Centre de recherche en Infectiologie du Centre de recherche du CHUL and Département de Microbiologie, Infectiologie et Immunologie, Faculté de Médecine, Université Laval, Québec, QC, Canada.
Antimicrob Agents Chemother. 2013 Oct;57(10):4911-9. doi: 10.1128/AAC.00418-13. Epub 2013 Jul 22.
The broad-spectrum fluoroquinolone ciprofloxacin is a bactericidal antibiotic targeting DNA topoisomerase IV and DNA gyrase encoded by the parC and gyrA genes. Resistance to ciprofloxacin in Streptococcus pneumoniae mainly occurs through the acquisition of mutations in the quinolone resistance-determining region (QRDR) of the ParC and GyrA targets. A role in low-level ciprofloxacin resistance has also been attributed to efflux systems. To look into ciprofloxacin resistance at a genome-wide scale and to discover additional mutations implicated in resistance, we performed whole-genome sequencing of an S. pneumoniae isolate selected for resistance to ciprofloxacin in vitro (128 μg/ml) and of a clinical isolate displaying low-level ciprofloxacin resistance (2 μg/ml). Gene disruption and DNA transformation experiments with PCR fragments harboring the mutations identified in the in vitro S. pneumoniae mutant revealed that resistance is mainly due to QRDR mutations in parC and gyrA and to the overexpression of the ABC transporters PatA and PatB. In contrast, no QRDR mutations were identified in the genome of the S. pneumoniae clinical isolate with low-level resistance to ciprofloxacin. Assays performed in the presence of the efflux pump inhibitor reserpine suggested that resistance is likely mediated by efflux. Interestingly, the genome sequence of this clinical isolate also revealed mutations in the coding region of patA and patB that we implicated in resistance. Finally, a mutation in the NAD(P)H-dependent glycerol-3-phosphate dehydrogenase identified in the S. pneumoniae clinical strain was shown to protect against ciprofloxacin-mediated reactive oxygen species.
广谱氟喹诺酮类药物环丙沙星是一种针对 DNA 拓扑异构酶 IV 和 DNA 回旋酶的杀菌抗生素，由 parC 和 gyrA 基因编码。肺炎链球菌对环丙沙星的耐药性主要通过喹诺酮类药物耐药决定区（QRDR）中 parC 和 GyrA 靶标突变的获得而发生。外排系统也被认为在低水平环丙沙星耐药中起作用。为了在全基因组范围内研究环丙沙星耐药性并发现其他与耐药性相关的突变，我们对体外选择的耐环丙沙星（128μg/ml）的肺炎链球菌分离株和显示低水平环丙沙星耐药性（2μg/ml）的临床分离株进行了全基因组测序。用包含在体外肺炎链球菌突变体中鉴定出的突变的 PCR 片段进行基因敲除和 DNA 转化实验表明，耐药性主要是由于 parC 和 gyrA 的 QRDR 突变以及 ABC 转运蛋白 PatA 和 PatB 的过度表达所致。相比之下，在对环丙沙星具有低水平耐药性的肺炎链球菌临床分离株的基因组中未发现 QRDR 突变。在流出泵抑制剂利血平存在的情况下进行的测定表明，耐药性可能是通过流出介导的。有趣的是，该临床分离株的基因组序列还揭示了编码 patA 和 patB 的突变，我们认为这些突变与耐药性有关。最后，在肺炎链球菌临床菌株中发现的 NAD(P)H 依赖性甘油-3-磷酸脱氢酶突变被证明可防止环丙沙星介导的活性氧。
