1a. Objectives (from AD-416):
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.
1b. Approach (from AD-416):
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.
3. Progress Report:
Cattle, the primary reservoirs of foodborne human pathogens Shiga toxin-producing Escherichia 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. In this context, under Objective 1: Subobjective 1A: Identify bacteria in the rectoanal junction (RAJ) microbiome that could effectively interfere with STEC colonization for possible use in probiotic applications, ARS researchers have sampled RAJ sites from cattle experimentally colonized with Escherichia coli O157 (O157) and recorded changes in O157 levels in the context of the normal bacteria found at this site over time. These data will be used to analyze fluctuations in specific bacteria at the RAJ in the presence of O157 using advanced genomics tools. Under Objective 1: Subobjective 1B: Identify bacterial ligands and tissue receptors involved in STEC adherence to the RAJ of the plan, ARS researchers at Ames, IA used a novel proteomics-based approach (interactomics) to identify 84 proteins in O157 that interact with bovine RAJ cells. Addition of these proteins to prevent O157 attachment to RAJ cells in an in vitro adherence assay, confirming their role in O157 attachment to these cells. This is a significant finding as these proteins can be used to develop intervention strategies that block O157 attachment to bovine intestinal cells. ARS researchers at Ames, Iowa have also delineated the function of two cell-surface proteins of O157 using mutant strains lacking these specific genes, under Objective 2. Of the two proteins, one is used for adherence to epithelial cells and the other is presumably involved in environmental survival (outside the animal) in biofilms. This differentiation is useful when developing novel and/or improving the current whole-cell killed vaccine for reducing fecal shedding of O157 bacteria by cattle. In support of Objective 3 of the plan, ARS researchers at Ames, Iowa in collaboration with colleagues at Iowa State University, used a novel approach to analyze the chemicals/metabolites (metabolomics) present or absent in blood to identify compounds that are uniquely present in Shiga toxin-producing Escherichia coli (STEC)-colonized cattle. By analyzing bovine blood samples, the researchers have identified 33 metabolites that change significantly only in STEC-colonized animals compared to non-colonized animals. This is a significant finding as these 33 metabolites can be used as biomarkers to identify cattle that are carrying STEC. The researchers are currently applying an alternate metabolomics analysis technique so that additional differentiating metabolites may be identified.
1. Identified Escherichia coli (E. coli) contributing to adherence to animal cells versus non-living surfaces. Since bacteria use specific proteins to interact with a host, and usually a different set of proteins to survive in the environment, this identification is important for the development of intervention strategies that either limit attachment to host cells or limit survival in the environment thus reducing contamination. Bacteria often adhere to non-living materials such as plastic and glass making them difficult to eliminate. ARS researchers at Ames, Iowa have identified that one protein promotes O157 adherence to epithelial cells and another protein contributes to O157 adherence to plastic and glass. This is a significant finding that delineates the function of these two bacterial surface proteins of O157, one for possibly binding epithelial cells during colonization of intestinal tract of cattle, and the other, presumably for survival in the environment outside the animal. Identification of these proteins that are present solely on the outside of the bacteria is the first step in developing novel and/or improving the current vaccines for reducing O157 bacteria in the cattle reservoir and the environment.
Sharma, V.K., Kudva, I.T., Bearson, B.L., Stasko, J.A. 2016. Contributions of EspA filaments and curli fimbriae in cellular adherence and biofilm formation of enterohemorrhagic Escherichia coli O157:H7. PLoS One. 11(2):e0149745. doi: 10.1371/journal.pone.0149745.