| Objective:To investigate the resistance of Shigella isolates against 16 antimicrobial agents in Anhui province and offer the resistance data to clinical therapy.To investigate the production of extended-spectrumβ-lactamases (ESBLs) and plasmid-mediated AmpCβ-lactamases in the clinical Shigella isolates in Anhui province.To investigate the correlation between the alterations of DNA gyrase gyrA and TopoisomeraseⅣparC genes and fluoroquinolone resistance in Shigella isolates.To detect the distribution of the energy-dependent multidrug efflux pump gene acrAB-tolC and expression level of acrAB gene in Shigella isolates.Materials and Methods:Isolates:From September 1 to September 30, 2005, a total of 91 Shigella isolates were collected in 31 hospitals in Anhui of China.Method:The MICs of the following agents were determined by the agar dilution method recommended by CLSI (Clinical Laboratory Standard Institute), 2005. Total DNA was extracted by suspending a few overnight colonies in 0.5 mL of double-distilled water and heating the mixture at 100°C for 10 min. Plasmid DNA was extracted and purified by Qiagen plasmid Mini Kit. The extracted DNA was subjected to amplification by PCR for the quinolone resistance-determining regions of gyrA, parC on 26 Shigella isolates (19strains showing ciprofloxacin≥4 ug/mL, and 7 strains selected showing ciprofloxacin < 4 ug/mL), the multidrug efflux pump acrAB-tolC genes on 91 strains were also identified by PCR. PCR analysis was also used to investigate the distribution ofβ-lactamases and Qnr present in Shigella strain. Reverse transcription PCR (RT-PCR) of acrAB genes were used to confirm the presence of acrAB.Results:There were 57 strains of S. flexnery, 31 strains S. sonnei and 2 strains S. boydii among 91 strains of Shigella isolates. The resistance rates of Shigella isolates to cefoperazone-sulbactam and piperacillin-tazobactam were remarkably lower than other third generation cephalosporins. The susceptible rates to carbopenems were 100%. The resistance rates to ciprofloxacin and pazufloxacin were 27.47% and 32.97%, respectively. 14 of 91 isolates were susceptible to all tested antimicrobial agents in this study. In this study we did not found the qnr genes.β-lactamases-producing Shigella isolates were 63.74% (58/91), the genes of blaTEM-1, blaCTX-M-1, blaCTX-M-13, blaOXA-1 group and ampC were detected in 62.07%(36/91), 17.24%(10/91), 24.14%(14/91), 36.21%(21/91) and 10.24% (6/91) among 91 isolates. 20 strains had a mutation at codon 83 of gyrA, 7 strains had a mutation at codon 87 of gyrA. 9 strains had a mutation at codon 80 (Ser80→Ile) of parC. Three of the ciprofloxacin-resistance isolates had a novel mutation at codon 64 (Ala64→Cys, Ala64→Asp) of parC, the mutation at codon 57 (Ser57→Thr) of parC and the mutations at codon 83 of gyrA (Ser→Ile) were firstly reported in Shigella isolates. The partial sequences of gyrA and parC genes reported in this article have been deposited in the GenBank database and assigned accession no. DQ898178, DQ898179, DQ898180, DQ898305. The strains with both gyrA and parC mutations were two to eight times more resistant than the strains which had only gyrA mutations. Isolates with a single gyrA mutation were less resistant to fluoroquinolones than those with an additional parC mutation, the novel mutation at codon 64 plus one other gyrA mutation conferred higher level resistance to fluoroquinolones than the mutation at codon 57 or 80 of parC together with one other gyrA mutation. The levels of mRNA expression of acrAB in multidrug-resistant strains were significantly higher than those in wild susceptible strains.Conclusion:There was a certain resistance rate of the Shigella isolates to fluoroquinolones and the third generation cephalosporins. More attention should be paid to the surveillance and control of such resistance. The prevalence ofβ-lactamases among clinical Shigella isolates was high, theβ-lactamase-genes possibly were the TEM-1, OXA-1 and CTX-M types in Shigella isolates in our locality.The mutations of gyrA at codon 83 and 87 were related to fluroquinolone resistance,and they woμLd make higher resistance together with additional parC mutation. The partial sequences of gyrA and parC genes reported in this article have been deposited in the GenBank database and assigned accession no. DQ898178, DQ898179, DQ898180, DQ898305.Overexpression of the acrAB genes was responsible for resistance to fluoroquinolones in Shigella isolates.
|
PDF Full Text Request