碳青霉烯类耐药雷氏普罗威登斯菌的出现及其耐药机制研究

Emergence of carbopenem resistant Providencia rettgeri and its resistance mechanisms

目的报告1株临床分离产NDM-1酶雷氏普罗威登斯菌FR90,研究其耐药机制。方法对菌株进行药物敏感实验,改良Hodge实验检测金属β内酰胺酶,PCR扩增碳青霉烯水解酶基因。采用质粒接合实验、质粒不相容群检测、S1酶切脉冲场电泳和bla NDM-1杂交、基因测序等分析携bla NDM-1质粒特征和基因环境。结果菌株FR90对氟喹诺酮类、多粘菌素B、β内酰胺类(包括碳青霉烯类)抗生素耐药,对氨曲南敏感。改良Hodge实验阳性,PCR检测几种常见碳青霉烯水解酶基因,结果仅bla NDM-1基因阳性。接合实验显示bla NDM-1基因位于一可接合性质粒上,质粒不相容群分型属于A/C型,Southern杂交结果显示质粒大小约为50 kb。对质粒上大小约为17 kb基因片段的测序结果示,ISAba125位于bla NDM-1基因两侧,下游依次为trpF、groS、groL基因和insE基因的一部分。结论中国大陆出现临床分离产NDM-1酶雷氏普罗威登斯菌,警示我国需对产NDM-1细菌进行严密监测,并采取感染控制措施以阻断耐药基因在细菌之间传播。

Objective To report a clinical isolated strain of Providencia rettgeri producing New Delhi metallo-β-1actamase （NDM-1）, FR90, and study its resistance mechanisms. Methods The bacterial strain was progressed with the antimicrobial susceptibility test. Production of metallo-β-lactamase was identified by modified Hodge test. PCR was used to detect several common resistance genes for carbopenemases. Plasmids conjugation, plasmid incompatibility group detection, Pulsed-field gel electrophoresis （PFGE） with S1 nuclease digestion of whole genomic DNA （S1-PFGE）, Southern blotting and gene sequencing were conducted to analyze the plasmid characteristics and the surrounding genetic structure of the blaNDM_1 gene. Results Providencia rettgeri FR90 was resistant to quinolone, Polymyxin B, and all β-lactas （including carbapenems ） except aztreonam. Modified Hodge test showed it was positive to metallo-β-lactamase. PCR displayed that only the blaNDM_1 gene was detected among several common resistance genes for carbopenemases. Further study revealed this blaNDM_1 was located on a broad host-range IncA/C-type conjugative plasmid FR90 in a size of 50 kb, confirmed by S1-PFGE and Southern blotting. A 17-kb fragment from the plasmid was obtained and sequenced, and the blaNDM_1gene was flanked by 2 copies of ISAba125, with a downstream gente of trpF, followed by groS, groL and a truncated insE gene. Conclusion We report a clinical isolation of NDM-1-producing Providencia rettgeri in the mainland of China, which reminds us to closely monitor the NDM-l-producing bacteria and take infection control measures to prevent the blaNDM-1 gene spreading among bacteria.