Location: Food Safety and Enteric Pathogens Research
2021 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
This is the final report for project 5030-32000-112-00D terminated in December 2020 and replaced with 5030-32000-225-00D.
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 slaughter and some significant advances were made in this regard.
Under Objective 1A, it was determined that the presence of O157 can result in subtle changes in the relative abundance of different bacterial communities at the rectoanal junction, suggesting microbial communities at this site were transiently impacted by O157 colonization. These insights into O157 colonization dynamics at the rectoanal junction may aid in development of probiotics-based strategies targeting O157 in cattle.
Under Objective 1B, bacterial cell surface proteins, EspA and Curlin, were determined to be involved in O157 adherence to epithelial cells and adherence to non-living materials, such as plastic and glass, respectively. Thus, EspA could influence colonization of cattle through adherence to intestinal epithelial cells. It was also demonstrated that Curlin limits O157 attachment to epithelial cells at the rectoanal junction and blocking Curlin expression results in increased O157 attachment. This data will need to be considered when designing anti-attachment therapies or vaccines, as any approach targeting Curlin may enhance attachment of O157 to the cattle rectoanal junction. Another O157 protein, carbon starvation-inducible lipoprotein (Slp) was determined to be involved in initial attachment to human epithelial cells. Slp interacts with a protein on the human intestinal cells called the polymeric immunoglobulin receptor or pIgR. Both Slp and pIgR maybe targeted for human therapeutic interventions that could interfere with O157 attachment. Slp may also play a role in initial attachment to bovine intestinal cells which needs further investigation.
Under Objective 1B, 84 O157 proteins involved in adherence to squamous epithelial cells and 89 O157 proteins that interact with the follicle-associated epithelial cells at the rectoanal junction were identified. The role of these proteins in adherence was confirmed by their ability to block O157 attachment to the cells in in vitro assays. This is a significant finding as these proteins are good candidates for intervention strategies to prevent O157 colonization in cattle. In the process of evaluating O157 proteins involved in adherence, a rectoanal junction-in vitro organ culture system was developed as an alternative to live animals to study O157 colonization at the rectoanal junction.
Some colonized animals, referred to as “super-shedders”, shed more O157 in their feces than most other cattle. Under Objective 1B, super-shed O157 isolates were determined to be hyperadherent to rectoanal junction cells, carrying antibiotic-resistance genes and demonstrating resistance to clinically (human and veterinary) relevant antibiotics. Evaluation of two super-shed O157 genomes demonstrated the presence of unique, recently acquired, genes associated with motility, adherence, and metabolism that may have influenced the persistent colonization of the bovine intestine by super-shed O157. Identifying unique characteristics of super-shed bacteria is important for the development of new methods to reduce super-shed O157 from cattle.
Under Objective 2, a killed whole-cell O157 vaccination strategy was improved to enhance immune responses in cattle by using two doses of the vaccine formulated with a chemically inactivated mutant strain of O157 and a commercially available adjuvant. The vaccine-adjuvant combination not only enhanced immunity against O157 but also efficacy with reduced fecal O157 shedding. These findings show that vaccine formulations for STEC must be tailored for optimal immune activation and memory response to reduce O157 colonization in cattle. Additionally, following vaccination with a specific vaccine formulation (inactivated mutant O157 with an immune stimulant), one of the immune response factors (immunoglobulin A) detected in the feces of vaccinated cattle, and specific immune cells and cytokines (interferon gamma) were shown to limit O157 attachment to cattle intestinal cells. These results provide a platform for developing improved vaccines for controlling O157; vaccines and adjuvants can be tested to determine if the levels of specific immune proteins can be enhanced to achieve greater reductions in colonization and fecal shedding of O157 in cattle.
Under Objective 2, sheep were used for rapid and cost-effective evaluation of a vaccine formulation. As expected, vaccination induced O157-specific antibody in the feces and blood. Adjuvant did impact responses, as did the modifications to the O157 vaccine strain. The data will be used to design a vaccine trial in cattle, selecting the vaccine expected to provide the greatest protection against challenge. Additionally, the genomes of two O157 bacterial strains were sequenced to identify how the genome and gene expression change during O157 colonization in cattle or during the O157 survival in the environment, such as feces or manure. The goal being to use the genome sequence as a guide for future therapeutic or vaccine discoveries.
Under Objective 2, the impact of O157 vaccination and O157 colonization on the diversity of the normal bacteria (microbiota) in cattle intestines was evaluated to gauge potential unforeseen consequences of O157 vaccination. Significant correlation between vaccination and shifts in intestinal microbiota was observed with a relative increase in bacteria known as Paeniclostridium spp. and Christenellaceae R7 in the microbiota of vaccinated cattle. While vaccination may be a strategy to limit O157 in cattle, changes in beneficial bacterial populations warrant further consideration. Stress can also lead to changes in normal behavior, growth, immunity, and impact intestinal microbiota. Two common stressors, dehorning and castration, were determined to cause changes to the intestinal microbiota regardless of analgesic administration. This work highlights the importance of reducing stress for overall animal health which in turn may influence efficacious immune responses to vaccines and other therapies.
Under Objective 3, a novel approach analyzing chemicals/metabolites (metabolomics) in cattle blood was used to identify those that are uniquely present only in STEC-colonized cattle. A total of 33 metabolites were identified that significantly changed only in STEC-colonized animals compared to non-colonized animals. Additional analysis performed with different mass spectrometry techniques, statistics, and screening of additional blood samples from cattle with and without STEC, enabled shortlisting 15 of 33 metabolites for use as potential biomarkers of bovine colonization with O157. In support of the development of a targeted assay, antibodies to 7 of the 15 metabolites were developed and are currently being evaluated in laboratory assays for their ability to accurately identify the specific metabolites. This is a significant finding as these metabolites can be used as biomarkers and incorporated into rapid pen-side assays for identifying STEC-colonized cattle. For future testing of the metabolite biomarkers in field samples, blood and fecal samples were collected from feedlot cattle in New Mexico. Accurate determination of the presence of viable STEC in the fecal samples required a combination of methods including culture, polymerase chain reaction and a cell toxicity assay to evaluate toxin production by the STEC. Of the 59 cattle tested, 19% were positive for viable STEC and the main STEC serotype isolated was O103. This data will be used to correlate with the presence of metabolites in matching blood samples.
Accomplishments
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. https://doi.org/10.1155/2021/8868151.
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. https://doi.org/10.1155/2021/6673202.
