Location: Characterization and Interventions for Foodborne Pathogens
2021 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 primary aims of this project are to understand better 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 human foodborne infection by STEC involve the binding of the bacteria to host intestinal cells.
In previous studies, we identified PchE (encoded by one of five pch genes in STEC O157:H7) as 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 to and invade host cells while avoiding host immune recognition. It was also learned that PchE represses biofilm formation while also regulating a number of adhesin genes. We found that PchE has a role in controlling the transition of STEC O157:H7 from motility (via control of flagellar genes) to cell attachment (via regulation of cell-binding proteins) and demonstrate that pchE is a general repressor of adhesion to both abiotic (food processing surfaces) and biotic (human host cells) surfaces. These studies also showed that PchE is a potential target for developing targeted interventions to manipulate biofilm formation in food processing environments and reduce host cell attachment to prevent or treat human infections. To this end, we initiated a study to identify chemical inducers of the negative regulation of biofilm formation and intestinal cell adhesion by PchE. We used 2 programs to perform computation searches for transcription factor binding sites. We mapped several potential binding sites, one that is positioned close to the pchE -35 transcriptional promoter region. We consider that site the top candidate for binding active regulators that might be used to lower biofilm formation and cell attachment. We are currently testing known compounds that control candidate transcription factors binding that site. As mentioned above, compounds the are involved in PchE regulation could serve in the development of targeted interventions to manipulate biofilm formation in food processing environments and reduce host cell attachment to prevent or treat human infections.
In addition to the 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. Both culture-independent and culture-dependent methods were used to examine the bacterial microbiota associated with beef. Over 1000 bacterial strains were isolated, and hundreds of them were identified by 16S rDNA sequencing. All of the isolates were tested for biofilm-forming ability, and a subset of biofilm-forming isolates was tested in mixed biofilms with STEC O157:H7 to determine a possible role in the persistence of STEC O157:H7. While preliminary results revealed that strains of some bacterial species, particularly Pseudomonas, increased the amount of E. coli attached to polystyrene surfaces by more than 10-fold, this year, a more rigorous study was undertaken to screen the biofilm-forming isolates in mixed biofilm assays with STEC O157:H7. These studies are underway and are expected to reveal candidate species that contribute to STEC O157:H7 persistence in foods.
Another aspect of the research being conducted involves studies of the movement of antibiotic resistance genes between bacteria. Antibiotic resistance in pathogenic bacteria is an urgent concern for public health.
Antibiotic resistance genes can be carried on circular DNAs called plasmids that can be transferred between bacteria in close contact with one another on surfaces. ARS scientists at Wyndmoor, Pennsylvania, and Athens, Georgia, worked together to identify large self-transmissible plasmids from the multi-drug resistant (MDR) Salmonella enterica and E. coli. Additional studies revealed several small plasmids carrying resistance to kanamycin and, in some cases, additional antibiotics. Here we report a study of how the large plasmids interact with smaller kanamycin resistance plasmids. We found that several distinct classes of the large plasmids were able to promote the movement of certain groups of small plasmids that were otherwise incapable of self-transfer. Little was known regarding the small plasmid transfer between bacteria and their reliance on different families of the conjugative plasmids. This research advances our knowledge on the interaction between different antibiotic resistance-encoding plasmid families and can help us focus research effort on those plasmids with a higher potential of transmitting the resistance genes. This research will augment our understanding of the transfer of antibiotic resistance plasmids between bacteria and ultimately lead to better control against the spread of antibiotic resistance genes in animals and the environment.
Accomplishments
Review Publications
Armstrong, C.M., Gehring, A.G., Paoli, G., Chen, C., He, Y., Capobianco Jr, J.A. 2019. Impacts of clarification techniques on sample constituents and pathogen retention. Foods. https://doi.org/10.3390/foods8120636.
Uhlich, G.A., Paoli, G., Kanrar, S. 2020. Escherichia coli serotype O157:H7 PA20R2R complete genome sequence. Microbiology Resource Announcements. 9(50): e01143-20. https://doi.org/10.1128/MRA.01143-20.
