Background:Although qnrA, encoding quinolone resistance protein, confers low-level resistance to fluoroquinolone, its role in quinolone resistance when associated with other resistant mechanisms remains unknown.Methods: One hundred extended-spectrum beta-lactamase (ESBL) producing Escherichia coli were collected from Siriraj Hospital (Bangkok, Thailand) and tested for qnrA by PCR. All qnrA positive isolates were investigated for gyrA mutations and a presence of class 1 integron by DNA sequencing and PCR. The locations of qnrA and intI1 genes were analyzed by Southern blot hybridization. Phenotypic characteristics were studied by antimicrobial susceptibility test and time kill method.Results: qnrA genes were found in 8% of ESBL-positive E. coli (8/100), confirmed to be qnrA1 by DNA sequencing. All qnrA1 positive isolates also harbored intI1 gene. Double mutations (S83L and D87N) in gyrA were found in 50% (4/8) of qnrA1 positive isolates. Specific qnrA1 and intI1 probes showed both qnrA1 and intI1 genes could be found on the chromosome and/or plasmids. Some isolates possessed two integron elements. The MIC (ciprofloxacin) against the isolate harboring both qnrA1 and gyrA with double mutations was 2-fold higher than that against the isolate with only gyrA, and was much higher than that against the isolate harboring only qnrA1 (MICs of 64 g/ml, 32 g/ml, and 0.12 g/ml, respectively). According to the time kill study, 0.5 g/ml of ciprofloxacin showed bactericidal activity after six hours of incubation against the isolate harboring only qnrA1. The bacteriostatic activity against the isolate harboring only gyrA with double mutations could be observed when the concentrations of ciprofloxacin were ≤256 g/ml. The regrowth of the isolate carried both genes in 64-128 g/ml at 24 hours of incubation were observed. Only 512 g/ml of ciprofloxacin showed bactericidal activity after four hours of incubation against the isolate harboring both qnrA1 and gyrA with double mutations.Conclusion: qnrA could integrate into the chromosome by class 1 integrons. Presence of fluoroquinolone resistance elements on both plasmid and chromosome indicated high selection pressure in E. coli and, may be, in other Gram-negative bacteria as well. The qnrA gene conferred higher-level ciprofloxacin resistance on double mutations in gyrA (S83L and D87N) background. http://dx.
intermediate strains of MRSA. Suscpetibility pattern has also been studied to formulate an effective, inexpensive and easily administered empirical therapy for GISA and hGISA. Methods: Three hundred and Forty seven MRSA isolated from the clinical specimens of Allied hospitals of AMC/NUST and PAEC General Hospital, Pakistan were subjected to the determination of Vancomycin minimum inhibitory concentration (MIC) and isolates having vancomycin MIC ≥ 1g/ml were subjected to determination of Glycopeptide resistance detection (GRD) using Etest. Susceptibility pattern of all the isolates were recorded using Kirby baur disc diffusion method and MIC for linezolid, daptomycin, chloramphenicol, minocycline and tigecycline using E-strips. MIC 50 and MIC 90 were calculated Results: All isolates were sensitive to vancomycin but 197 isolates showed higher MICs and 6 turned out to be heterogenous Glycopeptide intermediate (h GISA) strains. All the isolated organisms were highly susceptible to linezolid (98.3%), daptomycin (100%), chloramphenicol (96%), minocycline (95.7%) and tigecycline (94.8%). Conclusion: Significant number of isolates having MIC of vancomycin equal to or more than 1 ug/ml have been isolated and these can turn out to be GISA/h GISA. Vancomycin Susceptibility breakpoints as indicated by CLSI should be reevaluated because that doesn't cover GISA/h GISA isolates. This study suggests that linezolid, chloramphenicol, minocycline, daptomycin and tigecycline have high in vitro efficacy for MRSA infections. Prescribing antibiotics other than glycopeptides for MRSA infections will minimize the chances of emergence of GISA. Good hospital infection control measures prove to be the main stay against these infections because antibiotics can never be an effective alternative to good medical practice.
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