Project description:Campylobacter jejuni is the leading cause of campylobacteriosis in the developed world. Although most cases are caused by consumption of contaminated meat, a significant proportion is caused by consumption of contaminated water. Some C. jejuni isolates are better than others at surviving in water, which suggests that these strains are better adapted to transmission by water than others. The aim of this study is to investigate this phenomenon further. CFU counts and viability assays showed that strain 81116 survives better than strain 81-176 in a defined freshwater medium at 4°C. Comparative transcriptomic profiling using microarray revealed that these strains respond differently to water. This series presents the transcriptome of strain 81-176 in water.
Project description:Campylobacter, a major foodborne pathogen, is increasingly resistant to macrolide antibibotics. previous findings suggeted that development of macrolide resistance in campylobacter requires a multi-step process, but the molecular mechanisms involved in the process are not known. In our study, multiple seriess of macrolide-resistant C. jejuni mutants were selected in vitro by stepwise exposure of C. jejuni 81-176 to increasing concentrations of erythromycin and tylosin. A set of the selected resistance were subjected to microarray and the the global transcriptional profile was analyzed. In this sery, DNA microarray was used to compare the gene expression profiles of macrolide resistant strains (76E2, 76E8 and 76E64) with its parent wild-type strain C. jejuni 81-176. This assay identified a large number of genes that showed >1.5 fold changes (q-value<0.1) in expression in the macrolide resistant strains. The up-regulated genes are involved in surface structure,ribosomal, heat shock and some specific Misc memerbrane , while the majority of the down-regulated genes are involved in energy metabolism, amino acid biosynthesis. The over-expression of genes involved in surface structure and Misc memerbrance was associated with the development of intermedial-level resistance to macrolide in campylobacter 81-176. Keywords:intermedial-level macrolide resistant C. jejuni selected from C. jejuni 81-176. step-wise selection.
Project description:Campylobacter, a major foodborne pathogen, is increasingly resistant to macrolide antibibotics. previous findings suggeted that development of macrolide resistance in campylobacter requires a multi-step process, but the molecular mechanisms involved in the process are not known. In our study, multiple seriess of macrolide-resistant C. jejuni mutants were selected in vitro by stepwise exposure of C. jejuni 81-176 to increasing concentrations of erythromycin and tylosin. A set of the selected resistance were subjected to microarray and the the global transcriptional profile was analyzed. In this sery, DNA microarray was used to compare the gene expression profiles of macrolide resistant strains (76E2, 76E8 and 76E64) with its parent wild-type strain C. jejuni 81-176. This assay identified a large number of genes that showed >1.5 fold changes (q-value<0.1) in expression in the macrolide resistant strains. The up-regulated genes are involved in surface structure,ribosomal, heat shock and some specific Misc memerbrane , while the majority of the down-regulated genes are involved in energy metabolism, amino acid biosynthesis. The over-expression of genes involved in surface structure and Misc memerbrance was associated with the development of intermedial-level resistance to macrolide in campylobacter 81-176. Keywords:intermedial-level macrolide resistant C. jejuni selected from C. jejuni 81-176. step-wise selection. The design utilized an available two color microarray slide for the entire transcriptome of Campylobacter jejuni. Four hybridizations were performed each with independently extracted samples of either macrolide susceptible C. jejuni 81-176 cDNA samples or macrolide resistant C. jejuni cDNA samples. A dye swap was utilized to help minimize dye dependent bias. Thus there were three to four biological replicates of each sample.
Project description:Campylobacter jejuni is one of the most important zoonotic enteric bacterial pathogens able to colonize the gastrointestinal tract of various animals. The number of campylobacteriosis cases has increased worldwide and the particular concern has been the high level of fluoroquinolone resistance observed in C. jejuni isolates. In this study, we explored the effect of ciprofloxacin in Campylobacter jejuni by gene expression microarray analysis. The comparisons of transcriptional responses were performed in the absence and presence of ciprofloxacin with the wild-type strain 81-176 and its intermediate-resistant variant (P3). The resistant variant contained a single-nucleotide mutation causing amino acid change Asp-90-Asn in the gyrA gene instead of the most common Thr-86-Ile, causing a high-level resistance to ciprofloxacin. After the short-term exposure to a relatively high concentration of ciprofloxacin, the viability of C. jejuni 81-176 wild-type cells remained notably high when compared with other gram-negative bacteria. The general responses of short-term ciprofloxacin exposure were determined from both genetic backgrounds and resulted in the expression variation of genes participating in general cellular processes, e.g. , in carbon and amino-acid metabolism and protein transport.
Project description:The Cj1223c gene was cloned downstream of a strong promoter into the replicating plasmid pRY108, and was highly expressed in the wild type Campylobacter jejuni 81-176 strain (overexpressed). The Cj1223c gene was knocked out by allelic replacement in the 81-176 strain background (mutant). This mutant also carried an empty pRY108 vector. The Cj1223c overexpressing strain and the mutant were grown overnight in liquid broth, supplemented with kanamycin. The following morning, both cultures were diluted back to O.D.600=0.100, and allowed to continue to grow, shaking. At various time intervals (2, 6, 10, and 24 hours), samples of each culture were removed, combined with 1/10 stop solution, and the bacteria pelleted for RNA preparation.