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:
Progress was made on all objectives, all of which fall under National Program 108 – Food Safety, Component 1: Food Contaminants. Studies detailed in this project plan related to microbial community composition, biofilms, and persistence and transmission of antimicrobial resistance determinants will address specific needs as stated in: Problem Statement 1. - Population Systems, and Problem Statement 2. Systems Biology. During this past year, methods were investigated to concentrate foodborne organisms with a high recovery rate without altering the makeup of the test foods’ microbial flora using a state of the art filtration system. Using model systems, 90-100% recovery of E. coli was achieved without perturbing the makeup of the microbial flora. While bacterial sample concentration from foods was efficient and reproducible using vegetable washes (i.e., approximately 80% recovery with no effect on the original compositions), filter clogging impeded the concentration of bacteria from meat samples. In another study, the spinach microbiome was characterized using the MinION, a state of the art next-generation sequencing technology. 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. Preliminary analyses suggest that there was little or no effect on the native bacterial composition by the addition of the foodborne pathogen. 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, studies were conducted to evaluate the cluster formation under various growth conditions and time-lapsed light microscopic images were recorded to follow the growth and aggregate formation. Mathematical models are being developed to better quantify the bacterial growth. In addition, the closed whole genome sequences of two strains of Brochotrhix thermospacta were determined and submitted to GenBank. To aid in understanding biofilm (aggregates of microbial cells attached to a surface) formation by Shiga toxin producing E. coli O157:H7, the complete closed genome sequence of E. coli O157:H7 strain PA20 was determined. Gene expression studies were conducted when cells were challenged with an antibiotic at environmental temperatures to examine the differences in expression of biofilm- and virulence-associated genes. Studies were also conducted to determine the role of redundant DNA transcription factors on biofilm formation in E. coli O157:H7. Several redundant virulence gene regulators were cloned, expressed, and gene expression patterns were defined by qRT-PCR (quantitative real-time polymerase chain reaction). In addition, the genome sequences of biofilm-forming and non-biofilm-forming variants of E. coli O157:H7 were determined and comparative genome analyses are being conducted to define the mechanism(s) underlying a high frequency switch between biofilm and non-biofilm states. Lastly, preliminary studies were conducted to determine the role of E. coli O157:H7 biofilm components and properties in host cell attachment and adhesion under various conditions. These early studies focused primarily on method development for growth of human cultured cells, bacterial adhesion assays, and analysis of gene expression from adhered cells.
1. Genomic studies of pathogenic E. coli strain reveal distinct gene regulation. Some strains of E. coli (Shiga toxin producing E. coli; aka STEC) are harmful to humans and can be acquired through the consumption of contaminated foods. The persistence of these STEC in foods is aided by the ability of some strains to form or associate with microbial biofilms (aggregates of microbial cells attached to a surface). To aid in our understanding of biofilm formation by this pathogen, ARS researchers at Wyndmoor, Pennsylvania determined the complete closed genome sequence of a well characterized clinical isolate of STEC (strain PA20), and deposited the annotated genome in a publicly available DNA sequence database (GenBank). Using the complete genome as a reference, gene expression studies were conducted to examine the response of biofilm- and virulence-associated genes when the cells were subjected to antimicrobials. Under the applied antimicrobial stress, strain PA20 decreased the expression of biofilm genes while dramatically increasing the expression of numerous virulence genes in a time dependent manner. The decrease in their capacity to form biofilm suggests an additional mechanism by which antimicrobial or sanitation processes in the food industry might mitigate the presence of this important pathogen in foods.
2. First report of closed DNA sequences of Brochothrix thermospacta genomes, an important food spoilage organism. The ability of bacteria to associate in complex multicellular structures (biofilms and aggregates) is an important factor for their persistence in the environment. In food and food processing environments these complex bacterial structures can potentially trap pathogens and provide protection against intervention processes. ARS researchers at Wyndmoor, Pennsylvania, previously isolated strains of Brochothrix thermosphacta, an important food spoilage organism, some of which prefer to grow in aggregated clusters or webbed films in liquid cultures, in contrast to other strains that grow uniformly dispersed in solution. In order to provide a basis to study this unique growth, the complete genome sequences of two strains of Brochothrix thermosphacta were reported and deposited in the GenBank public DNA sequence database. One strain forms complex multicellular structures, and one strain that grows normally. These two DNA sequences are the first two complete and closed genome sequences for this species and provide an important resource for us and other scientists in studies on food spoilage and the potential for this organism to contribute to pathogen persistence in foods.
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Zhang, D., Coronel-Aguilera, C., Romero, P., Perry, L., Minocha, U., Rosenfield, C., Gehring, A.G., Paoli, G., Bhunia, A.K., Applegate, B. 2016. The use of a novel nanoLuc-based reporter phage for the detection of Escherichia coli O157:H7. Scientific Reports. doi: 10.1038/srep33235.
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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. doi: 10.1093/femsle/fnw123.
Edlind, T., Brewster, J.D., Paoli, G. 2017. Enrichment, amplification, and sequence-based typing of Salmonella enterica and other foodborne pathogens. Journal of Food Protection. 80(1):15-24.
Uhlich, G.A., Paoli, G., Zhang, X., Dudley, E.G., Figler, H.M., Cottrell, B.J., Androzzi, E. 2017. Whole-genome sequence of Escherichia coli serotype O157:H7 strain PA20. Genome Announcements. doi: 10.1128/genomeA.01460-16.
Uhlich, G.A., Chen, C., Cottrell, B.J., Andreozzi, E., Irwin, P.L., Nguyen, L.T. 2017. Gene duplication and promoter mutation expand the range csgD-dependent biofilm responses in a STEC population. Microbiology. 163:611-621.