Project description:Pathogenic biofilms have been associated with persistent infections due to their high resistance to antimicrobial agents. To identify non-toxic biofilm inhibitors for enterohemorrhagic Escherichia coli O157:H7, indole-3-acetaldehyde was used and reduced E. coli O157:H7 biofilm formation. Global transcriptome analyses revealed that indole-3-acetaldehyde most repressed two curli operons, csgBAC and csgDEFG, and induced tryptophanase (tnaAB) in E. coli O157:H7 biofilm cells. Electron microscopy showed that indole-3-acetaldehyde reduced curli production in E. coli O157:H7. Together, this study shows that Actinomycetales are an important resource of biofilm inhibitors as well as antibiotics.
Project description:Escherichia coli O157:H7 strains have been classified into different genotypes based on the presence of specific shiga toxin-encoding bacteriophage insertion sites. Genotypes that are predominant in clinical isolates are named clinical genotypes and those that are isolated mostly from bovine sources are bovine-biased genotypes. To determine whether inherent differences in gene expression could possibly explain the variation in infectivity of these genotypes, we compared the expression patterns of O157:H7 strains isolated from cattle, which belonged to either clinical genotype 1 or bovine-biased genotype 5. Important virulence factors of O157, including locus of enterocyte effacement, enterohemolysin, and pO157 plasmid encoded genes, showed increased expression in clinical genotype. Genes essential for acid resistance such as gadA, gadB, and gadC and other stress fitness-associated genes were up-regulated in the bovine-biased genotype 5. Overall, these results suggest that clinical genotype 1 strains more commonly cause human illness because of an enhanced ability to express O157 virulence factors known to be important for disease pathogenesis. By contrast, strains of the bovine-biased genotype 5 appear to be more resistant to adverse environmental conditions, which enable them to survive well in bovines without causing disease.
Project description:Comparative genomic hybridization between Escherichia coli strains to determine core and pan genome content of clinical and environmental isolates Two color experiment, Escherichia coli Sakai (reference), clinical and environmental Escherichia coli strains (testers): At least two replicates including a single dye swap for each reference-tester comparison
Project description:Multiple infection sources for enterohemorrhagic Escherichia coli O157:H7 are known, including food of animal origin and produce. The ecology of this pathogen outside its human host is largely unknown. One third of its annotated genes still are hypothetical. To identify genetic determinants expressed under environmental factors, we applied strand-specific RNA-sequencing of strain E. coli EDL933 under 11 different biotic and abiotic conditions: LB medium at pH4, pH7, pH9, or at 15°C; LB with nitrite or trimethoprim-sulfamethoxazole; LB-agar surface, M9 minimal medium, spinach leaf juice, surface of living radish sprouts, and cattle feces. Of 5379 annotated genes, only 144 are transcriptionally completely inactive under all conditions. Of 1,771 hypothetical genes, 1,672 exhibit significant transcriptional signals under at least one condition. The pathogenicity island LEE showed highest transcriptional activity in LB medium, minimal medium, and after treatment with antibiotics. Unique sets of genes, including many hypothetical genes, are highly up regulated on radish sprouts, cattle feces, or in the presence of antibiotics. For instance, azoR is biotechnologically important, but its environmental function has been elusive. This gene is highly active on radish sprouts. Further, we observed induction of the shiga-toxin carrying phages by antibiotics and confirmed active biofilm related genes on radish sprouts, in cattle feces, and on agar plates. Thus, environmental transcriptomics uncovers hitherto unknown gene functions and regulatory patterns of Escherichia coli O157:H7.
Project description:Comparative genomic hybridization between Escherichia coli strains to determine core and pan genome content of clinical and environmental isolates
Project description:The existence of two separate lineages of Escherichia coli O157:H7 has previously been reported, and research indicates that one of these lineages (lineage I) might be more pathogenic towards human hosts. We have previously shown that the more pathogenic lineage expresses higher levels of Shiga toxin 2 (Stx2) than the non-pathogenic lineage II. To evaluate why lineage 2 isolates do not express appreciable levels of toxin, two lineage 2 isolates (FRIK966 and FRIK2000) were chosen as representatives of lineage 2 and whole genome microarrays were performed using Agilent microarrays using the E. coli O157:H7 EDL933 lineage I clinical type isolate as a reference. Microarray results were utilized to evaluate what genes and pathways might be missing or differentially expressed. Quantitative RT-PCR was utilized to validate the microarray data.
Project description:We compared the transcriptional profiles of 12 E. coli O157:H7 isolates grown to stationary phase in LB broth. These isolates possess the same two enzyme PFGE profile and are related temporally or geographically to the above outbreak. These E. coli O157:H7 isolates included three clinical isolates, five isolates from separate bags of spinach, and single isolates from pasture soil, river water, cow feces, and a feral pig.
Project description:Multiple infection sources for enterohemorrhagic Escherichia coli O157:H7 are known, including food of animal origin and produce. The ecology of this pathogen outside its human host is largely unknown. One third of its annotated genes still are hypothetical. To identify genetic determinants expressed under environmental factors, we applied strand-specific RNA-sequencing of strain E. coli EDL933 under 11 different biotic and abiotic conditions: LB medium at pH4, pH7, pH9, or at 15°C; LB with nitrite or trimethoprim-sulfamethoxazole; LB-agar surface, M9 minimal medium, spinach leaf juice, surface of living radish sprouts, and cattle feces. Of 5379 annotated genes, only 144 are transcriptionally completely inactive under all conditions. Of 1,771 hypothetical genes, 1,672 exhibit significant transcriptional signals under at least one condition. The pathogenicity island LEE showed highest transcriptional activity in LB medium, minimal medium, and after treatment with antibiotics. Unique sets of genes, including many hypothetical genes, are highly up regulated on radish sprouts, cattle feces, or in the presence of antibiotics. For instance, azoR is biotechnologically important, but its environmental function has been elusive. This gene is highly active on radish sprouts. Further, we observed induction of the shiga-toxin carrying phages by antibiotics and confirmed active biofilm related genes on radish sprouts, in cattle feces, and on agar plates. Thus, environmental transcriptomics uncovers hitherto unknown gene functions and regulatory patterns of Escherichia coli O157:H7. Eleven different conditions were sequenced on the SOLiD system. Of two of the condtions, spinach medium and LB-nitrite, technical replicates were sequenced. Of LB medium and radish sprouts, biological replicates were sequenced on an Illumina MiSeq.
Project description:Escherichia coli O157:H7 strains have been classified into different genotypes based on the presence of specific shiga toxin-encoding bacteriophage insertion sites. Genotypes that are predominant in clinical isolates are named clinical genotypes and those that are isolated mostly from bovine sources are bovine-biased genotypes. To determine whether inherent differences in gene expression could possibly explain the variation in infectivity of these genotypes, we compared the expression patterns of O157:H7 strains isolated from cattle, which belonged to either clinical genotype 1 or bovine-biased genotype 5. Important virulence factors of O157, including locus of enterocyte effacement, enterohemolysin, and pO157 plasmid encoded genes, showed increased expression in clinical genotype. Genes essential for acid resistance such as gadA, gadB, and gadC and other stress fitness-associated genes were up-regulated in the bovine-biased genotype 5. Overall, these results suggest that clinical genotype 1 strains more commonly cause human illness because of an enhanced ability to express O157 virulence factors known to be important for disease pathogenesis. By contrast, strains of the bovine-biased genotype 5 appear to be more resistant to adverse environmental conditions, which enable them to survive well in bovines without causing disease. The results are based on O157:H7 clinical and bovine-biased genotype cultures grown in DMEM medium to exponential phase. Four strains were selected from each genotype and strains were considered as biological replicates. A double loop microarray design was used for comparing the samples. Differences in transcript levels were determined using a mixed model ANOVA in R/MAANOVA which tested for significant differences due to strain (clinical or bovine-biased) using the following linear model: array+dye+sample (biological replicate)+strain+error. We incorporated the dye-swaps among the biological replicates.