Location: Food Safety and Enteric Pathogens Research
2017 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 are the primary reservoir of the foodborne human pathogen Shiga toxin-producing Escherichia coli (STEC) and remain asymptomatic over the course of STEC colonization. The lack of disease presentation in cattle makes it difficult to detect and apply interventions to eliminate the bacterium. Understanding the complex dynamics between animal, bacteria, and intestinal environment 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.
To better characterize the bacteria in the environment that STEC occupies in cattle, under Objective 1: Subobjective 1A: Identify bacteria in the bovine rectoanal junction (RAJ) microbiome that can interfere with STEC colonization, a study was completed comparing the abundance and diversity of bacteria at the RAJ and feces of healthy cattle. Using 16S ribosomal ribonucleic acid (rRNA) gene sequences for microbiome analysis, preliminary results indicate differences in identity of bacteria specific to the RAJ versus the feces. In addition, studies are ongoing to assess changes in RAJ bacterial communities during STEC serotype O157 infection. Collectively, this information is necessary for development of an intervention strategy to limit O157 colonization in cattle.
In support of Objective 1: Subobjective 1B: Identify bacterial ligands and tissue receptors involved in STEC adherence, a unique protocol was standardized that allows for identifying bacterial proteins that interact with animal cells, which allows for the bacteria to remain in the intestinal tract. This assay has been applied to STEC and different cells from the RAJ of cattle, and work is ongoing to identify the specific bacterial proteins involved in attachment of STEC using protein sequencing methods. Once identified, these proteins can be used as vaccine antigen, or to develop other counter-measures to block the attachment of O157 to the bovine RAJ.
Vaccination is one potential mechanism to limit STEC colonization in cattle, and work has continued towards developing a modified strain of STEC to use as a vaccine. Under Objective 2 researchers have preliminarily validated two sets of short STEC genome sequences. Genetic modification has been completed, and lab assays are ongoing to assess which of these two genome sequences is best for increasing the expression of STEC specific proteins. This modified strain will then be tested as a vaccine to limit colonization and fecal shedding of O157 in cattle.
In addition to modifying the bacteria to enhance vaccine efficacy, adjuvants can be added to vaccines to increase the cattle immune response. Pairing of the right adjuvant with vaccine antigen (STEC bacteria) is critical for the success of a vaccine. Thus, under Objective 2 immunogenicity and efficacy of inactivated, whole-cell STEC vaccine was tested when formulated with and without adjuvant. Vaccination of cattle with an emulsion prepared by mixing inactivated bacteria with an adjuvant (oil-in-water emulsions) induced higher levels of antibody, an important immune mediator, against O157. This increased immune response was associated with significant reductions in fecal shedding of O157. These data are significant and suggest that future vaccine testing will require formulation with adjuvant for ultimate protection.
Accomplishments
1. Identified Escherichia coli O157 protein that limits its attachment to bovine intestinal cells. Cattle are the primary reservoir of the human foodborne pathogen Escherichia coli O157 (O157). O157 bacteria attach to bovine cells at the rectoanal junction (RAJ), thus, identifying bacterial proteins that allow O157 attachment at the RAJ is critical for developing non-antibiotic methods to limit attachment and colonization and reduce O157 in cattle. Since the well-characterized O157 adherence protein, intimin, does not contribute to O157 attachment to the cells at the RAJ, ARS researchers in Ames, Iowa examined another common protein and discovered that this protein limits O157 attachment to RAJ cells. Removing, disrupting or interfering with this protein resulted in better O157 attachment to the RAJ cells. These data will need to be considered, particularly by pharmaceutical or vaccine companies, when designing anti-attachment therapies or vaccines, as any approach targeting the protein may actually enhance attachment of O157 to the cattle RAJ.
2. Identified genetic features that contribute to survival of Escherichia coli O157. The genetic mechanisms employed by Escherichia coli O157 (O157) bacteria to colonize and persist in the digestive tract of cattle are not fully understood. Bacterial genome characterization allows for interrogating the mechanisms used by the bacteria to colonize cattle, and thus, is useful for identifying therapeutic targets. ARS researchers in Ames, Iowa performed full genome sequence of two O157 bacterial strains. Fine-tune analysis of the genetic make-up allowed identification of genetic features that contribute to survival of O157 in cattle and the environment outside cattle. The practical application extends to utility of these genomes as a reference tool to identify how the genome and gene expression change during the course of colonization of O157 in cattle or during survival in the environment, such as feces or manure. Ultimately, full genome sequence is the platform to which comparisons and site-specific query of gene function can begin and guide future therapeutic or vaccine discoveries.
Review Publications
Sharma, V.K., Bayles, D.O., Alt, D.P., Looft, T.P. 2016. Complete genome sequences of curli-negative and curli-positive isolates of foodborne Escherichia coli O157:H7 strain 86-24. Genome Announcements. 4(6):e01323-16. doi: 10.1128/genomeA.01323-16.
Sharma, V.K., Bayles, D.O., Alt, D.P., Looft, T.P., Brunelle, B.W., Stasko, J.A. 2017. Disruption of rcsB by a duplicated sequence in a curli-producing Escherichia coli O157:H7 results in differential gene expression in relation to biofilm formation, stress responses, and metabolism. BMC Microbiology. 17(1):56. doi: 10.1186/s12866-017-0966-x.
Kudva, I.T., Carter, M.Q., Sharma, V.K., Stasko, J.A., Giron, J.A. 2016. Curli temper adherence of Escherichia coli O157:H7 to squamous epithelial cells from the bovine recto-anal junction in a strain-dependent manner. Applied and Environmental Microbiology. 83(1):e02594-16. doi: 10.1128/AEM.02594-16.
