Location: Characterization and Interventions for Foodborne Pathogens
2019 Annual Report
Objectives
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.
Approach
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.
Progress Report
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.
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. In previous studies, we examined the effect of varying antibiotic concentrations on the biofilm-forming and virulence gene expression in STEC; revealing that virulence regulatory pathways affect the regulation of biofilm formation as well as virulence genes. Curli fimbriae (encoded by the gene csgD) serve as adhesins (i.e., a binding protein) that are essential for biofilm formation and can also contribute to adhesion to eukaryotic cells. We have shown that clinical strains of E. coli O157:H7 contain transcriptional regulators (e.g., Pch and GlrA) that affect csgD expression. However, the role that these regulators play in controlling biofilm formation and virulence in the host is not fully understood. To determine the role these regulators play in curli (CsgD) expression numerous mutant strains were constructed and the 5 pch genes (pchA, pchB, pchC, pchD, pchE) as well as the glrA were cloned and over-expressed. These experiments revealed the contributions of each regulator to cell attachment, confirmed their effect on specific adhesion proteins (curli and others), and showed the attachment phenotypes unique for the clinical and non-clinical backgrounds. Further gene expression studies using RT-PCR and cell adhesion assays were conducted to study the adhesins controlled by PchE. In this study, we showed that there are several adhesins, previously shown to bind host or animal cells, that are controlled by PchE. Strong regulation of flagella was shown but expression of flagella past early exponential phase strongly repressed cell adhesion. In general, pchE seems to be an overall strong repressor of adhesion as well as biofilm formation. The study revealed a highly orchestrated mechanism of adhesin gene expression that appears to help the pathogen attach and invade host cells while avoiding host immune recognition. Additional proteomic studies of the 5 Pch proteins are being conducted using STEC O157:H7 grown at host conditions in order to better understand their functions and regulation. Furthermore, based on the results of these studies, a reporter system was constructed that will be used to identify compounds that drive expression of pchE. The identified compounds will have potential as antibiofilm or antimicrobial agents.
The whole genome sequence was determined for Escherichia coli serotype O157:H7 strain ATCC 43888, a Shiga toxin-deficient human fecal isolate. Due to its reduced toxicity and availability from a curated culture collection, the strain has been used extensively by us and others in numerous applied research studies. The complete genome sequence of E. coli O157:H7 ATCC43888 determined, assembled, annotated, and submitted to the GenBank public database. Genome comparison of this strain with genomes of other laboratory strains with variable biofilm forming capabilities are being conducted to determine novel factors or regulatory pathways involved in STEC biofilm formation that may lead to persistence on foods and in food processing environments.
In addition to regulation of intrinsic factors related to biofilm formation the role of extrinsic factors (e.g., the population of other microbes) in foods may have a significant effect on the persistence of STEC. These studies aim to determine if STEC association with other biofilm-forming bacteria present on beef or processing surfaces may play an important role in STEC persistence. To address this question studies were conducted to examine the microbial populations associated with a variety of beef products. A culture-independent 16S-targeted microbiome sequencing approach is being applied to determine the population of organisms associated with these beef products. The culturable population from these beef products was isolated and are being tested for a possible role in the persistence of STEC O157:H7 via the formation of mixed culture biofilms.
An additional study was initiated aimed at determining the bacterial community associated with ground chicken. The aim of this study is to identify organisms or community profiles that could be used to predict the presence of Salmonella. Ground chicken samples were acquired from the USDA Food Safety and Inspection Service and tested for the presence of Salmonella. The microbial communities present in 52 Salmonella-positive samples and 107 Salmonella-negative samples are being determined for both pre- and post-enrichment. The samples are being sequenced and comparative analyses of the microbial communities is being done. It is expected that indicator organisms (or populations) will be identified in the pre-enrichment samples, precluding the need for culture enrichment. If successful, this method could be used as a predictive screen for the presence of Salmonella in ground chicken.
Antibiotic resistance in pathogens is a pressing public health issue. Genes coding for antibiotic resistance are often carried on circular DNA called plasmids and can be transferred into naïve hosts via transfer events such as conjugation (bacterial mating). Working with a scientist from ARS in Athens, Georgia, large conjugative plasmids were identified from the multi-drug resistant (MDR) Salmonella enterica and E. coli in an interest to study how they interact with other small high-copy-number resistance plasmids. The conjugation capability of the strains was evaluated, and the conjugative plasmids were then characterized on their ability to transfer other resistance plasmids into naïve hosts. Further analysis such as sequencing will be carried out. Although quite a lot of the MDR plasmids have been sequenced, the conjugation capability of these plasmids was seldom evaluated. This approach can help us focus effort on those strains and plasmids with higher potential of transmitting resistance genes. Little is known regarding the small plasmid transfer between bacteria and their reliance on different families of the large conjugative plasmids. This research is augmenting our understanding of resistance plasmid transfer between bacteria which could ultimately lead to better control against the spread of resistance genes in animals and the environment.
Accomplishments
1. Antibiotic stress increases virulence in E. coli. Antibiotic stress increases virulence in E. coli. The ability to attach to surfaces and form biofilms may be a contributing factor in the persistence of pathogenic E. coli O157:H7 in foods and food processing environments. ARS scientists at the USDA-ARS Eastern Regional Research Center in Wyndmoor, Pennsylvania, are studying biofilm in E. coli in order to understand their role in environmental persistence and human pathogenesis. Genome-wide gene expression was examined in E. coli O157:H7 in the presence of antibiotics with the potential to affect the capacity for biofilm formation. The results of this study indicated that, when subjected to DNA damaging antibiotic stress, E. coli responds by increasing expression of virulence genes, but genes involved in biofilm formation are repressed. In this study key regulators of virulence gene expression were identified. The results provide new insights into the regulation and expression of biofilm and virulence genes that have important implications for understanding the environmental persistence and human pathogenesis of E. coli O157:H7.
Review Publications
Rotundo, L., Amagliani, G., Carloni, E., Omiccioli, E., Magnani, M., Paoli, G. 2018. Evaluation of PCR-based methods for the identification of enteroaggregative hemorrhagic escherichia coli in sprouts. International Journal of Food Microbiology. 291:59-64. https://doi.org/10.1016/j.ijfoodmicro.2018.11.011.
Andreozzi, E., Gunther, N.W., Reichenberger, E.R., Cottrell, B.J., Rotundo, L., Nunez, A., Uhlich, G.A. 2018. Pch genes control biofilm and cell adhesion in a clinical serotype O157:H7 isolate. Frontiers in Microbiology. 9(2829). https://doi.org/10.3389/fmicb.2018.02829.
Perez Jr, J.J., Chen, C. 2018. Detection of acetyltransferase modification of kanamycin, an aminoglycoside antibiotic, in bacteria using ultra-high performance liquid chromatography tandem mass spectrometry. Journal of Rapid Communications in Mass Spectroscopy. 32:1549-1556.
Perez Jr, J.J., Chen, C. 2018. Rapid detection and quantification of aminoglycoside phosphorylation products using direct infusion high resolution and ultra-high performance liquid chromatography-mass spectrometry. Rapid Communications in Mass Spectrometry. 32:1822-1828.
Wang, R., Vega, P., Xu, Y., Chen, C., Irudayaraj, J. 2018. Exploring the anti-quorum sensing activity of a D-limonene nanoemulsion for Escherichia coli O157:H7. Biomedical Materials Research. https://doi.org/10.1002/jbm.a.36404.
Peritz, A., Chen, C., Paoli, G., Gehring, A.G. 2018. Serogroup-level resolution of the “Super-7” Shiga toxin-producing Escherichia coli using nanopore single-molecule DNA sequencing. Analytical and Bioanalytical Chemistry. https://doi.org/10.1007/s00216-018-0877-1.
