Kim Soo-Young, Lee Si-Kyung, Park Myeong-Soo, Na Hun-Taek
Department of Biological Sciences, Konkuk University, Seoul 05029, Republic of Korea.
Department of Biosources and Food Science, Konkuk University, Seoul 05029, Republic of Korea.
J Microbiol Biotechnol. 2016 Sep 28;26(9):1605-12. doi: 10.4014/jmb.1602.02063.
Quinolone-resistant Salmonella strains were isolated from patient samples, and several quinolone-sensitive strains were used to analyze mutations in the quinolone resistance-determining region (QRDR) of gyrA, gyrB, parC, and parE and to screen for plasmid-mediated quinolone resistance. Among the 21 strains that showed resistance to nalidixic acid and ciprofloxacin (MIC 0.125-2.0 μg/ml), 17 strains had a mutation in QRDR codon 87 of gyrA, and 3 strains had a single mutation (Ser83 → Phe). Another cause of resistance, efflux pump regulation, was studied by examining the expression of acrB, ramA, marA, and soxS. Five strains, including Sal-KH1 and Sal-KH2, showed no increase in relative expression in an analysis using the qRT-PCR method (p < 0.05). In order to determine the genes involved in the resistance, the Sal-9 isolate that showed decreased susceptibility and did not contain a mutation in the gyrA QRDR was used to make the STM (MIC 8 μg/ml) and STH (MIC 16 μg/ml) ciprofloxacin-resistant mutants. The gyrA QRDR Asp87 → Gly mutation was identified in both the STM and STH mutants by mutation analysis. qRT-PCR analysis of the efflux transporter acrB of the AcrAB-TolC efflux system showed increased expression levels in both the STM (1.79-fold) and STH (2.0-fold) mutants. In addition, the expression of the transcriptional regulator marA was increased in both the STM (6.35-fold) and STH (21.73-fold) mutants. Moreover, the expression of soxS was increased in the STM (3.41-fold) and STH (10.05-fold) mutants (p < 0.05). Therefore, these results indicate that AcrAB-TolC efflux pump activity and the target site mutation in gyrA are involved in quinolone resistance.
从患者样本中分离出喹诺酮耐药性沙门氏菌菌株，并使用几种喹诺酮敏感菌株分析gyrA、gyrB、parC和parE的喹诺酮耐药决定区（QRDR）中的突变，并筛选质粒介导的喹诺酮耐药性。在对萘啶酸和环丙沙星耐药（MIC为0.125 - 2.0μg/ml）的21株菌株中，17株在gyrA的QRDR密码子87处发生突变，3株发生单一突变（Ser83→Phe）。通过检测acrB、ramA、marA和soxS的表达来研究另一种耐药原因——外排泵调节。包括Sal-KH1和Sal-KH2在内的5株菌株在使用qRT-PCR方法进行的分析中相对表达未增加（p<0.05）。为了确定与耐药性相关的基因，使用对环丙沙星敏感性降低且gyrA的QRDR中未发生突变的Sal-9分离株制备环丙沙星耐药突变体STM（MIC 8μg/ml）和STH（MIC 16μg/ml）。通过突变分析在STM和STH突变体中均鉴定出gyrA的QRDR Asp87→Gly突变。对AcrAB-TolC外排系统的外排转运蛋白acrB进行qRT-PCR分析显示，STM（1.79倍）和STH（2.0倍）突变体中的表达水平均增加。此外，转录调节因子marA的表达在STM（6.35倍）和STH（21.73倍）突变体中均增加。此外，soxS的表达在STM（3.41倍）和STH（10.05倍）突变体中增加（p<0.05）。因此，这些结果表明AcrAB-TolC外排泵活性和gyrA中的靶位点突变与喹诺酮耐药性有关。
