Location: Characterization and Interventions for Foodborne Pathogens
2020 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 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 O157:H7; revealing that virulence regulatory pathways affect the regulation of biofilm formation as well as virulence genes. In follow-up studies PchE (encoded by one of five pch genes in STEC O157:H7) was identified 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 now report the role of PchE in controlling the transition of STEC O157:H7 from motility (via control of flagellar genes) to cell attachment (via regulation of cell adhesin 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 show that PchE is a potential target for the development of targeted interventions to manipulate biofilm formation in food processing environments and reduce host cell attachment to prevent or treat human infection. Proteomic studies of the 5 Pch proteins were also conducted using STEC O157:H7 grown at host conditions to better understand their functions and regulation. Additional studies testing potential cellular adhesins in STEC showed that subtle differences in early attachment might be masked by the expression of intimin in the cultured cell assays; therefore, mutant strains with a deletion of the eae gene are being constructed to eliminate this artifact. This would allow further testing of cellular/abiotic adhesins.
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. In addition, a CRISPR-based antimicrobial system was constructed targeting unique regions of the STEC genome that would function as antimicrobial and/or an intervention to prevent hemolytic uremic syndrome, one of the most deadly illnesses caused by STEC. While this technology awaits development of a suitable delivery system, single-walled carbon nanotubules (SWCNT) are being tested as a delivery vehicle for the CRISPR-based STEC intervention system. Transformation efficiencies of only 103/ml were attained, which is too low to be effective. Studies are underway to increase the efficiency of the SWCNT delivery system and to evaluate other mechanisms for CRISPR delivery.
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. Four different cuts of beef from three different stores (total 36 independent beef samples) were tested for their microbial population and the transfer of bacteria to the stainless steel (SS) and high-density polyethylene (HDPE cutting board material) coupons (72 coupon samples) upon contact at 10-degree C. A culture-independent 16S-targeted microbiome sequencing approach was applied to determine the population of organisms associated with these beef products and populations of bacteria that were transferred from beef products to SS and HDPE coupons. The culturable population from these beef products (over 1000 isolates in total) and a subset of those that were transferred to these food processing surfaces were isolated, identified by 16S rDNA sequencing, and tested for biofilm forming ability in both single and mixed biofilms with STEC O157:H7 to determine a possible role in the persistence of STEC O157:H7. Strains of some bacterial species, particularly Pseudomonas, were able to increase the amount of E. coli attached to polystyrene surfaces by more than 10-fold.
Two separate studies were undertaken to determine the bacterial community associated with chicken. The aim of these studies was to identify organisms or community profiles that could be used to predict the presence of Salmonella. One study involving chicken carcass rinse samples has been completed and resulted in an accurate, sensitive and specific predictor of Salmonella presence/absence. A second study employed ground chicken samples acquired from the USDA Food Safety and Inspection Service and tested for the presence of Salmonella. The microbial communities present in 53 Salmonella-positive samples and 105 Salmonella-negative samples were determined for both pre- and post-enrichment. The samples were sequenced and comparative analyses of the microbial communities is now being conducted. It is expected that indicator organisms (or populations) will be identified in the pre-enrichment samples, precluding the need for culture enrichment. With larger data sets it is expected that this method could be used as a routine predictive screen for the presence of Salmonella in chicken products and of other foodborne pathogens.
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 entericaand 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 transfer by themselves. Little was known regarding the small plasmid transfer between bacteria and their reliance on different families of the conjugative plasmids. This approach can help us focus effort on those with 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
1. Other bacteria may help E. coli O157:H7 persist in foods. Shiga toxin-producing E. coli (STEC) O157:H7 is an important foodborne pathogen, contributing significantly to the $15.6 billion annual economic burden resulting from food recalls and human foodborne illness. To address questions of how STEC O157:H7 persist on beef products, bacteria from several beef cuts and the bacteria that can experimentally transfer from beef to two common food contact surfaces, stainless steel (SS) and high-density polyethylene (HDPE; cutting board material) were identified and isolated. Studies were also carried out to evaluate bacterial isolates for biofilm formation and if these biofilms contribute to binding of STEC O157:H7. A total of 134 bacterial types were identified among beef cuts while 205 different kinds of bacteria were identified from the SS and HDPE surfaces. While there were no bacteria common to all beef cuts, a few bacteria (Caulobacter and Pseudomonas) were found on all SS and HDPE surfaces. Sixty-one of 962 beef isolates, 31 of 211 SS isolates, and 29 of 199 HDPE isolates were strong biofilm-formers. Some of bacterial isolates increased the amount of STEC O157:H7 in biofilms by more than 10-fold. The identification of beef-associated bacteria that form biofilms and increase the persistence of STEC O157:H7 will lead to more rational approaches to the design of targeted intervention technologies for beef processing facilities.
2. Bacterial microbiome predicts the presence of pathogens in food. Traditional microbiological testing methods are slow, but recent advancements in sequencing technologies and fast computing power make it possible to use machine learning to identify microbiomes that predict the presence or absence of specific pathogens. ARS scientists in Wyndmoor, Pennsylvania, in collaboration with an industry partner, analyzed 299 poultry wash samples using the microbiome to predict the risk of a sample containing Salmonella. The predictive analysis had high accuracy (87%), sensitivity (80%), and specificity (90%). This result shows new ways to use microbiome data to predictive food safety.
3. Food spoilage microbe forms highly complex multicellular structures. The food spoilage bacterium Brochothrix thermosphactais capable of growth at refrigeration temperatures and contributes to significant economic losses for the meat, poultry, and seafood industries. ARS scientists in Wyndmoor, Pennsylvania, previously isolated several strains of B. thermosphactafrom retail chicken meat exhibiting different growth patterns, with some strains growing in very large cell aggregates forming tight “balls” that are composed of up to 10,000 individual cells and are visible to the naked eyes. Time-lapse microscopic imaging revealed that these strains form simple filaments and looped helices, that are further twisted into multi-stranded cables that are then organized into intertwined cables comprised of 20 or more individual bacterial filaments. These complex aggregates are formed at the upper end of the permissible growth temperature for B. thermosphacta (30°C). Although Brochothrix is non-pathogenic, these large cellular structures may provide an environment favorable to the growth of foodborne pathogens and may protect pathogens from disinfectant treatments. A better understanding of the conditions that favor aggregate formation will provide more rational approaches for the development of interventions to reduce food spoilage and improve food safety.
Review Publications
Chen, C., Nguyen, L.T., Paoli, G., Irwin, P. 2020. The complex multicellular morphology of the food spoilage bacteria Brochothrix thermosphacta strains isolated from ground chicken. Canadian Journal of Microbiology. 66(4):303-312.
Zhou, M., Li, X., Hou, W., Wang, H., Paoli, G., Shi, X. 2019. Incidence and characterization of Salmonella from raw meat products sold at small markets in Hubei province China. Frontiers in Microbiology. 10(2265). Available: https://doi.org/10.3389/fmicb.2019.02265l. https://doi.org/10.3389/fmicb.2019.02265.
Uhlich, G.A., Paoli, G., Zhang, X., Andreozzi, E. 2019. Whole-genome sequence of Escherichia coli serotype O157:H7 strain ATCC 43888. Genome Announcements. Available: Microbiol Resources Announcement 8:e00906-19. https://doi.org/10.1128/MRA.00906-19.
