Project description:In past, resistance mechanisms have been identified by analysis of resistant isolates or defined mutants. Recently, high-throughput transposon mutagenesis coupled with sequencing (TraDIS-Xpress) is another approach proving useful for elucidating the roles of genes involved in the overall cellular response to a particular stress. In this study, we used TraDIS-Xpress to determine the role played by genes following exposure to colistin stress. Approximately 10^7 cells from the mutant library were inoculated into LB broth at a range of doubling concentrations of colistin ( 0.25 x MIC, 0.5 x MIC, 1 x MIC, 2 X MIC). Experiments were performed with no induction, or with induction using 0.2 or 1 mM of Isopropyl β-D-1-thiogalactopyranoside (IPTG). All experiments were performed in duplicate.
Project description:Temperate bacteriophages play a pivotal role in the biology of their bacterial host. Of particular interest are bacteriophages infecting enterohemorrhagic E. coli (EHEC) due to their significant contribution in the pathogenicity of these pathogens, most notably by encoding the key virulence factor of this pathogen, the Shiga toxin. To better understand the role of EHEC phages on the functionality of its host, we isolated eight temperate phages from clinical EHEC isolates and characterized their genomic composition, morphology and receptor targeting. Morphological analysis identified one long-tailed member from the Siphoviridae family, targeting the OmpC receptor for host recognition, while the other seven phages are short-tailed (Podoviridae) and target the essential BamA protein. Genomic characterization revealed significant variation between the long- and short-tailed phages. Five of the eight isolated phages encode the potent Shiga toxin. Comparative analysis displays the typical lambdoid mosaicism, indicative of horizontal gene transfer driving evolution. These findings provide insights into the genetic and morphologic diversity and receptor specificity of EHEC phages, highlighting their role in evolution and pathogenicity of clinical EHEC strains
Project description:Previous studies have shown serotonin and indole downregulate expression of virulence genes in EHEC, here we demonstrate the combinatorial effect of serotonin and indole on EHEC transcriptome
Project description:Contamination with enterohemorrhagic Escherichia coli O157:H7 (EHEC) is a worldwide problem but there is no effective therapy available for EHEC infection. Biofilm formation is closely related with EHEC infection and is one of the mechanisms of antimicrobial resistance. Antibiofilm screening of 560 plant secondary metabolites against EHEC shows that ginkgolic acids C15:1 and C17:1 at 5 μg/ml and Ginko biloba extract at 100 μg/ml significantly inhibited EHEC biofilm formation on the surface of polystyrene, nylon membrane, and glass. Importantly, the working concentration of ginkgolic acids and G. biloba extract did not affect bacterial growth and has been known to be non-toxic to human. Transcriptional analyses showed that ginkgolic acid C15:1 repressed curli genes and prophage genes in EHEC, which were corroborated by reduced fimbriae production and biofilm reduction in EHEC. Interestingly, ginkgolic acids and G. biloba extract did not inhibit the biofilm formation of commensal E. coli K-12 strain. The current study suggests that plant secondary metabolites are important resource of biofilm inhibitors, as well as other bioactive compounds.
