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United States Department of Agriculture

Agricultural Research Service

Research Project: Prevention and Characterization of Persistent Colonization by E. Coli O157:h7 and Other Shiga Toxin-Producing E. Coli (Stec) in Cattle
2013 Annual Report


1a.Objectives (from AD-416):
1) Determine and characterize molecular mechanisms promoting colonization, effective adherence, and persistence of E. coli O157:H7 and other STECs in cattle;.
2)Understand the impact of bovine intestinal environment and immune responses on growth, adherence, and persistence of E. coli O157:H7 and other STECs in cattle;.
3)Conduct comparative analysis of bovine E. coli O157:H7 and STEC isolates of public health significance to identify components for use in developing rapid diagnostic tools and effective interventions; and.
4)Develop and test efficacy of chemical, biological, subunit proteins, and whole cell vaccines to prevent or reduce colonization of cattle intestines by E. coli O157:H7 and STECs.


1b.Approach (from AD-416):
Experimental animal models, tissue cultures, and specific mutants will be used to describe molecular mechanism(s) enabling E. coli O157:H7 bacteria to grow, adhere, and colonize the cattle intestine. Reporter gene fusions and global gene analysis technologies will be used to determine effects of host gastrointestinal environment and innate immune system on the expression of specific bacterial genetic systems and metabolic pathways that promote E. coli O157:H7 persistence in cattle intestine. Emerging non-O157 STEC serotypes will be compared with E. coli O157:H7 to identify genetic and molecular features unique to these serotypes. Bacterial genes or gene products identified in these studies will be used, based on their importance in colonization, for developing whole-cell or protein/subunit protein vaccines for reducing or eliminating E. coli O157:H7 and non-O157 STEC colonization and shedding in cattle.


3.Progress Report:
The goal of this project is to use molecular approaches to determine mechanism promoting the adherence of Escherichia coli (E. coli) O157:H7 and other Shiga toxin-producing E. coli (STEC) to the large intestine of cattle. The adherence to intestinal tissues promotes fecal shedding of E. coli O157:H7 and STEC in cattle causing contamination of cattle hides, carcasses, and beef. In support of objective #1, ARS researchers at Ames, IA, compared the fecal shedding of specific mutants in experimentally infected cattle to determine genes that regulated or participated in the adherence of E. coli O157:H7 in cattle intestines. The results allowed design of vaccines for reducing the fecal shedding of E. coli O157:H7 in animal trials. The current work has also confirmed that a mutant (sdiA mutant) of E. coli O157:H7 unable to sense specific chemical signals in the rumen (cow’s four-compartment stomach) could not survive in the rumen and was shed poorly in the feces. These findings have important implications for screening the rumen contents to identify specific chemicals or rumen bacteria for reducing the survival of E. coli O157:H7 in the rumen. Proteomic approaches were used to decipher protein networks expressed by E. coli O157:H7 grown in the rumen fluid. The identification of proteins implicated in the survival of E. coli O157:H7 in the rumen would allow the development of interventions for reducing the survival of these bacteria in cattle. It has been known that many bacteria afflicting disease in humans and animals produce highly specialized bacterial “aggregates” (biofilms) for coping with stressful external environments. Analysis of a mutant lacking a specific genetic regulator (Hha) revealed increased production of hair-like structures (curli fimbriae) on bacterial cell surfaces and increased biofilm formation on non-living solid surfaces. These findings may assist in investigating the role of curli in E. coli O157:H7 colonization of cattle intestines, determining if E. coli O157:H7 would produce specialized bacterial “aggregates” in the environment of cattle production premises, and correlating the presence of bacterial “aggregates” to the number of animals colonized with E. coli O157:H7 in cattle production facilities. In addition to the requirement of a specialized protein secretion system that secrete specific proteins (intimin and Tir) for the adherence of E. coli O157:H7 to a specific tissue (follicular epithelium) in the large intestine, novel factors have been implicated in the adherence of E. coli O157:H7 and six other non-O157:H7 STEC (declared food adulterants by the Food Safety and Inspection Service) to other tissue (squamous epithelium) types in the terminal portion (recto-anal junction or RAJ) of the large intestine. Proteomic approaches were used to identify genes affecting the adherence of E. coli O157:H7 and non-O157 STEC to RAJ. These findings would provide tools for blocking the adherence of these bacteria to RAJ. Testing of a non-antibiotic alternative, a small metabolic inhibitor molecule, has demonstrated a strong inhibitory effect on the fecal shedding of E. coli O157:H7 in cattle.


4.Accomplishments
1. Molecular mechanisms promoting fecal shedding of Escherichia coli (E. coli) O157:H7 in cattle. An understanding of the mechanisms that promote intestinal colonization and increase fecal shedding of E. coli O157:H7 bacteria in cattle are critical for developing vaccines and other interventions for reducing the fecal shedding of these harmful bacteria in cattle. By infecting cattle with a mutant strain of E. coli O157:H7 unable to produce a specific protein (SdiA), ARS researchers at Ames, IA, confirmed that the SdiA protein is essential for the survival of E. coli O157:H7 bacteria in a cow’s rumen (four-compartment stomach) before reaching and colonizing the large intestine of these animals. The other studies have shown that the SdiA protein senses specific chemical signals (quorum-sensing signals) to enhance survival of E. coli O157:H7 in the rumen. The importance of the SdiA protein in the survival of E. coli O157:H7 in cow’s rumen may contribute to the development of procedures to identify bacteria and/or chemicals that might counteract SdiA. Inhibiting the functions of SdiA would reduce survival of E. coli O157:H7 in the rumen, the outcome needed for reduced intestinal colonization and fecal shedding of E. coli O157:H7 in cattle as well as for subsequently reducing the risk of E. coli O157:H7-related human illnesses though enhancing safety of foods produced from cattle.

2. Bacterial proteins are required for adherence of six, non-O157:H7 Escherichia coli (E. coli) types to cattle intestinal tissues. Besides E. coli O157:H7, cattle could also be colonized by six other E. coli types (E. coli O16, O103, O111, O121, O145, and O45) recently declared as adulterants in beef by the Food Safety and Inspection Service (FSIS). These six E. coli types are as deadly as E. coli O157:H7 because of their ability to cause bloody diarrhea, kidney failure, and even death in humans. Human infections with these six E. coli types result from the consumption of beef, water, and produce contaminated with feces of cattle colonized with any of these six bacterial types. Thus, understanding of the mechanisms of intestinal colonization by these six E. coli types are essential for developing interventions for reducing the colonization of cattle intestines by these harmful E. coli. ARS researchers at Ames, IA, used specific types of cells (squamous cells) from the terminal portion (recto-anal junction) of the large intestine of cattle to demonstrate that these six E. coli adhere to and colonize intestinal tissues using protein factors different from those used by E. coli O157:H7. These findings are important for designing of vaccines/therapies to target these six E. coli types to enhance both the safety of foods produced from cattle and to mitigate the risk of transfer of these bacteria to humans and resulting human illnesses.

3. Determining the role of specific filamentous structures in adherence and aggregation of Escherichia coli (E. coli) O157:H7 bacteria on solid materials. Many bacterial types afflicting disease in humans and animals produce highly specialized bacterial “aggregates” (biofilms) on solid surfaces in the environment external to their human or animal host. These bacterial “aggregates” enable bacteria to survive in the harsh external environments and ensure their transmission to the appropriate host when presented with the opportunity. In order to produce specialized “aggregates”, bacteria must synthesize hair-like structures (fimbriae) and/or coatings of “sticky” substances at their cell surfaces. Production of these hair-like structures and sticky coatings is governed by specific signals such as low temperatures and limited nutrient supply in the external environment. ARS researchers at Ames, IA, determined that a mutant of E. coli O157:H7 lacking a specific genetic regulator (Hha) produced increased amounts of hair-like structures which correlated with the increased formation of bacterial “aggregates” on solid surfaces. These findings may assist in investigating the role of curli in E. coli O157:H7 colonization of cattle intestines, determining if E. coli O157:H7 would produce specialized bacterial “aggregates” in the environment of cattle production premises, and correlating the presence of bacterial “aggregates” to the number of animals colonized with E. coli O157:H7 in cattle production facilities. Since curli enhances adherence of E. coli O157:H7 to animal and human tissues, including curli proteins in vaccines for cattle vaccination could potentially reduce colonization of cattle by E. coli O157:H7. Any reductions achieved in the level of E. coli O157:H7 colonization in cattle by these methods would lower the risk of contamination of farm environment, meat produced from cattle, and the human illnesses due to E. coli O157:H7 infections.

4. Identification of factors produced by Escherichia coli (E. coli) O157:H7 bacteria to promote their growth and survival in cows stomach (rumen). E. coli O157:H7 is presumed to be equipped with a variety of mechanisms that allow it to overcome and breach defense systems of cow’s stomach. By breaching these defenses, E. coli O157:H7 bacteria gain access to cow’s intestine which is more hospitable for growth and colonization by E. coli O157:H7. One of our goals is to understand the kind and types of factors unique to these mechanisms so that we could design interventions for inhibiting these mechanisms and preventing E. coli O157:H7 bacteria to reach and colonize cattle intestine. ARS researchers at Ames, IA, would use a modern technology (proteomics) to identify specific factors that E. coli O157:H7 would produce under conditions resembling cow’s stomach. These factors would then be obliterated from E. coli O157:H7 bacteria to validate that the lack of these factors would prevent survival of E. coli O157:H7 in cow’s stomach. The results of these studies would provide us with the information to design novel means, such as vaccine or chemical or dietary supplements, to counter attack the mechanisms used by E. coli O157:H7 bacteria to survive and grow in cow’s stomach and intestine. The far reaching benefits of these studies would be the elimination and/or reduction of E. coli O157:H7 burden in cattle that in turn would enhance safety of foods produced from cattle and lower the incidence of human infections by E. coli O157:H7.


Review Publications
Sharma, V.K., Bearson, S.M. 2013. Evaluation of the impact of quorum sensing transcriptional regulator SdiA on long-term persistence and fecal shedding of Escherichia coli O157:H7 in weaned calves. Microbial Pathogenesis. 57:21-26.

Kudva, I.T., Hovde, C.J., John, M. 2013. Adherence of non-O157 Shiga-toxin Escherichia coli to bovine recto-anal junction squamous epithelial cells appears to be mediated by mechanisms distinct from those used by O157. Foodborne Pathogens and Disease. 10(4):375-381.

Sharma, V.K., Bearson, B.L. 2013. Hha controls Escherichia coli O157:H7 biofilm formation by differential regulation of global transcriptional regulators FlhDC and CsgD. Applied and Environmental Microbiology. 79(7):2384-2396.

Kudva, I.T., Davis, M.A., Griffin, R.W., Garren, J., Murray, M., John, M., Hovde, C.J., Calderwood, S.B. 2012. Polymorphic amplified typing sequences (PATS) and pulsed-field gel electrophoresis (PFGE) yield comparable results in the strain typing of a diverse set of bovine Escherichia coli O157:H7 isolates. International Research Journal of Microbiology. 10.1155. Available: http://www.hindawi.com/journals/ijmb/2012/140105/.

Last Modified: 7/22/2014
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