Project description:To distinguish between Helicobacter pylori isolates that may cause greater disease in patients, we used whole genome expression profiling as a platform to study host-pathogen interactions and identify gene signatures associated with isolates from patients with higher cancer risk. Expression profiles were studied for 3 clinical isolates from a region of high gastric cancer incidence (PZ5056, PZ5080, PZ5086) in Colombia and 3 isolates from a region with low gastric cancer incidence in Colombia (PZ5004, PZ5024, PZ5026). Each experiment was done in triplicate by infecting monolayers of gastric epithelial cells for 1 hour with the isolates.
Project description:Microarray was used to analyze azole resistance of Candida glabrata oropharyngeal isolates from 7 hematopoietic stem cell transplant recipients receiving fluconazole prophylaxis. Transcriptional profiling of the sequential-paired clinical isolates by microarray revealed 19 genes upregulated in the majority of resistant isolates compared to their paired-susceptible isolates. All seven resistant isolates had greater than two fold upregulation of CgPDR1, a master transcriptional regulator of PDR network, and all 7 resistant isolates showed upregulation of known CgPDR1-target genes. The altered transcriptome can be explained in part by the observation that all 7 resistant isolates had acquired a single nonsynonymous mutation in their CgPDR1 ORF. Four mutations occurred in the regulatory domain (L280P, L344S, G348A, S391L) and one in the activation domain (G943S) while two mutations (N764I, R772I) occurred in an undefined region. Association of azole resistance and the CgPDR1 mutations was investigated in the same genetic background by introducing the CgPDR1 sequences from one sensitive and five resistant isolates into a laboratory azole-sensitive strain (cgpdr1) via integrative transformation. The cgpdr1 strain was restored to wild-type fluconazole susceptibility when transformed with CgPDR1 from the susceptible isolate but became resistant when transformed with CgPDR1 from the resistant isolates. However, despite the identical genetic background, upregulation of CgPDR1 and CgPDR1-target genes varied between the 5 transformants, independent of the domain locations in which the mutations occurred. In sum, gain-of-function mutations in CgPDR1 not only contributed to the clinical azole resistance but different mutations had varying degrees of impact on the CgPDR1-target genes.
Project description:Precise definition of porin profiles is of critical importance to understand the role of porins in antimicrobial resistance. In this study, the outer membrane proteins (OMP) profiles of 26 clinical isolates of Klebsiella pneumoniae and of strain ATCC 13883 (wild-type) and ATCC 700603 (producing SHV-18) have been determined using both sodium-dodecyl-sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and matrix-assisted laser desorption/ionization–time of flight/mass spectrometry (MALDI-TOF/MS). SDS-PAGE was performed using both homemade and commercial gels, and protein bands were identified by liquid chromatography coupled to mass spectrometry. A rapid extraction method was used to analyse OMPs by MALDI-TOF/MS. The sequences of porin genes were obtained by whole genome sequencing (WGS) and mutations were defined by BLAST. Same results were obtained for all strains either using SDS-PAGE or MALDI-TOF/MS. SDS-PAGE showed protein bands of ~35, ~36, and ~37 KDa, identified as OmpA, OmpK36 and OmpK35, respectively. By MALDI-TOF/MS, peaks at ~35700 (OmpA), ~37000 (OmpK35), and ~38000 (OmpK36) m/z were detected. ompK35 was intact in nine wild-type isolates and was truncated in 13 isolates, but OmpK35 was not observed in 3 isolates without mutations in ompK35. One point mutation was detected in another isolate and multiple mutations were detected in the remaining isolate. ompK36 was truncated in two isolates lacking this protein and presented one point mutation (n=1) or multiple mutations in the remaining isolates. In conclusion, MALDI-TOF/MS was reliable for porin detection, but because of the complex regulation of porin genes, WGS cannot always anticipate protein expression, as observed with SDS-PAGE and MALDI-TOF/MS.
Project description:Comparative genomic hybridization between Escherichia coli strains to determine core and pan genome content of clinical and environmental isolates
Project description:Replicates of strains used in comparison of microarray to RAPD analysis for the publication. Findings from use of an open-reading frame-specific Campylobacter jejuni DNA microarray to investigate genetic diversity among clinical isolates associated with 5 independent clusters of infection were compared with data from random amplified polymeric DNA (RAPD) and Penner serotyping analyses. The DNA microarray provides a highly specific epidemiological typing tool for analysis of C. jejuni isolates and reveals both divergent and highly conserved gene classes among isolates Set of arrays that are part of repeated experiments Keywords: Biological Replicate
