated with beef slaughter facilities (and their correlation with the presence of STEC). 3.3 16S rDNA-targeted metagonomics studies of biofilm forming bacteria associated with beef and beef slaughter facilities. 3.4 Quantitative computational analyses of microbiomes by whole-genome metagenomics.
1b. Approach (from AD-416):
Microbes rarely exist in the environment as a monoculture but in complex microbial communities that are often attached to solid surfaces. The association of pathogenic bacteria within these biofilm communities is known to lead to their persistence in food processing environments, ultimately resulting in the contamination of foods and foodborne illness. The goal of this research project is to better understand microbial communities and community structures by which Shiga toxin-producing Escherichia coli (STEC) persist in beef and result in human illness by 1) determining the unique mechanisms of biofilm formation in STEC; 2) evaluating the role of antibiotic resistance in biofilm formation and persistence; 3) determining the composition of microbial communities in beef and beef processing facilities; 4) testing for a correlation between community composition and the presence of STEC; 5) determining the presence of biofilm forming bacteria in beef and beef processing facilities; and 6) testing if biofilm-forming flora from beef and beef processing facilities can contribute to the association and persistence of STEC with mixed biofilms.
3. Progress Report:
This report documents progress for the parent project 8072-42000-076-00D, Shiga Toxin-Producing Escherichia coli in Biofilms and within Microbial Communities in Food. The aims of this project are to better understand the persistence of pathogens on foods and in food systems through studies of food-associated microbial communities, the association of pathogens with these communities, and the capacity for pathogens to form biofilm. This past year, studies were completed to concentrate foodborne organisms with a high recovery using a state of the art filtration system. The goal was to find methods to concentrate bacteria from food without altering the makeup of the test foods’ microbiome. Using model systems, 90-100% recovery of E. coli was achieved without perturbing the makeup of the food microbiota. While bacterial sample concentration from vegetable washes was efficient and reproducible, filter clogging impeded the concentration of bacteria from meat samples. The composition of the native microbial population was characterized with or without the addition of the foodborne pathogen, E. coli O157:H7. Shotgun metagenomics and 16S rDNA sequencing were evaluated quantitatively. Analyses suggest that there was little or no effect on the native bacterial composition by the addition of the foodborne pathogen. In a related study the MinION long-read next-generation sequencing technology was used to study the spinach microbiome using both shotgun whole genome sequencing (WGS) and 16S ribosomal RNA gene (“16S”) analyses. WGS results were analyzed using a commercial, custom-curated database and cloud-based analysis platform CosmosID, and the 16S results were analyzed using EPI2ME. The two approaches showed different microbial compositions (in bacterial types and in proportions) from the same DNA source, underlining the importance of method and database selection in evaluating the microbial communities. The food spoilage bacterium Brochothrix thermophacta contributes to significant economic loss in meat, poultry, and seafood. We previously isolated a strain of Brochothrix thermospacta that prefers to grow in complex multicellular clusters or webbed films that could potentially trap pathogens and provide protection against intervention processes. This year we published the closed genome sequences of two Brochothrix strains (one strains that grows normally and a second that displays this complex morphology) and submitted the sequences to GenBank. These were the first closed genomes submitted to the GenBank database. The growth of organisms was studied to evaluate the cluster formation under various growth conditions and time-lapsed light microscopic images were recorded to follow the growth and aggregate formation. Models of bacterial growth were developed to better understand the number of cells that make complex morphological structures. Studies were conducted to identify growth conditions that were favorable to multicellular cluster formation. The two Brochothrix strains were grown under conditions that were both unfavorable and favorable to cell cluster formation. Currently, comparative genomic analysis and gene expression analysis (RNA-Seq) of the two strains grown under various conditions are being done to identify genetic factors that contribute to this unusual growth pattern. Shiga toxin-producing E. coli (STEC) O157:H7 is an important foodborne pathogen. The persistence of this deadly bacterium in foods is aided by its ability to bind to surfaces and form or associate with biofilms. Similarly, the initial stages of foodborne human infection by STEC involve the binding of the bacteria to host intestinal cells. This year a study was completed and published that detailed the effect of varying antibiotic concentrations on the biofilm-forming and virulence gene expression in STEC at different temperatures. This study revealed virulence regulatory pathways affect the regulation of biofilm formation. Follow-up studies were conducted to examine a number of redundant virulence regulatory genes in STEC prophage regions to better understand the effect these regulators have on the expression of cell surface binding proteins that are important for binding to environmental surfaces (e.g., food and food processing surfaces) and host cells during infection. Strains of serotype O157:H7 from different sources vary in their expression of csgD, a regulator controlling formation of curli fimbriae. Curli are an essential component of biofilms and can contribute to adhesion to eukaryotic cells. We have shown that clinical strains have barriers restricting curli expression and that transcriptional regulators (e.g., Pch and GlrA) whose genes are located in the laterally transfer regions of the chromosome control csgD expression when activated. However, the role that these regulators play in controlling biofilm formation and virulence in the host has not been determined. To determine the role these regulators play in regulating curli (CsgD) expression, pchA, pchB, pchC, pchD, pchE and glrA were cloned and over-expressed in 2 strains of O157:H7 (with csgD-dependent properties typical of either clinical or non-clinical isolates) and with their ler-, csgBA- or espA-deficient progeny. Single deletions of the 5 pch genes and glrA were also constructed in both the clinical and non-clinical strain. All strains were compared to each other and to parent strains for adhesion to cultured Hep2 cells at 37°C and for biofilm formation at 30°C. We determined the contributions of each regulator to cell attachment, confirmed their effect on specific adhesion proteins such as curli and EspA, and showed the attachment phenotypes unique for the clinical and non-clinical backgrounds. We also discovered novel effects of the regulators on biofilm formation and regulation, and discovered a new strong suppressor of csgD, which could prove a target to disrupt biofilm formation. In addition, the whole genome sequences were determined for several STEC strains with varying biofilm-forming capabilities. The whole genome sequence for strain B6914-ARS, a variant of the well characterized strain B6914 with an interesting biofilm phenotype, was completed, published and deposited in the GenBank public genome database. Currently assembly, annotation and comparative genomic analyses of the other sequenced STEC genome sequences are underway to determine novel factors or regulatory pathways involved in STEC biofilm formation that may lead to persistence on foods and in food processing environments. Antibiotic resistance in pathogenic bacteria is a major concern in both the food production and medical industries. It has long been established that antibiotic resistant bacteria can transfer their resistance genes to other bacteria in a process called conjugation. The conjugation mechanism for moving antibiotic resistance to other bacteria often resides on the large extrachromosomal circular DNA molecules called “conjugative plasmids”. In order to better understand the occurrence of multi-drug resistance in bacteria and the movement of antibiotic resistance between bacteria, ARS scientists at Wyndmoor, Pennsylvania, and Athens, Georgia have worked together to identify several large conjugative plasmids from MDR Salmonella enterica and E. coli isolates collected from the National Antibiotic Resistance Monitoring System (NARMS) and other programs. The conjugation capability of the strains is being evaluated, and the conjugative plasmids identified will be further analyzed using next-generation sequencing techniques if the plasmid sequence is not already available. In addition, several much smaller plasmids were also identified from bacteria in the NARMS collection. Several of these small plasmids have been isolated, fully sequence and characterized. This year, preliminary experiments were also carried out to better understand the contribution of these much less studied smaller plasmids on the movement of antibiotic resistance between bacteria.
1. Citrus oil spray disrupts E. coli biofilm formation. Bacteria live in complex communities in the environment and bacteria in these communities use chemical signals to communicate their presence to one another. These chemical signals regulate various behaviors such as motility, biofilm formation, and virulence characteristics of harmful bacteria (pathogens). Compounds capable of inhibiting bacterial cell-to-cell communication have been explored for their potential to reduce the persistence of pathogens in foods, or as potential alternatives to antibiotics. ARS scientists at Wyndmoor, Pennsylvania worked with scientists at Purdue University (West Lafayette, Indiana) to study a fine emulsion of a chemical found in citrus peels (D-limonene) for its ability to disrupt one pathway of intercellular communication in pathogenic E. coli. Very low concentrations of D-limonene were able to interfere with cell-to-cell signaling and alter cellular physiology including disrupting formation of biofilms. These results indicate that D-limonene emulsion could be applied to food processing surfaces to minimize bacterial biofilm formation, reduce virulence, or as an alternative when antibiotics are not suitable.
2. Small plasmids contribute to antibiotic resistance in Salmonella. Antibiotic resistance in harmful bacteria (pathogens) is a major concern in both food production and medicine. While the role of large mobile DNA molecules, called plasmids, in the expression and spread multi-drug resistance in pathogens has been conclusively demonstrated, small plasmids carrying antibiotic resistance genes are often over-looked. ARS scientists at Wyndmoor, Pennsylvania and Athens, Georgia, had previously developed a method to screen for the presence of small plasmids in multidrug resistant isolates of the pathogenic bacteria Salmonella. These isolates, collected by the 2005 National Antibiotic Resistance Monitoring System (NARMS), were further characterized by using molecular techniques and DNA sequence analysis. A follow-up study was conducted using NARMS isolates collected during the 2010-2011 period, resulting in the isolation and characterization of additional novel small plasmids. The results indicate that these small plasmids are widespread in pathogens Salmonella (and pathogenic E. coli) isolated from animals raised for food, that their overall population is changing over time, and underscores the important roles they may play in the harboring and transmission of antibiotic resistance genes between pathogenic bacteria.
3. Field portable DNA sequencing for pathogen detection. In order to prevent the distribution of contaminated foods and reduce the burden of foodborne illness, food producers and regulatory agencies need rapid, accurate and cost-effective methods for the identification of bacterial foodborne pathogens. Recently, bacterial genome sequencing (i.e., determining the sequence of a bacterium’s complete set of DNA) has been broadly adopted by regulatory and public health agencies to characterize bacterial pathogens and track outbreaks of foodborne illness. Nevertheless, due to expense and technical limitations, these genomic technologies have not been adopted for rapid foodborne pathogen detection and identification. Recently, an inexpensive and portable DNA sequencing device, the Oxford Nanopore MinION DNA sequencer, which overcomes several of these limitations, was introduced. We used the MinION to identify the 7 different types of pathogenic E. coli that are currently not allowed in foods in the US within a complex mixture. The MinION sequencer therefore demonstrated the potential for inexpensive, rapid, specific, and field -portable detection and identification of foodborne bacterial pathogens
Chen, C., Strobaugh Jr, T.P., Nguyen, L.T., Abley, M.J., Lindsey, R.L., Jackson, C.R. 2018. Isolation and characterization of two novel groups of Kanamycin-resistance ColE1-like plasmids in Salmonella enterica serotypes from food animals. PLoS One. https://doi.org/10.1371/journal.pone.0193435.
Uhlich, G.A., Reichenberger, E.R., Cottrell, B.J., Fratamico, P.M., Andreozzi, E. 2017. Whole-genome sequence of Escherichia coli serotype O157:H7 strain B6914-ARS. Genome Announcements. https://doi.org/10.1128/genomeA.01191-17.