Location: Food Safety and Enteric Pathogens Research
2020 Annual Report
Objectives
Since antibiotics are neither recommended nor used for STEC treatment in humans or carrier-animals, strategies as described in the National Strategy for Combating Antibiotic Resistant Bacteria (CARB; 1) are needed and will be investigated for STEC control in cattle as exemplified in the following objectives:
Objective 1: Understand the impact of the bovine intestinal environment, especially at the rectoanal junction, and the molecular mechanisms that promote or inhibit colonization, adherence, and persistence of STEC in cattle and develop intervention strategies to control STEC colonization.
Subobjective 1A: Identify bacteria in the rectoanal junction (RAJ) microbiome that could effectively interfere with STEC colonization for possible use in probiotic applications.
Subobjective 1B: Identify bacterial ligands and tissue receptors involved in STEC adherence to the RAJ.
Objective 2: Formulate and assess the efficacy of vaccines for controlling STEC colonization of cattle based on whole-cell and subunit vaccines and identify proteins and epitopes conserved in STEC.
Objective 3: Define potential biomarkers using systems-based approaches that will allow the development of rapid diagnostic tests to identify STEC-colonized cattle.
Approach
Experimental animal and animal organ/tissue culture models will be used for determining qualitative and quantitative changes in bacterial communities constituting the rectoanal junction (RAJ) and fecal microbiomes, in response to colonization of cattle intestines by STEC serotype O157:H7 (O157). These microbiome changes will be correlated with the duration and magnitude of fecal O157 shedding by colonized cattle and compared to fecal/RAJ microbiome of non-colonized cattle. The results from these comparative studies will be used for identifying bacterial species that are part of the cattle intestinal microbiome and can compete effectively with O157 for bovine intestinal colonization. In vitro organ and tissue culture systems will be used for evaluating effects of these bacteria on O157 colonization at the RAJ. Proteomics-based techniques will be used for identifying proteins that are used by O157 to adhere to RAJ epithelial cells. Analogs mimicking these proteins will be designed and evaluated for their ability to interfere with O157 adherence to the RAJ epithelial cells. The existing O157 mutant vaccine strain will be modified and will be used as a whole-cell killed vaccine for determining if vaccination of cattle with the modified vaccine would show increased efficacy in controlling O157 colonization of cattle compared to the unmodified mutant strain-based vaccine. Metabolomics-based techniques will be used to identify metabolites present in blood samples of O157-colonized and non-colonized cattle. Few metabolites that are uniquely present in O157-colonized cattle will be evaluated for use as biomarkers to differentiate such cattle from the non-colonized animals.
Progress Report
Cattle carry the foodborne, human disease-causing bacteria Escherichia coli O157:H7 (O157) in their intestines and shed these bacteria in their feces. Foods contaminated with O157-containing cattle feces is a source of human infections. In the cattle intestine, O157 persists mainly at the recto-anal junction (RAJ) found close to the anal canal. Understanding the complex dynamics between animal, bacteria, and intestinal environment is important for developing intervention strategies to effectively reduce O157 in cattle prior to and/or at slaughter.
Super-shedding cattle release more O157 in their feces than the majority of other cattle and play a prominent role in the prevalence and transmission of O157. The bacteria, host, and/or the environment could contribute to the super-shedding phenomenon. A study was completed, under the broader goals of Objective 1, to evaluate genetically diverse (as determined by pulsed field gel electrophoresis profiles) super-shed O157 isolates for unique genetic features and determine if differences in adherence patterns on RAJ cells would be observed as a result of the genetic differences. Adherence assays performed with RAJ squamous epithelial (RSE) cells confirmed that all super-shed O157 isolates shared a hyperadherent binding pattern that may contribute to the persistence of these bacteria at the bovine RAJ. However, the hyperadherent phenotype was not dependent on any specific genetic features across different super-shed O157 strains.
Cattle vaccines targeting O157 have potential to reduce O157 shedding in feces. However, both vaccination and colonization with O157 can impact numbers and kinds of bacterial populations (microbiota) normally found in cattle intestines. Shifts in the intestinal microbiota could affect immune responses and health of cattle. Under Objective 1: Subobjective 1A, we evaluated the impact of vaccination and O157 colonization on the diversity of intestinal microbiota and immune responses of cattle. Microbiota analysis of fecal samples (which contains intestinal bacteria) collected from these animals over a 30-day period indicated a significant correlation between vaccination and changes in the intestinal bacterial populations. Vaccinated cattle had higher representation of certain populations (families) of bacteria in their microbiota. Vaccinated cattle also had significantly greater fecal antibodies against O157 and showed higher O157-specific cellular immune responses compared to non-vaccinated cattle. Further investigation of these changes in microbiota and immune responses due to vaccination may assist in the development of optimal vaccine strategies for reducing O157 colonization in cattle.
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 an/the animal experiences a stress (nutritional, environmental, or physical), can ‘spill’ into the intestine. O157, like other intestinal foodborne bacterial pathogens, can sense/metabolize these signals to alter its gene expression for successful colonization and persistence in the animal intestine. In accordance with the broader goals outlined under Objective 1, 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 sequenced via short-read Illumina sequencing. The output 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 non-exposed 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 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 stressed animals are more susceptible to O157 colonization compared to non-stressed animals. If NE treated O157 were verified to contain higher levels of proteins involved in O157 attachment to cattle intestine, these 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.
The cattle RAJ-in vitro organ culture (RAJ-IVOC) system developed as an alternative to a live animal to study O157 colonization of the RAJ, under Objective 1: Subobjective 1A, was evaluated using histological analysis to determine a time point at which tissue morphology and function is compromised. Laboratory conditions for culturing the tissue sections were optimized to maximize cell viability and perform short-term bacterial adherence assays.
Under Objective 2, RAJ biopsy samples were collected to evaluate live-animal samples for follicle formation at the RAJ in vaccinated animals. The follicle region of the RAJ is a site of O157 attachment in ruminants, including cattle and sheep. Vaccination for O157 was associated with increased follicle formation and understanding the immune status in this region of the RAJ can provide insight on immune responses associated with protection. RAJ biopsies can be repeatedly collected from the same animal, and therefore limit the number of animals required for tissue collection. Collection of RAJ biopsies from O157 vaccinated sheep was performed and used in assays to assess immune cell populations present in the region. Given the association of vaccine efficacy with follicle formation (from bovine study), collection of FAE and associated lymphoid tissue may be used for future assessment of anti-O157 mucosal cellular immune status of the animal without needing to euthanize the animal for collection of intestine.
Accomplishments
1. Super-shed Escherichia coli O157:H7 hyperadhere to bovine recto-anal junction cells. E. coli O157:H7 (O157) readily colonizes the recto-anal junction (RAJ) of the cattle intestinal tract, which is at the rectum. Some colonized animals, referred to as “super-shedders,” release more O157 in their feces than the majority of other cattle, and play a prominent role in the prevalence and transmission of O157. The bacteria, host, and/or the environment could contribute to the super-shedding phenomenon. ARS researchers at Ames, Iowa, evaluated 101 genetically diverse super-shed O157 isolates in their ability to attach to bovine RAJ cells. Results indicate all isolates displayed a hyperadherent binding pattern to RAJ cells, despite the genetic diversity across the super-shed isolates. In addition, when 53 of the 101 super-shed O157 isolates were tested for antibiotic resistance, 94 percent were found to be resistant to clinically (human and veterinary) relevant antibiotics and all carried genes associated with antibiotic resistance. The RAJ-based attachment assay was used as a proxy of attachment between bovine intestinal cells and O157, thus reducing the number of animals needed for research. Identifying unique characteristics of super-shed bacteria is important for the development of new methods to reduce super-shed O157 from cattle and to eliminate increased O157 release in the environment.
2. O157 colonization of the bovine recto-anal junction (RAJ) has minimal impact on resident RAJ bacterial communities. Cattle are the primary ruminant reservoir of E. coli O157:H7 (O157), a foodborne human pathogen. In cattle, O157 preferentially colonizes the RAJ but causes no disease in colonized cattle. The diverse resident microbes (microbiota) normally found at the RAJ could impact or be impacted by O157, thus influencing O157 colonization at the RAJ. To get a better understanding of O157 interaction with the RAJ microbiota, ARS researchers in Ames, Iowa, compared changes in the microbiota at the RAJ and in feces of animals experimentally inoculated with O157. The presence of O157 resulted in subtle changes in the relative abundance of different bacterial communities at the RAJ, suggesting RAJ microbial communities were transiently impacted by O157 colonization. These insights into O157 colonization dynamics at the RAJ may aid in development of better strategies targeting O157 in cattle.
3. Vaccination of cattle against O157 led to shifts in the intestinal microbiota. Vaccines targeting E. coli O157:H7 (O157) in cattle have potential to reduce O157 colonization and thus, reduce carcass contamination. However, non-O157 E. coli is a member of a normal microbiota, and vaccination against O157 E. coli may impact numbers and kinds of normal bacteria (microbiota) found in cattle intestines. As the microbiota has a role in animal health, shifts in the intestinal microbiota induced by vaccination could affect immune responses and health of cattle. ARS researchers in Ames, Iowa, evaluated the impact of O157 vaccination and O157 colonization on the diversity of intestinal microbiota to gauge potential unforeseen consequences of O157 vaccination. Microbiota analysis of fecal samples (which contains intestinal bacteria) from vaccinated and non-vaccinated cattle indicated a significant correlation between vaccination and shifts in intestinal bacterial populations. While vaccination may be a strategy to limit O157 in cattle, unforeseen consequences of changes in beneficial bacterial populations warrant further consideration.
Review Publications
Mir, R.A., Brunelle, B.W., Alt, D.P., Arthur, T.M., Kudva, I.T. 2020. Super-shed Escherichia coli O157:H7 has potential for increased persistence on the recto-anal junction squamous epithelial cells and antibiotic resistance. International Journal of Microbiology. 2020(2368154):1-16. https://doi.org/10.1155/2020/2368154.
Mir, R., Schaut, R.G., Looft, T.P., Allen, H.K., Sharma, V.K., Kudva, I.T. 2020. Recto-anal junction (RAJ) fecal microbiomes of cattle experimentally challenged with Escherichia coli O157:H7. Frontiers in Microbiology. 11:693. https://doi.org/10.3389/fmicb.2020.00693.
Mir, R.A., Schaut, R.A., Allen, H.K., Looft, T.P., Loving, C.L., Kudva, I.T., Sharma, V.K. 2019. Cattle intestinal microbiota shifts following Escherichia coli O157:H7 vaccination and colonization. PLoS One. 14(12):e0226099. https://doi.org/10.1371/journal.pone.0226099.
