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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

2021 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
Cattle, the primary reservoirs of foodborne human pathogens Shiga toxin-producing E. coli (STEC), remain asymptomatic over the course of STEC colonization making it difficult to detect and apply control measures. Understanding the complex dynamics of host-bacterial factors that allow STEC to colonize cattle is important for developing intervention strategies that can effectively reduce STEC in cattle prior to and/or at slaughter. STEC O157 (O157) persist at the rectoanal junction of the cattle intestine. Some cattle shed O157 in greater numbers than other cattle and are referred to as super-shedding cattle. O157 strains, including those that are shed by super-shedding cattle, demonstrate varied attachment patterns on rectoanal junction cells. Under Objective 1, Sub-objective 1.A, genes likely contributing to the attachment phenotypes of O157 and super-shed O157 strains were identified. The analysis demonstrated a role for multiple adherence factors, including those of type 1 fimbriae, that impacted cell attachment and were expressed in a strain and host cell dependent manner. The findings were noteworthy since previous dogma suggested that the type 1 fimbrial operon is genetically “switched off” in O157, and hence was not suspected to play a role in adherence of O157 to host cells. The proteins encoded by all genes identified as playing a role in attachment may be developed into blocking therapies aimed at preventing O157 and super-shed O157 colonization of cattle. As O157 asymptomatically colonizes the bovine gastrointestinal tract, it is shed into the environment by dairy and feedlot cattle posing a broad contamination risk. Foodborne outbreaks of O157 were initially attributed to contaminated beef, but many recent outbreaks have increasingly been associated with fresh produce, possibly due to manure contamination of irrigation water. Bovine commensal E. coli may be the evolutionary source of human intestinal pathogenic strains, including produce outbreak isolates. Specific genotypes of O157 may have altered niche adaptation in the bovine host, impacting the amount shed into the environment and thus the risk of produce contamination. In addition, there is a possibility that some bovine O157 isolates may have increased pathogenic potential in humans and pose a greater risk than other isolates. Identification of genetic signatures, including polymorphic markers and virulence genes, correlated with colonization or shedding dynamics from animal reservoirs can inform surveillance data on potential risk. In support of Objective 1, Sub-objective 1.A, to better understand the association between phylogenetic type and bovine colonization and shedding dynamics in the asymptomatic host, a trial was performed in which groups of cattle were inoculated with O157 isolates of different lineages, representing isolates from both produce and beef outbreaks. Colonization and shedding were the same across groups, regardless of the challenge isolate. Genetic differences between the isolates were identified, but it’s unlikely they alone contributed to factors impacting colonization and shedding from calves. Work is ongoing to include additional isolates into the genetic analysis, as well as transcriptomic studies to understand if gene expression between isolates is significantly different. In addition, work aligned with Objective 1, Sub-objective 1.B1 was completed, including the assessment of stress hormone norepinephrine (NE) on O157 gene expression. O157 can exploit a variety of factors (nutrients and chemical signals) produced by the animal and bacterial communities in the animal intestine. Some of the chemical signals, such as stress hormone norepinephrine (NE) produced in larger amounts as animals experience a stress (nutritional, environmental, or physical), can ‘spill’ into the intestine. O157, like other intestinal foodborne bacterial pathogens, may sense/metabolize these signals to alter its gene expression for successful colonization and persistence in the animal intestine. O157 was cultured in a medium containing NE to assess the expression of genes that could potentially promote colonization and persistence of O157 in the cattle intestine. The ribosomal-free RNA prepared from NE-exposed and unexposed O157 was subjected to RNA-Seq analysis. The output sequencing data was mapped to the reference genome of O157 and analyzed by a pipeline of bioinformatics programs to identify genes and quantify the expression of these genes in NE-exposed O157 relative to NE-unexposed control O157 cultures. The major findings of this data were that of the 581 differentially expressed genes, several of these genes encoded pathways that have been shown by other studies to be directly involved in enhancing survival of O157 in the highly acidic environment of the stomach and attachment of O157 to cattle intestine. In addition, several other pathways, such as those involved in nitrogen, sulfur, amino acid and iron metabolism were impacted by NE suggesting that O157 tailors its metabolic machinery in response to NE for presumably increasing its competitive growth advantage/survival in the cattle intestine. The potential practical implication of this research would be to show whether the animals that were not stressed were less susceptible to O157 colonization compared to the stressed animals. If NE-treated O157 bacteria were verified to contain higher levels of proteins involved in O157 attachment to cattle intestine, these bacterial cells could be evaluated for use as whole cell vaccine or source of proteins for use as subunit vaccines in reducing O157 colonization in cattle. In support of Objective 1, Sub-objective 1.B2, biopsy sections of cattle rectoanal junction (RAJ) were collected and processed to single cells suspensions to assess viability and phenotype of recovered cells by flow cytometry. The ability to perform single-cell RNA-sequencing of RAJ cells depends on the ability to isolate viable cells or isolation of nuclei. Sections of RAJ were also snap frozen and nuclei isolation protocols are currently under testing. The pipeline to analyze scRNA-seq data for bovine was developed from our pipeline for swine, using the bovine genome and annotation. RAJ biopsy collection procedure was developed and will be used in an upcoming transportation trial. Collectively, the protocols necessary for the proper collection of samples have been developed and are ready to be deployed. One gut microbiota modulation strategy of particular interest is the application of probiotics with the ability to kill or outcompete specific members of the gut ecosystem, including outcompeting colonization by STEC. Probiotics can act via competitive exclusion, occupying the ecological niche, often due to competition for use of the same nutrient sources. To support isolation and testing of a non-STEC isolate to serve as a non-pathogenic commensal to outcompete STEC as outlined in Objective 2, Sub-objective 2A, more than 80 non-STEC isolates of E. coli recovered from cattle inoculated with O157 were isolated and subjected to short-read DNA sequencing. Currently, a computational pipeline is being developed to interrogate metabolic pathways of the E. coli in comparison to multiple O157 STEC isolates. Data analysis thus far suggests some of the isolates express E. coli virulence genes associated with pathogenic E. coli. However, many of the isolates do not express many common E. coli virulence genes and have metabolic pathways comparable to foodborne outbreak STEC isolates. Data analysis is ongoing to further characterize the potential probiotic isolates, as well as development of a computational pipeline to apply already sequenced isolates of E. coli from other ARS colleagues. While probiotics may not shift the microbiota on a large scale, their ability to exclude O157 from regions of attachment and persistence may limit food and environment contamination. In support of Objective 3, protocols were developed and validated to characterize bacterial populations at the swine intestinal mucosa via 16S rRNA gene sequencing. In addition, single-cell RNA-sequencing of peripheral immune cells and intestinal cells was performed. Unique populations of T cells were identified in peripheral blood of pigs compared to humans, as well as in the intestine of pigs when compared to intestines of humans and mice. Given that foodborne Salmonella does not cause overt disease in swine but does cause disease in humans, work is ongoing to understand if the swine-specific populations may have a role in minimizing Salmonella-induced disease in the pig. The tools and protocols developed will be applied to upcoming trials for Objective 3. In addition, in support of Objective 3, butyrate-producing commensal organisms isolated from the swine intestinal tract were further characterized using genotypic and phenotypic attributes. Long-read sequencing was performed, and with already existing short-read sequencing, the genomes of two organisms were closed. Phylogenetic analysis indicated one organism as Roseburiathe and the other falling into a genus of Lachnospiraceae, both representing new species. The genomes suggest the potential for spore formation, a feature important for development of a probiotic of anaerobic organisms. Both organisms are able to utilize starch as a substrate, suggesting they may serve as a butyrate-producing probiotic when delivered with a prebiotic, such as dietary resistant starch.

1. Identified bacterial surface structures involved in O157 attachment to cattle intestinal cells. O157 causes significant disease in humans, but does not impact cattle health. Cattle carry O157 in their intestines and some cattle, called ‘super shedders’, excrete O157 in numbers greater than most cattle. The O157 strains recovered from super shedding cattle exhibit a distinct strong aggregative pattern of attachment to cells from the rectal region of the cattle intestine. ARS researchers in Ames, Iowa, and Clay Center, Nebraska, along with collaborators at the Pennsylvania State University and University of Washington, used a set of bacterial mutants, and super shedder and non-super shedder isolates to determine the mechanisms associated with this striking pattern of attachment. Our results show that bacterial surface structures, referred to as the type 1 fimbriae, may play a role in this attachment by some O157 strains and hence need to be targeted to prevent O157 attachment to bovine intestinal cells. These findings are noteworthy since it was previously suggested that the type 1 fimbriae may not be present on O157, and hence cannot play a role in adherence of O157 to intestinal cells. These findings are useful for development of intervention strategies to limit colonization in cattle.

2. Specific type of immune response in vaccinated cattle may play a role in reducing fecal shedding of O157. Healthy cattle are a major carrier of O157 bacteria in their intestines and fecal shedding of these bacteria by these cattle is a frequent source of contamination of beef, milk, and produce. Consumption of contaminated foods results in several O157-linked human disease outbreaks each year and thus, vaccination has been considered a potentially effective means to reduce O157 colonization in cattle. However, a vaccine would be effective in reducing shedding only if it would induce a specific type of immune response in cattle intestines capable of inhibiting O157 colonization. ARS researchers in Ames, Iowa, examined specific intestinal sites in cattle administered a vaccine prepared by mixing of a type of inactivated O157 bacteria and an immune response-stimulating substance. Analysis of these intestinal sites resulted in the identification of specific immune cells that produced high levels of a protein (interferon gamma) in vaccinated cattle compared to non-vaccinated animals. Additional studies demonstrated that treatment of cells derived from cattle intestine with interferon gamma reduced the ability of O157 to attach to these cells. In addition, increased production of interferon gamma in the intestine of vaccinated animals correlated with a significantly reduced fecal shedding of O157. Development of newer vaccines which could further enhance the level of interferon gamma in the intestine might result in higher reductions in the fecal shedding of O157 in vaccinated cattle.

3. Reference transcriptomes of porcine peripheral immune cells for improved genome to phenome. A major goal of biological research is to use genetic information (ie, genotype) to predict the complex physical composition or function of an individual or individual cells (ie, phenotype). Identifying and cataloging the genes expressed in specific cell types is thus critical to correlating a specific genotype to a specific phenotype and development of methods to screen for complex traits such as disease resilience. Cells in the blood are expected to represent the physiological state of an animal and may potentially predict outcomes when animals face stress. Thus, blood immune cell gene expression is valuable in development of tools for predictive modeling of immune response to stress or infection. ARS researchers in Ames, Iowa, Clay Center, Nebraska, and Beltsville, Maryland teamed up with Iowa State University collaborators to deeply characterize porcine blood immune cell gene expression. Common sorting methods resulted in deep sequencing of 8 blood immune cell populations, whereas new technology resulted in sequence data from more than 14,000 individual cells falling into more than 20 blood immune cell populations. Gene expression in pig immune cells was relatively comparable to mouse and human immune cells, though some unique differences highlight the importance of studying pigs directly. The data is publicly available to the international research community as a resource for further exploration, and several queries by international groups have already been received to utilize the data for annotation of pig immune cells in other tissues and develop improved genotype to phenotype analysis pipelines.

Review Publications
Moreau, M.R., Kudva, I.T., Katani, R., Cote, R., Li, L., Arthur, T.M., Kapur, V. 2021. Non-fimbrial adhesin mutants reveal divergent Escherichia coli O157:H7 adherence mechanisms on human and cattle epithelial cells. International Journal of Microbiology. 2021. Article 8868151.
Katani, R., Kudva, I.T., Srinivasan, S., Schilling, M., Cote, R., Li, L., DebRoy, C., Arthur, T.M., Kapur, V., Stasko, J.A. 2021. Strain and host-cell type dependent role of type 1 fimbriae genes in the adherence phenotype of super-shedder strains of Escherichia coli O157:H7. International Journal of Medical Microbiology. 311(4).
Kudva, I.T., Oosthuysen, E.R., Wheeler, B.K., Loest, C.A. 2021. Evaluation of cattle for naturally colonized Shiga toxin-producing Escherichia coli requires combinatorial strategies. International Journal of Microbiology. 2021. Article 6673202.
Schaut, R.G., Palmer, M.V., Boggiatto, P.M., Kudva, I.T., Loving, C.L., Sharma, V.K. 2021. Mucosal IFN-¿ production and potential role in protection in Escherichia coli O157:H7 vaccinated and challenged cattle. Infection and Immunity. 11. Article 9769.
Herrera-Uribe, J., Wiarda, J., Sivasankaran, S.K., Daharsh, L., Liu, H., Byrne, K.A., Smith, T.P., Lunney, J.K., Loving, C.L., Tuggle, C.K. 2021. Reference transcriptomes of porcine peripheral immune cells created through bulk and single-cell RNA sequencing. Frontiers in Genetics. 12. Article 689406.