Shiga Toxin-Producing Escherichia coli in Biofilms and within Microbial Communities in Food

Goals / 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.

Project Methods
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 01/04/16 to 01/03/21

Outputs
PROGRESS REPORT 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. 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. 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. Record of Any Impact of Maximized Teleworking Requirement: Though all milestones were at least partially met, the limited access to the lab has had an obvious impact on the pace of research progress.

Impacts
(N/A)

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.

Progress 10/01/19 to 09/30/20

Outputs
Progress Report 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. 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. 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 01 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. 02 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. 03 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.

Impacts
(N/A)

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.2019.02265.
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.

Progress 10/01/18 to 09/30/19

Outputs
Progress Report 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. 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. 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 01 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.

Impacts
(N/A)

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.
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).
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.
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.

Progress 10/01/17 to 09/30/18

Outputs
Progress Report 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. 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. 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. Accomplishments 01 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. 02 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. 03 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

Impacts
(N/A)

Publications

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.
He, S., Cui, Y., Zhang, F., Shi, C., Paoli, G., Shi, X. 2018. Influence of ethanol adaptation on Salmonella enterica serovar Enteritidis survival in acidic environments and expression of acid tolerance-related genes. Food Microbiology. 72:193-198.
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.

Progress 10/01/16 to 09/30/17

Outputs
Progress Report 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. 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. 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. Accomplishments 01 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. 02 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.

Impacts
(N/A)

Publications

Ghatak, S., He, Y., Reed, S.A., Strobaugh Jr, T.P., Irwin, P.L. 2017. Whole genome sequencing and analysis of Campylobacter coli YH502 from retail chicken reveals a plasmid-borne type VI secretion system. Genomics Data. 11:128-131.
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.
Gehring, A.G., Fratamico, P.M., Lee, J., Ruth, L., He, X., He, Y., Paoli, G., Stanker, L.H., Rubio, F.M. 2017. Evaluation of ELISA tests specific for Shiga toxin 1 and 2 in food and water samples. Food Control. 77:145- 149.
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.
Uhlich, G.A., Paoli, G., Chen, C., Cottrell, B.J., Zhang, X., Yan, X. 2016. Whole-genome sequence of Escherichia coli serotype O157:H7 strain EDL932 (ATCC 43894). Genome Announcements. doi: 10.1128/genomeA.00647-16.
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.

Progress 10/01/15 to 09/30/16

Outputs
Progress Report 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. 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. This new Project Plan was recently certified through ARS Office of Scientific Quality Review (OSQR). For further details on current work see the 2016 annual report for project 8072-42000-067-00D.

Impacts
(N/A)

Publications