水环境分离细菌及临床分离弗劳地柠檬酸杆菌中喹诺酮耐药基因qnr及aac（6’）-Ib-cr的检测

Prevalence of qnr and aac（ 6＇）- I b-cr genes in water-borne environmental bacteria and clinical isolates of Citrobacter freundii in China

目的调查水环境分离细菌及临床分离弗劳地柠檬酸杆菌中喹诺酮耐药基因qnr和aac（6’）-Ib-cr的流行情况及qnr基因型分布。方法从杭州10个不同的水域中分离细菌，从4个城市的多家医院收集弗劳地柠檬酸杆菌临床菌株。琼脂稀释法测定环丙沙星、左氧氟沙星和萘啶酸对细菌的最低抑菌浓度（MIC）。PCR方法检测细菌中qnrA、qnrB、qnrS及aac（6’）-Ib-cr基因。对PCR产物进行测序及序列分析，确定各基因型别。结果从水环境分离到78株革兰阴性杆菌（包括33株肠杆菌科细菌、21株气单胞菌属、10株不动杆菌属、10株假单胞菌属、2株产碱杆菌属和2株邻单胞菌属细菌）。10株弗劳地柠檬酸杆菌中有8株qnrB基因阳性；9株大肠埃希菌中qnrS1和aac（6’）-Ib-cr基因阳性各1株；1株斑点气单胞菌（Aeromonaspunctata）qnrS2阳性。临床分离的103株弗劳地柠檬酸杆菌中，qnr基因检出75株（72．8％），其中qnrA1有3株（2．9％），qnrB有65株（63．1％），qnrS2有1株（1．0％），qnrA1合并qnrB阳性5株（4．8％），qnrS1合并qnrB阳性1株；aac（6’）-Ib检出33株（32．0％），其中12株（11．6％）存在-cr变异体。qnrB的基因型以qnrB9、qnrB8和qnrB6为主。结论首次在欧洲以外地区分离到携带qnrS2基因的气单胞菌属细菌。水环境及临床分离的弗劳地柠檬酸杆菌中qnrB基因的携带率非常高，后者同时有较高的aac（6’）-Ib-cr携带率。弗劳地柠檬酸杆菌中qnrB基因的亚型以qnrB9、qnrB8和qnrB6最为常见。

Objective To＇investigate the prevalence of qnr and aac（6＇）- I b-cr genes in waterborne environmental bacteria and clinical isolates of Citrobacter freundii, and the subtypes of qnr gene. Methods Environmental bacteria were isolated from surface water samples obtained from 10 distinct locations in Hangzhou city, and clinical isolates of C. freundii were isolated from several hospitals of 4 cities in China. Minimum inhibitory concentrations （MICs） of ciprofloxacin, levofloxacin and nalidixic acid were determined by agar dilution method, qnrA, qnrB, qnrS and aac （6＇）- I b-cr genes were screened by PCR, and the genotypes were analyzed by DNA sequencing. Results Seventy-eight gram negative bacilli （ including 33 Enterobacteriaceae, 21 Aeromonas spp. , 10 Acinetobacter spp. , 10 Pseudomonas spp. , 2 Alcaligenes spp. , and 2 Plesiomonas spp. ） were isolated from water samples. Among these isolates, 8 of 10 C. freundii were positive for qnrB gene. qnrS1 and aac（6＇）- I b-cr were detected in two distinct Escherichia coli, and qnrS2 was detected in a Aeromonas punctata qnr and aac（6＇）- I b-cr genes were present in 75 （72.8%） and 12 （11.6%） of 103 clinical isolates of C. freundii, respectively. Three （2.9%） C. freundii isolates were positive for qnrA1 gene, 65 （63.1%） qnrB, 1 （1.0%） qnrS2, 5 （4.8%） were positive for both qnrA1 and qnrB, and 1 was positive for both qnrS1 and qnrB. Within the subtypes of qnrB gene, qnrB9 predominated, followed by qnrB8 and qnrB6. Conclusion It was the first isolate of Aeromonas spp. harboring qnrS2 gene outside Europe. The prevalence of qnrB in water-borne environmental and clinical isolates of C. freundii was particularly high and qnrB9, qnrB8 and qnrB6 were the most common subtypes, aac（6＇）- I b-cr gene widely spread in clinical isolates of C. freundii.