No abstract

No abstract

Mechanisms of drug resistance in Campylobacter jejuni were investigated. Mutant strains 34PEF r , which was resistant to pefloxacin (128-fold increase in the MIC), and 34CTX r , which was resistant to cefotaxime (32-fold increase in the MIC) and which was derived from the susceptible parent 34 s , were obtained by serial passages on pefloxacin and cefotaxime gradient plates, respectively. Both mutants showed cross-resistance to erythromycin, chloramphenicol, tetracycline, ␤-lactams, and quinolones. While the quinolone resistance of strain PEF r could be explained by a mutation at codon 86 of the gyrA gene, the multidrug resistance phenotype of both strains was further investigated. Accumulation of pefloxacin, ciprofloxacin, and minocycline was measured by fluorometry and was found to be lower in the mutant strains than in the parent strain. Preincubation of the cells with carbonyl cyanide m-chlorophenylhydrazone, however, completely abolished this difference. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of outer membrane preparations from both mutant strains showed overexpression of two proteins of 55 and 39 kDa which were absent from the outer membranes of the wild-type strain. These results indicate that in C. jejuni 34PEF r and 34CTX r , multidrug resistance is associated with an efflux system with a broad specificity.Campylobacter jejuni is recognized as a leading cause of diarrhea in the world (3, 5). Typically, C. jejuni diarrhea is a self-limiting disease lasting up to 5 days, but it may persist longer and may develop into severe forms, especially in children and immunocompromised patients (2, 3).Fluoroquinolones are powerful antimicrobial agents against many gram-negative enteric bacteria including C. jejuni (4,35,37). Besides their potent in vitro activity against susceptible organisms (37), quinolones can also achieve high concentrations in the gastrointestinal tract (11). Nevertheless, the resistance of C. jejuni to fluoroquinolones during therapy has been reported (1,12,15,30,31).In gram-negative bacteria three mechanisms of quinolone resistance have been described, including alterations in the DNA gyrase structure (17,26,38), decreased outer membrane permeability (10), and export through an active efflux system (10,18,21).Quinolone-resistant mutants of C. jejuni selected in vitro by single-step quinolone exposure have been shown to be due to point mutations in the gyrA gene, the molecular target of these agents (16,36). However, little is known about another possible resistance mechanism, namely, elimination of antibiotics through an active efflux system. This mechanism was investigated by stepwise in vitro selection of C. jejuni mutants resistant to the quinolone pefloxacin or to the ␤-lactam cefotaxime. Study of the cross-resistance of the generated mutants provided evidence for an active antibiotic efflux system in C. jejuni. MATERIALS AND METHODSBacterial strains and selection of resistant mutants. The pefloxacin-susceptible strain 34 s was isolated from a stool specimen...

A total of 27 strains of Campylobacter jejuni (24 clinical strains and three laboratory strains) were examined for the presence of point mutations in the quinolone resistance determining region (QRDR) of gyrA gene by nonradioisotopic single‐strand conformation polymorphism (non‐RI SSCP) analysis with silver stain. Direct DNA sequencing of the polymerase chain reaction (PCR)‐amplified DNA fragments confirmed the results obtained by non‐RI SSCP analysis and revealed that in clinical strains high‐level quinolone resistance [minimal inhibitory concentration (MIC) to ciprofloxacin ≥ 16 μg/ml] was closely associated with one type of single‐point mutation at codon 86 (Thr‐lle). Two strains with MICs of 8 and 1 μg/ml showed point mutations at codons 86 and 70, respectively. Furthermore, transitions at codon 119 of the gyrA QRDR were identified in 17 strains. Six types of bands were separated in a single electrophoretic step with silver stain within 2 hours after PCR amplification of the gyrA QRDR as follows: type I associated to mutation at codon 70 (Ala‐Thr), type II to mutation at codon 90 (Asp‐Asn), type III to variant with transition at 119, type IV to wild‐type, type V to mutation at codon 86 (Thr‐lle), and type VI to mutation at codon 86 (Thr‐lle) and transition at codon 119. Using four DNA extracts from Cambylobacter coli organisms as templates for amplification of the gyrA QRDR, no PCR products were obtained. Non‐RI SSCP was proved to be a simple, rapid, and useful screening method for detecting gyrA mutations associated with ciprofloxacin resistance in C. jejuni. © 1996 Wiley‐Liss, Inc.

The ThinPrep cervical smear is widely used in clinical practice for the cytological and molecular screening against abnormal cells and Human Papillomavirus (HPV) infection. Current advancements made to LC-MS proteomics include the use of stable isotope labeling for the in-depth analysis of proteins in complex clinical specimens. Such approaches have yet to be realized for ThinPrep clinical specimens. In this study, an LC-MS method based on isobaric (iTRAQ) labeling and high-resolution FT-Orbitrap mass spectrometry was used for the proteomic analysis of 23 human ThinPrep smear specimens. Tandem mass spectrometry analysis was performed with both nitrogen high collision dissociation (HCD MS/MS) and helium collision induced dissociation (CID MS/MS) peptide fragmentation modes. The analysis of three 8-plex sample sets yielded the identification of over 3200 unique proteins at FDR < 1%, of which over 2300 proteins were quantitatively profiled in at least one of the three experiments. The interindividual variability served to define the required sample size needed to identify significant protein expression differences. The degree of in-depth proteome coverage allowed the detection of 6 HPV-derived proteins including the high-risk HPV16 type in the specimens tested. The presence of the HPV strains of origin was also confirmed with PCR-hybridization molecular methods. This proof-of-principle study constitutes the first ever report on the nontargeted analysis of HPV proteins in human ThinPrep clinical specimens with high-resolution mass spectrometry. A further testament to the sensitivity and selectivity of the proposed study method was the confident detection of a significant number of phosphopeptides in these specimens.