耐环丙沙星鲍氏不动杆菌中喹诺酮耐药相关基因分析

Screening of quinolone resistance determinants in ciprofloxacin-resistant Acinetobacter baumannii isolates

目的研究鲍氏不动杆菌(ABA)临床分离株对喹诺酮类抗生素耐药机制。方法收集来自6家医院的114株耐环丙沙星ABA临床分离菌株,PCR扩增喹诺酮类抗生素结合靶基因(gyrA、gyrB、parC和parE),并进行序列测定,与参考菌株ATCC17978基因组进行比对,确定其喹诺酮耐药决定区是否存在耐药相关突变;PCR扩增9个质粒介导的喹诺酮类耐药基因[qnrA、qnrB、qnrC、qnrD、qnrS、qepA、aac(6')-Ⅰb-cr、oqxA和oqxB],并对阳性扩增产物进行测序以确定基因亚型。结果绝大部分菌株(113/114,99.1%)的gyrA基因发生了Ser83Leu突变,其中67株菌(67/114,58.8%)同时发生了parC基因的Ser80Leu突变,以上两种突变是常见的喹诺酮耐药相关突变。与标准菌株进行比对,受试菌株gyrB基因Arg393Ser、Arg393Cys、Thr401Ala、Pro406Ser、Val430Phe、Cys440Ser和Gly480Arg突变率分别为95.6%、0.9%、96.5%、96.5%、100%、96.5%和96.5%;parE基因在7个位点发生了同义突变,突变率>96%。83.3%(95/114)的菌株中检出aac(6')-Ⅰb基因,但不存在"cr"突变,其余质粒介导的喹诺酮类耐药基因扩增均为阴性。结论该研究耐环丙沙星ABA中未发现质粒介导的喹诺酮耐药基因,在gyrB和parE基因中发现的突变可能与环丙沙星耐药性无关,实验菌株的环丙沙星耐药性主要与染色体上的喹诺酮耐药决定区GyrASer83Leu和ParC-Ser80Leu两种突变相关。

Objective To investigate the quinolone resistance determinants in ciprofloxacin-resistant Acinetobacter baumannii（ABA） clinical isolates.Methods One hundred and fourteen ciprofloxacin-resistant ABA strains were collected from six Chinese hospitals.The quinolone resistance determining region（QRDR） of 4 target genes（gyrA,gyrB,parC and parE） was amplified,sequenced and compared with the reference genome of ATCC17978 to identify possible resistancerelated mutations.Nine plasmid-mediated quinolone resistance（PMQR） genes（qnrA,qnrB,qnrC,qnrD,qnrS,qepA,aac（6＇）-Ⅰ b-cr,oqxA and oqxB） were also amplified,and the amplicons were then sequenced to determine their characteristics.Results Almost all isolates（113/114,99.1%） harbored a substitution in codon 83 of gyrA gene,leading to a Ser83 Leu mutation.Meanwhile,58.8%（67/114） of the isolates possessed dual mutations of GyrA-Ser83 Leu and GyrASer80 Leu,which were known determinants for ciprofloxacin resistance.There were also multiple non-synonymous substitutions in gyrB,leading to Arg393 Ser,Arg393Cys,Thr401 Ala,Pro406Ser,Val430 Phe,Cys440Ser and Gly480 Arg mutations with prevalence rates of 95.6%,0.9%,96.5%,96.5%,100%,96.5% and 96.5%,respectively.For parE,all the seven mutations were synonymous and found in more than 96% of the tested isolates.For PMQR genes,although 83.3%（95/114） of the isolates were positive for aac（6＇）-Ⅰ b,no ＂cr＂ mutations were identified.None of the other eight PMDR genes were found in our strain collection.Conclusion Although multiple mutations are identified in gyrB and parE,these mutations might be the characteristic SNP markers for specific clones,unlikely linked to quinolone resistance.No PMQR is found in the tested isolates.Mutations in chromosomal QRDR（GyrA-Ser83 Leu and ParC-Ser80Leu） are the main determinants of ciprofloxacin resistance in our ABA collection.