临床分离气单胞菌喹诺酮类的耐药机制研究

Mechanism of resistance to quinolones in Aeromonas species isolated from clinical patients

目的探讨临床分离气单胞菌属细菌(Aeromonas spp.)喹诺酮类药物耐药机制。方法收集从北京友谊医院2017年腹泻患者粪便标本中分离的气单胞菌,采用微量肉汤稀释法检测其对喹诺酮类药物的敏感性,提取菌株核酸进行PCR扩增,测序分析气单胞菌喹诺酮耐药决定区(quinolone-resistance-deter mining region,QRDR)及喹诺酮耐药质粒相关(plasmid-mediated quinolone resistance,PMQR)基因。结果共收集111株气单胞菌,其中7株(6.3%)对萘啶酸(NAL)和环丙沙星(CIP)均耐药,54株(48.6%)对NAL耐药但对CIP敏感,50株(45.0%)对NAL和CIP均敏感。QRDR测序结果显示,NAL耐药菌株gyrA基因第83位发生丝氨酸(Ser)突变,突变为异亮氨酸(Ile)、缬氨酸(Val)或酪氨酸(Tyr)。对NAL-CIP耐药的菌株中,除了gyrA基因突变外,parC基因第87位Ser突变为Ile。CIP敏感的104株菌株中,仅有7株parC基因第87位Ser突变。PMQR基因检测结果显示,QnrS基因检出率为7.2%(8株),aac(6′)-Ib-cr基因检出率为6.3%(7株),且QnrS和aac(6′)-Ib-cr基因检出主要集中在CIP耐药菌株中,CIP耐药菌株PMQR基因检出率为85.7%。结论气单胞菌喹诺酮低水平耐药主要与gyrA基因第第83位突变或QnrS基因有关。gyrA基因第83位突变和parC基因第87位突变Ser87Ile联合PMQR基因存在时,可能与气单胞菌对喹诺酮的高水平耐药相关。

Objective To investigate the mechanism of quinolone resistanceof Aeromonas spp.isolated from clinical patients.Methods The Aeromonas strains isolated from feces specimen of diarrheal outpatients in Beijing Friendship Hospital during 2017 were subjected to the susceptibility test to quinolones by microbroth dilution method.PCR were performed to extract the nucleic acids of the strains.The genes of quinolone-resistance-determining region(QRDR)of Aeromonas species and plasmid-mediated quinolone resistance(PMQR)on chromosome were analyzed by sequencing.Results Of the 111 collected strains,7(6.3%)strains were resistant to ciprofloxacin(CIP)-nalidixic acid(NAL),54(48.6%)were resistant to NAL,but sensitive to CIP,and 50(45.0%)were sensitive to both CIP-NAL.The sequence of QRDR showed that Ser83Ile/Val/Tyr mutation on gyrA was screened in NAL-resistant strains.In addition to mutation of gyrA,Ser87Ile mutation of gene parC was identified in all NAL-CIP-resistant strains.In the 104 CIP-sensitive strains,Ser87Ile mutation of parC was only found in 7 strains.The results of PMQR gene sequencing for these strains showed that detectable rate of gene QnrS was 7.2%(8 strains),aac(6′)-Ib-cr was 6.3%(7 strains).Most of QnrS and aac(6′)-Ib-cr were found in CIP-resistant strains.PMQR-positive strains constituted 85.7%of the CIP-resistant strains.Conclusion The Ser83 mutation of gene gyrA or QnrS may relate with low levels of quinolone resistance of these strains.The gene PMQR coupled with Ser83Ile/Val/Tyr mutation in gyrA and Ser87Ile mutation in parC may give rise to high level of quinoloneresistance for Aeromonas app.