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ARS Home » Midwest Area » Ames, Iowa » National Animal Disease Center » Food Safety and Enteric Pathogens Research » Research » Research Project #438679

Research Project: Intestinal Microbial Ecology and Non-Antibiotic Strategies to Limit Shiga Toxin-Producing Escherichia coli (STEC) and Antimicrobial Resistance Transmission in Food Animals

Location: Food Safety and Enteric Pathogens Research

2022 Annual Report

Objective 1: Identify host and bacterial factors contributing to attachment and colonization of outbreak-associated Shiga toxin-producing Escherichia coli at gastrointestinal mucosa under various environmental conditions. Sub-objective 1.A: Characterize unique genetic features of outbreak-associated O157 promoting attachment and colonization in food animals. Sub-objective 1.B1: Ascertain the impact of stress conditions on O157 to identify factors impacting virulence gene expression. Sub-objective 1.B2. Ascertain impact of stress on host cells and local microbiota affecting O157 colonization at the rectoanal junction. Objective 2: Develop novel non-antibiotic intervention strategies to limit Shiga toxin-producing E. coli colonization, persistence and/or shedding from food animals. Sub-objective 2.A: Identify novel E. coli with encoded bacteriocins, or competitive nutritional networks that interfere with O157 viability, growth, or attachment to food animal intestinal mucosa. Sub-objective 2.B: Evaluate efficacy of host and/or bacterial proteins to limit O157 mucosal attachment or colonization. Objective 3: Identify intervention strategies to mitigate stress-induced dysbiosis, improve mucosal immunity, and minimize antimicrobial resistance gene transfer from food animal commensals to human pathogens. Sub-objective 3.A: Define changes in host intestinal immune status, intestinal bacterial membership and function, and AMR mobile elements associated with weaning stress. Sub-objective 3.B: Identify non-antibiotic intervention strategies to minimize negative impact on gut dysbiosis and antimicrobial resistance gene transmission to human pathogens.

The goal of this project is to research practical solutions for food safety problems important to food production and public health sectors in the United States and globally. The research addresses food safety at the first link in the food production chain, namely the food animals on the farm. The research investigates the bacterial communities and the animal’s physiological response in the intestinal tract, as well as the interactions between microbiota and intestinal cells that lead to colonization with foodborne organisms and mobility of antimicrobial resistance genes into foodborne pathogens. Some microbiota members confer benefits to the host. Still others are benign to the animal but are harmful foodborne pathogens. The gut microbial consortium comprises a reservoir of antibiotic resistance genes of undefined composition and risk potential. Environmental factors, particularly stress, can modulate both host and bacteria, impacting the symbiotic relationship. Experiments are planned to: 1) examine the environmental, bacterial, and immunological factors affecting Shiga toxin-producing Escherichia coli (STEC) colonization of cattle; 2) identify unique genetic features of STEC impacting colonization and attachment; 3) define the impact of stress on bacterial and immunological events impacting bacterial colonization and antibiotic resistance gene transfer; and 4) examine novel, intervention strategies to reduce foodborne pathogen carriage and antibiotic resistance gene transfer in food animals. The combination of basic and applied research will supply knowledge and tools, as well as applicable strategies to control foodborne pathogens and antibiotic resistance gene mobility.

Progress Report
Foodborne outbreaks of Shiga toxin-producing Escherichia coli (STEC) serotype O157:H7 (O157) were initially attributed to contaminated beef, but many recent outbreaks have increasingly been associated with fresh produce. Identification of genetic signatures correlated with attachment to bovine cells may inform surveillance data on potential risk of O157 survival or shedding into the environment. In support of Objective 1, Sub-objective 1.A, to better understand the association between phylogenetic type and colonization and shedding dynamics in the asymptomatic bovine host, a series of adherence assays were performed using O157 isolates from various outbreaks. Specifically, bovine rectoanal junction (RAJ) squamous epithelial (RSE) cells and bovine small intestinal epithelial cells (BIEC) were used to evaluate differential attachment of O157 isolates. In addition, to explore multiple phenotypes, biofilm formation and toxin production was assessed. Overall, differences in phenotypes were characterized across the tested isolates, particularly biofilm formation. Data analysis is ongoing to identify genetic features associated with specific O157 phenotype across the various assays. Prior findings indicate that colonization and shedding from cattle was not different across the four tested O157 isolates; thus, phenotypes outside cattle colonization are critical to informing outbreak risk. A broad phylogenetic analysis on all O157 isolates in the NCBI Pathogen Detection Database in the last five years is being performed to select an additional 20-30 isolates to perform indicated phenotype assays. Aligned with Objective 1, Sub-objective 1.B2.: Ascertain impact of stress on host cells and local microbiota affecting O157 colonization at the rectoanal junction, a trial was performed in which cattle were subjected to transportation stress and samples collected to assess impact on the animal and microbiota. Specifically, a group of cattle were transported approximately 2 hours, with a non-transported control cohort included in the trial. Samples were collected prior to transport, and 30 min, 5 hours, and 1-, 7-, and 14-days post-transportation. Samples collected included blood, feces, rectoanal mucosal swabs (RAMS), and RAJ biopsies. Flow cytometry was used to assess changes in leukocyte population frequency, as well as expression of cell surface markers indicative of cell activation. DNA extraction from feces and RAMS is ongoing to perform 16S rRNA amplicon sequencing to assess impact of transportation stress on microbial communities. Methods to limit O157 attachment to bovine intestinal epithelial cells are highly desired as an intervention strategy to limit colonization. Previous data from our group suggested bovine anti-STEC IgA in feces was elicited by experimental O157 vaccination. However, sample quantity was limited for deeper characterization of anti-IgA responses and cross-reactivity against various O157 isolates. Thus, a trial was performed under Sub-objective 2.B.: Evaluate efficacy of host and/or bacterial proteins to limit O157 mucosal attachment or colonization, in which groups of cattle were parenterally vaccinated with one of two different inactivated mutant strains of O157 to assess peripheral and mucosal immunogenicity. Blood and feces were collected prior to vaccination, and at 3-, 4-, and 6-weeks post-vaccination. Feces were processed in a manner to minimize IgA degradation (fecal extracts), and blood used for sera isolation. Vaccination induced robust peripheral anti-O157 IgG responses, and assessment of anti-O157 IgA in fecal extracts is ongoing. Also, under Sub-objective 2.B.: Evaluate efficacy of host and/or bacterial proteins to limit O157 mucosal attachment or colonization, a unique protocol for identifying RAJ cell proteins that interact with O157 proteins was standardized. The protocol involved the development of ‘bacterial columns’ with O157 proteins, passage of proteins extracted from cells comprising the RAJ and then identifying all RAJ cell proteins that directly interacted with O157 proteins using tandem mass spectrometry, bottom-up proteomics. After proteomic analysis, 232 RAJ squamous epithelial (RSE) cell and follicle-associate epithelial (FAE) cell proteins interacting with O157 have been shortlisted from a total of 5420 proteins for further evaluation. The 232 proteins are currently being analyzed in silico to shortlist proteins specific to each cell type at the RAJ. In support of Objective 1, Sub-objectives 1.A. and 2.B., additional standardization, and optimization of the RAJ-in vitro organ culture (RAJ-IVOC) model system was completed to allow bacterial adherence studies, without loss of tissue integrity and with adherence results like that observed in vivo. RAJ tissues, collected at necropsies of cattle in unrelated studies and assembled into IVOCs, were tested for tissue integrity by staining for viable cells, cell markers and histopathological examination. Different E. coli strains (O157 and non-O157) were used to standardize the adherence assay. Adherence was evaluated by immunofluorescent staining of tissue sections and E. coli recovery. E. coli attached as predicted based on prior results with primary RSE cells. The RAJ-IVOC model developed here provides a convenient pre-screening system to evaluate interactions of multiple bacteria with the RAJ, prior to in vivo experiments, allowing for reduction of animal usage. Collateral effects of swine production practices on the gut microbial community are not well understood, especially as it relates to antibiotic resistance gene (ARG) transfer from commensal bacteria into foodborne pathogens. The incidence of foodborne, multi-drug resistant Salmonella has increased in the past decade, and factors contributing to its emergence have not been fully defined. Identifying factors limiting ecological disturbances and ARG transfer is a critical first step to develop microbiota modulation strategies to limit negative impacts on human and animal health. In support of Objective 3, Sub-objective 3.A.: Define changes in host intestinal immune status, intestinal bacterial membership, and function, and ARG mobile elements associated with weaning stress, several separate studies were performed including assessment of the impact of weaning age on the microbiota, immune status of pigs and assessment of ARG transfer into foodborne Salmonella. To understand ARG mobility into Salmonella, a series of experiments were performed with Salmonella enterica serovar Typhimurium 4232 strain (Salmonella 4232 strain) acting as an ARG recipient. To assess the ability of the Salmonella 4232 strain to acquire ARGs from commensal bacteria, Salmonella 4232 was spiked into swine cecal contents in vitro. Salmonella 4232 transconjugants resistant to tetracycline and ampicillin were observed. Further, preliminary trials in which swine were inoculated with Salmonella 4232 demonstrated transfer of ampicillin resistance genes to the strain. Together, these data demonstrate ARGs can be transferred to Salmonella 4232 in vitro and in vivo at a low frequency. Ongoing work includes characterizing the types of plasmids that can be acquired by Salmonella 4232 and determining if the plasmid types are of relative importance in the transfer of ARGs in swine. In support of Objective 3, Subobjective 3.A., a large trial was performed in which groups of piglets were weaned at the standard 18-21 days of age or late weaned at 28 days of age. Necropsies were performed at 0, 2-, 7-, 14-, 21-, 28-, and 35-days post-weaning to isolate various intestinal tissues to identify changes in intestinal immune cell abundance and phenotype and collect intestinal contents to determine changes in microbial communities as well as ARG abundance and mobility. Weaning age did impact the functional phenotype of immune cells in the jejunum (as determined using flow cytometry on single-cell suspensions created from jejunum), especially in the first week after weaning, and it was not confounded by age. Overall, management practices, including weaning age, can impact intestinal stability and changes to common practices may limit intestinal disturbances that negatively impact human health. In support of Objective 3 as it relates to understanding ARG in swine commensal populations, an analysis of publicly available fecal metagenomes from post-weaning piglets was performed, which led to the identification and characterization of nearly 50,000 plasmid sequences. Plasmid community diversity changed over the five weeks post weaning and antibiotic treatment after weaning led to an increased abundance of conjugative and broad host-range plasmids. These data and ongoing work will inform how plasmid populations that often transfer ARGs between bacteria are changing during the critical post-weaning phase and how antibiotic treatment during this period can influence the population of plasmids that may be acquired by foodborne pathogens.

1. The stress hormone norepinephrine enhances expression of genes associated with attachment and survival in the foodborne pathogen E. coli O157. The stress hormone norepinephrine (NE) is produced in larger amounts as animals experience a stress (nutritional, environmental, or physical) and can be secreted into the intestine (gut-brain axis). The foodborne E. coli O157 (O157) that is pathogenic in humans is present in cattle intestine but does not cause overt disease in the animal. Stressful events such as transportation cause increased O157 shedding by cattle although it is unclear how stress effects O157. Thus, a series of studies was performed to assess changes in gene expression in O157 in response to NE. Researchers in Ames, Iowa discovered more than 500 genes differentially expressed by O157 in response to NE. Several of the genes altered are associated with enhanced O157 survival in the highly acidic environment of the stomach and attachment of O157 to cattle intestine. In addition, several other gene pathways, such as those involved in nitrogen, sulfur, amino acid uptake were upregulated while those involved in iron metabolism were downregulated following NE exposure suggesting O157 tailors its metabolic machinery in response to NE.

2. E. coli O157 differentially express proteins associated with survival and adaptation in the rumen of cattle fed contrasting diets. Cattle are carriers of human pathogenic E. coli O157 (O157) and understanding how it survives within the rumen of cattle will help identify possible targets for O157 control. ARS researchers in Ames, Iowa utilized the in-house developed, novel method of introducing and recovering O157 from the rumen of cattle, without having to euthanize the animal at the end of the study, to determine proteins expressed by O157 within the rumen. Cattle were fed either a high protein lactation or a high fiber maintenance diet to generate biochemically contrasting rumen environments and three different O157 strains experimentally introduced into their rumens. Comparative analysis of all proteins (proteomes) produced by the three O157 strains indicated that the differences in protein expression were mainly influenced by the animal’s diet and growth conditions, irrespective of the strain being tested. Following stringent analysis, 89 and 87 proteins were differentially expressed by the O157 in rumen of animals on lactation and maintenance diet, respectively, including those with putative roles in metabolism (ie. nitrogen metabolism), transport (ie. iron uptake), and environmental adaptation (ie. motility). A subset of these proteins involved in O157 survival in the bovine rumen could be targeted for developing control modalities to reduce O157 carriage in cattle and farm to fork contamination.

Review Publications
Biernbaum, E.N., Kudva, I.T. 2022. AB5 Enterotoxin-Mediated Pathogenesis: Perspectives gleaned from Shiga toxins. Toxins. 14(1). Article 62.
Sharma, V.K., Akavaram, S.N., Bayles, D.O. 2022. Genomewide transcriptional response of Escherichia coli O157:H7 to norepinephrine. BMC Genomics. 23. Article 107.
Kudva, I.T., Trachsel, J.M., Biernbaum, E.N., Casey, T. 2022. Novel reusable animal model for comparative evaluation of in vivo growth and protein-expression of Escherichia coli O157 strains in the bovine rumen. PLoS ONE. 17(5). Article e0268645.
Trachsel, J.M., Bearson, B.L., Kerr, B.J., Shippy, D.C., Byrne, K.A., Loving, C.L., Bearson, S.M. 2022. Short chain fatty acids and bacterial taxa associated with reduced Salmonella enterica serovar I 4,[5],12:i:- Shedding in swine fed a diet supplemented with resistant potato starch. Microbiology Spectrum. 10(3). Article e0220221.
Hoeffler, R.S., Kudva, I.T. 2021. EDL933 Strains of Escherichia coli O157 can demonstrate differential adherence to bovine recto-anal junction squamous epithelial cells. Letters in Applied Microbiology. 15(1):129-138.