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Characterization of a Novel Contact-Dependent Inhibitory Mechanism in Calf Adapted Escherichia Coli


<OL> <LI> Determine the in vitro culture conditions that influence the phenotypic expression of bactericidal activity of SSuT E. coli against E. coli O157:H7. <LI>Identify the efficacy of bactericidal activity against different bacterial species. <LI>Identify the gene(s) responsible for the expression and regulation of the contact-dependent bactericidal activity. </ol>

Outputs: The information generated from this project will be published in peer-reviewed journals and the preliminary data will be used to actively solicit extramural funding from USDA-AFRI under Biological Approaches for Food Safety program addressing different aspects of food-safety including antibiotic resistance related to colonization potential, increased pathogen load, persistence of bacteria and their viability in food animals. If we are successful in designing an effective probiotic based on this novel bacterial inhibitory mechanism, we will actively solicit funding from USDA-AFRI Practical Approaches for Food Protection that focuses on finding new and improved ways to control food-borne contaminants.

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NON-TECHNICAL SUMMARY: The selection pressure caused by the extensive use of antibiotics in animal agriculture has lead to the selection and widespread of antibiotic resistant bacteria. This is a major veterinary and public health problem. This is compounded by the fact that certain resistance bacteria can persist in environment even when antibiotic use is curtailed. The current project tries to understand the fundamental biology that play a role in providing competitive advantage for certain bacteria in a given environment. This project tries to understand the molecular mechanism of a novel contact dependent inhibition (bactericidal) trait observed in multi-drug resistant bacteria from dairy cattle. The project will analyze the influence of different experimental conditions including varying pH, temperatures, nutritional supplement on the efficacy of bactericidal activity. It is expected from these experiments to define the nature of the bactericidal activity. This knowledge will provide new opportunities to capitalize on a novel bacterial inhibitory mechanism that might be applicable directly or through engineered probiotics to target the elimination of antibiotic resistant and/or pathogenic bacteria in a given environment.


APPROACH: SSuT E. coli exhibiting bactericidal activity will be competed with susceptible E. coli in different environmental conditions including different temperatures (25, 37, and 42); different pH (3.5,5.5,6.5, and 7.5; in presence of different amino acids; presence of different carbon sources. Competition index will be calculated based on CFU/ml at 24 hrs. Difference in CI values between control and experimental conditions will be assessed using student t test and a P<0.05. Representative SSuT E. coli with bactericidal activity will be competed against different pathogenic E. coli including ETEC, STEC including E. coli O157:H7 from animal and human sources, methicillin resistant Staphylococcus aureus, Salmonella enterica, cephalosporin resistant E. coli. A CI value >0.5 will be used to define the presence of inhibitory activity of selected E. coli against competing bacterial species. To identify gene(s) that play a role in contact dependent inhibition phenotype, random disruption of genes in SSuT E. coli will be conducted using transposon mutagenesis. Transposon EZTn5: Knr will be then used to transform SSuT E. coli to create random insertional mutations. A total of 5000 mutant colonies will be screened and competed with susceptible E. coli strain. Mutants that show CI of <0 will be selected for sequence analysis to identify genes that have been disrupted.

Besser, Thomas; Sawant, Ashish
Washington State University
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