全基因测序法分析肺炎克雷伯菌JM45株对喹诺酮类药物耐药基因

Analysis of quinolone-resistance genes in Klebsiella pneumoniae JM45 by whole genome sequencing

目的分析泛耐药肺炎克雷伯菌JM45株携带的喹诺酮类药物耐药基因,研究其对喹诺酮类药物的耐药机制。方法采用Roche454高通量测序技术对肺炎克雷伯菌JM45株做全基因组测序(完成图),分析喹诺酮类耐药基因携带状况,再将gyrA与parC全长基因与其他6株肺炎克雷伯菌做分子进化分析。结果最终得到JM45株一条完整的基因组(染色体)序列及两条质粒序列;基因组(染色体)序列大小为5 273 812bp(GC含量65.8%),质粒1序列大小为317 156bp(GC含量53.0%),质粒2序列大小为12 209bp(GC含量55.3%);在基因组(染色体)序列中携带了gyrA基因(全基因组Feature ID:KPN_1614),parC基因(Feature ID:KPN_0743);与肺炎克雷伯菌喹诺酮类药物敏感株相比较,gyrA基因第83位密码子由TCC→ATC,翻译成氨基酸序列后丝氨酸(Ser)→异亮氨酸(Ile),parC基因第80位密码子由AGC→ATC,翻译成氨基酸序列后丝氨酸(Ser)→异亮氨酸(Ile)。结论肺炎克雷伯菌JM45株喹诺酮类药物耐药是gyrA基因和parC基因喹诺酮耐药决定区(QRDR)突变所致;gyrA与parC全长基因的分子进化分析提示JM45株与肺炎克雷伯菌HS11286关系最为接近(序列相同),与肺炎克雷伯菌342关系最远。

OBJECTIVE To investigate the quinolone-resistance genes in pandrug-resistant Klebsiella pneumoniae JM45 and study the drug resistance mechanisms of the strain to the quinolones antibiotics. METHODS The whole genome sequencing （ completed graph）was performed by using high throughput Roche 454 sequencing approach, then the quinolone-resistance genes were analyzed, and the molecular evolutionary analysis was performed with full-length gyrA and parC between JM45 and other 6 K. pneumoniae isolates. RESULTS A complete gerlome （chromosome） sequence and 2 plasmids sequences were obtained in JM45. The size of chromosome was 5 273 812 bp （GC content： 65.8%）, the size of plasmid 1 was 317 156 bp （GC content.. 53.0%）, and the size of plasmid 2 was 12 209 bp （ GC content ： 55.3 % ）. gyrA （ Feature ID.. KPN_ 1614） and parC （ Feature ID： KPN_0743） were positive in chromosome. As compared with the K. pneumoniae strains which were sensitive to quinolor^es, the 83 rd codon of gyrA in JM45 changed from TCC to ATC（Ser-~Ile）, and the 80th codon of parC in JM45 changed from AGC to ATC（Ser--~Ile）. CONCLUSION Mutations of gyrA and parC in quinolone resist- anee-determining region （QRDR） play a key role in quinolone resistance of JM45. And the moIecular evolutionary analysis of full-length gyrA and parC suggests that the closest relationship exists between JM45 and K. pneumon- iae subsp, pneumoniae HSl1286 （the same sequence）, and that the farthest relationship exists between JM45 and K. pneumoniae 3 4 2.