嗜水气单胞菌对喹诺酮类药物耐药的分子机制

摘要:【目的】调查从浙、苏、皖等地水产动物中分离的23 株致病性嗜水气单胞菌的耐药谱并探索喹诺酮类抗生素耐药菌株的耐药分子机制。【方法】根据美国临床实验室标准化协会药敏判断标准(2011 版)测定23株嗜水气单胞菌耐药谱并筛选喹诺酮类抗生素耐药株;对筛选的耐药菌株和体外诱导耐药菌株的gyrA和parC基因耐药决定区进行分析;用二倍稀释法测定加入多重耐药外排泵抑制剂碳酰氰基-对-氯苯腙前后耐药菌株对恩诺沙星最小抑菌浓度的变化,同时检测喹诺酮类药物相关的外排泵基因qepA、oqxA和mdfA;并检测质粒介导的喹诺酮耐药的qnr家族基因qnrA、qnrB、qnrC、qnrD和qnrS。【结果】所有菌株都存在对5种以上药物的耐药性;39.1%(9/23)的嗜水气单胞菌对喹诺酮类药物耐药,其中55.6%(5/9)对恩诺沙星耐药。耐恩诺沙星的5株菌株均携带qnrS,而不携带qnrA、qnrB、qnrC、qnrD基因,也不携带外排泵基因qepA、oqxA和mdfA。其中耐药菌株AH19同时存在gyrA与parC基因双突变、质粒介导的耐药基因qnrS和主动外排泵三种耐药机制,菌株AH4、AH7和AH20存在gyrA与parC基因双突变和质粒介导的耐药基因qnrS两种耐药机制,AH6存在gyrA基因突变和质粒介导的耐药基因qnrS机制,体外诱导耐药菌株ATCC7966-QR相对于原始菌株ATCC7966发生了gyrA与parC基因双突变。【结论】喹诺酮类药物作用靶位的改变和质粒介导的耐药基因qnrS的存在是本研究中涉及的嗜水气单胞菌对喹诺酮类药物耐药的主要作用机制,主动外排泵机制是个别菌株存在的耐药机制。     

嗜水气单胞菌对喹诺酮类药物耐药的分子机制

【目的】调查从浙、苏、皖等地水产动物中分离的23株致病性嗜水气单胞菌的耐药谱并探索喹诺酮类抗生素耐药菌株的耐药分子机制。【方法】根据美国临床实验室标准化协会药敏判断标准(2011版)测定23株嗜水气单胞菌耐药谱并筛选喹诺酮类抗生素耐药株;对筛选的耐药菌株和体外诱导耐药菌株的gyrA和parC基因耐药决定区进行分析;用二倍稀释法测定加入多重耐药外排泵抑制剂碳酰氰基-对-氯苯腙前后耐药菌株对恩诺沙星最小抑菌浓度的变化,同时检测喹诺酮类药物相关的外排泵基因qepA、oqxA和mdfA;并检测质粒介导的喹诺酮耐药的qnr家族基因qnrA、qnrB、qnrC、qnrD和qnrS。【结果】所有菌株都存在对5种以上药物的耐药性;39.1%(9/23)的嗜水气单胞菌对喹诺酮类药物耐药,其中55.6%(5/9)对恩诺沙星耐药。耐恩诺沙星的5株菌株均携带qnrS,而不携带qnrA、qnrB、qnrC、qnrD基因,也不携带外排泵基因qepA、oqxA和mdfA。其中耐药菌株AH19同时存在gyrA与parC基因双突变、质粒介导的耐药基因qnrS和主动外排泵三种耐药机制,菌株AH4、AH7和AH20存在gyrA与parC基因双突变和质粒介导的耐药基因qnrS两种耐药机制,AH6存在gyrA基因突变和质粒介导的耐药基因qnrS机制,体外诱导耐药菌株ATCC7966-QR相对于原始菌株ATCC7966发生了gyrA与parC基因双突变。【结论】喹诺酮类药物作用靶位的改变和质粒介导的耐药基因qnrS的存在是本研究中涉及的嗜水气单胞菌对喹诺酮类药物耐药的主要作用机制,主动外排泵机制是个别菌株存在的耐药机制。     

多药耐药铜绿假单胞菌氨基糖苷类修饰酶和16S rRNA甲基化酶基因分析

目的了解临床分离的铜绿假单胞菌对氨基糖苷类、β-内酰胺类、喹诺酮类抗菌药物耐药情况、氨基糖苷类耐药相关基因和16S rRNA甲基化酶基因存在情况以及菌株之间的亲缘性。方法采用琼脂稀释法测定临床分离的30株铜绿假单胞菌对7种临床常用于治疗铜绿假单胞菌感染的抗菌药物的敏感性,采用聚合酶链反应分析氨基糖苷类修饰酶、16S rRNA甲基化酶基因型及其他基因型,运用SPSS统计分析软件对菌株样本亲缘性做聚类分析。结果30株铜绿假单胞菌对临床常用抗生素的耐药率分别是奈替米星70%、妥布霉素63.3%、庆大霉素63.3%、环丙沙星53.3%、亚氨培南40%和阿米卡星13.3%,而多黏菌素B的耐药率为0。21株氨基糖苷类耐药菌株中（其中20株为多药耐药菌株）,氨基糖苷类耐药基因型aac（6＇）-Ⅰ阳性13株（61.9%）、aac（6＇）-Ⅱ阳性13株（61.9%）、ant（2＇＇）-Ⅰ阳性10株（47.6%）、ant（3＇＇）-Ⅰ阳性9株（42.9%）、aac（3）-Ⅱ阳性1株（4.8%）,另有1株菌oprD2基因缺失,未检出基因型aac（6＇）-Ⅰae、aph（3＇）-Ⅲ、aac（6＇）-aph（2＇＇）和ant（4＇）-Ⅰ;16S rRNA甲基化酶基因rmtA基因型阳性19株（90.4%）、armA基因型阳性有8株（38.1%）,未检出基因型rmtC、rmtD。聚类分析结果显示分离的菌株中存在克隆传播。结论大部分测试的铜绿假单胞菌对临床常用的铜绿假单胞菌抗感染药物已产生广泛耐药,尤其对氨基糖苷类抗生素。这些菌株的氨基糖苷类修饰酶常见耐药基因型检出率高,16SrRNA甲基化酶基因型rmtA和armA的检出率亦较高。30株测试菌株中存在克隆传播。     

铜绿假单胞菌β-内酰胺类药物耐药相关基因(12种)研究

目的了解临床分离的铜绿假单胞菌β-内酰胺类药物耐药相关基因（12种）存在状况。方法自临床分离40株对铜绿假单胞菌。采用聚合酶链反应（PCR）检测耐药基因（TEM、SHV、OXA-10群、CTX-M-1群、PER、VEB、GES、CARB、IMP、VIM、DHA和oprD2）。结果40株中CARB阳性18株（45．0％）、35株oprD2基因缺失（87．5％）．其余基因均阴性。结论临床分离的铜绿假单胞菌CARB基因携带率高。     

铜绿假单胞菌parC基因突变与耐氟喹诺酮的关系

研究了成都地区临床分离的铜绿假单胞菌拓扑异构酶ⅣparC基因突变与耐氟喹诺酮类药物的关系。测定临床分离的55株铜绿假单胞菌的MIC值,从中筛选出1株敏感菌和8株耐药菌,以标准敏感菌株ATCC27853作为质控菌株。用PCR反应扩增parC基因的喹诺酮耐药决定区(QRDR),扩增产物片段长度为396bp,同时对上述10株菌的PCR产物进行测序分析。临床分离敏感菌和标准菌株ATCC27853的parC基因序列与国外报道的序列相同,而R25,R42,R43,R44等4株耐药菌株在87位(TCGCG→TTG)均有突变,该单位点突变引起氨基酸由Ser→Leu的改变,此外,新发现在所有耐药菌株115位有一静止突变(GCT→GCG),该突变未引起氨基酸的改变。拓扑异构酶ⅣparC基因突变是铜绿假单胞菌对氟喹诺酮类药物产生耐药性的机制之一,以87位的突变最为常见。     

肺炎克雷伯杆菌对氟喹诺酮耐药的机制研究

目的研究肺炎克雷伯杆菌对氟喹诺酮类药物（FQNs）的耐药机制。方法筛选临床分离的对环丙沙星耐药的肺炎克雷伯杆菌共10株,采用微量肉汤稀释法检测菌株对5种氟喹诺酮类药物的MIC值;采用PCR方法检测菌株染色体和质粒携带的喹诺酮耐药基因（gyrA基因、parC基因和qnr基因）并测序;质粒接合试验验证qnr基因的转移性。结果 10株肺炎克雷伯杆菌对5种氟喹诺酮类药物均产生耐药性。扩增产物经测序发现10株肺炎克雷伯杆菌染色体的gyrA基因和parC基因均有突变;有2株菌株（K79和K107）携带qnrA基因,这2株菌的接合菌对喹诺酮抗菌药的MIC值上升了5～30倍;未检测到qnrB阳性的菌株。结论 gyrA和parC基因突变是肺炎克雷菌对氟喹诺酮类产生耐药机制的主要原因,质粒上qnrA基因的存在,也是产生喹诺酮耐药的一个重要因素。     

多重耐药铜绿假单胞菌群体感应系统与主动外排基因表达的研究

目的研究临床多重耐药铜绿假单胞菌群体感应(QS)系统与主动外排泵MexAB-OprM系统基因表达水平与抗生素耐药关系。方法收集苏州市立医院和上海市江湾医院2011年2月至6月间临床标本中分离的铜绿假单胞菌,定量分析细菌生物被膜形成能力;MIC法检测细菌抗生素耐药性,用多重聚合酶链反应(PCR)扩增群体感应系统lasI、lasR及主动外排泵系统mexA基因,实时定量逆转录RT-PCR检测lasI、lasR和mexA基因的相对表达量。结果临床样本分离出84株铜绿假单胞菌,其中产生物被膜菌58株,占比69%;多重耐药菌共24株,占比28.6%;多重耐药菌株中产生物被膜有11株,占45.8%;多重耐药菌中mexA基因表达上调有18株,占75%;lasI基因表达上调有8株,占33.3%。结论多重耐药菌株的生物被膜形成率显著低于非多重耐药组,多重耐药铜绿假单胞菌的主动外排泵MexAB-OprM系统基因表达出现显著上调,生物被膜菌的lasI基因表达显著上调而lasR基因的表达无明显变化。     

对一株铜绿假单胞菌22种耐药基因的研究

目的研究铜绿假单胞菌多重耐药情况和相关机制。方法采用聚合酶链反应（PCR）法对一株多重耐药铜绿假单胞菌进行β-内酰胺酶基因、氨基糖苷类修饰酶（AMEs）基因、喹诺酮类耐药基因、耐消毒剂基因（qacE△1-sul1）和整合酶基因检测,并对VIM基因进行了测序。结果PCR扩增结果显示该菌株aac（6′）-Ⅰb、blaCARB、gyrA、oprD2、ant（2″）-Ⅰ、qacE△1-sul1、blaIMP-I、blaTEM、blaVEB、aac（3）-Ⅱ、ant（3″）-Ⅰ、intⅠ1、blaVIM基因均为阳性,而aac（3）-Ⅰ、aac（6′）-Ⅱ、blaGES、blaGIM、blaOXA-10群、blaPER、blaSPM、blaSHV、blaDHA基因均为阴性,VIM基因扩增产物测序后经BLAST同源性分析表明为VIM-2型。结论铜绿假单胞菌存在多重耐药基因。管壁涂有季胺类、双胍类消毒剂和磺胺的II代导管的抑菌效果需重新评价。     

铜绿假单胞菌对碳青霉烯类药物的耐药机制研究

目的 探讨铜绿假单胞菌对碳青霉烯类药物的耐药机制.方法 收集2008年11月至2009年4月我院临床分离的铜绿假单胞菌31株,根据药敏结果分为碳青霉烯类耐药组(21株)和碳青霉烯类敏感组(10株).另设1株标准株ATCC 27853,用亚胺培南-EDTA(乙二胺四乙酸)抑制试验检测菌株是否产生金屑酶,采用PCR法检测各菌株的外膜孔道蛋白oprD2基因,探讨铜绿假单胞菌对碳青霉烯类抗生素耐药机制.结果 21株耐药株有7株产生金属酶;21株耐药株经oprD2基因扩增,15株阴性,6株阳性,10株敏感株全部阳性.统计学检验结果表明,碳青霉烯类耐药组与敏感组oprD2基因阳性率的差异有极显著性(P＜0.01).结论 oprD2基因缺失和金属酶是本院铜绿假单胞菌对碳青霉烯类抗生素耐药的重要机制.     

耐亚胺培南铜绿假单胞菌相关耐药基因的检测

目的对耐亚胺培南（IMP）的铜绿假单胞菌（IRPa）相关耐药基因进行检测。方法 2003年至2009年从临床标本中分离到（P.aeruginosa）共220株,采用三维试验筛选产β-内酰胺酶的铜绿假单胞菌,应用普通PCR和多重PCR分别检测碳青霉烯酶基因和质粒携带的C类头孢菌素酶（AmpC酶）耐药基因,应用荧光定量RT-PCR的方法检测oprD2基因的表达情况。结果共检出43株产β-内酰胺酶的菌株,其中产AmpC酶、超广谱β-内酰胺酶（ESBLs）、金属β-内酰胺酶（MBLs）和未知酶菌株的构成比分别58.14%（25/43）、18.60%（8/43）、4.65%（2/43）和16.28%（7/43）。74株耐亚胺培南的铜绿假单胞菌中,有2株菌携带IMP-9基因,1株菌携带DHA质粒型AmpC酶基因,其他碳青霉烯酶基因检测为阴性。40株菌株oprD2基因表达蛋白量降低,34株oprD2基因表达蛋白量正常。结论 oprD2基因的突变或蛋白表达量降低是IRPa对亚胺培南耐药的主要原因,AmpC酶可水解亚胺培南可能与铜绿假单胞菌对亚胺培南的耐药有一定的关系,而KPC-1酶和MBLs在铜绿假单胞菌对亚胺培南耐药机制中不是主要因素。     

肺炎克雷伯菌gyrA基因与parC基因的突变研究

目的探讨肺炎克雷伯菌对环丙沙星和左氧氟沙星的药物敏感性,及对喹诺酮敏感和耐药菌株中gyrA与parC基因的突变情况。方法收集肺炎克雷伯菌临床分离株231株,采用K-B纸片法测定肺炎克雷伯菌对环丙沙星和左氧氟沙星的敏感性,随机选取对环丙沙星和左氧氟沙星均耐药菌株4株和均敏感的菌株3株,分别PCR扩增gyrA基因和parC基因的耐药决定区,扩增片段长度分别为625、319bp,PCR扩增产物经纯化后测序并做序列分析。结果肺炎克雷伯菌对环丙沙星和左氧氟沙星的耐药率分别为51.1%（118/231）和45.9%（106/231）;gyrA和parC基因经序列分析显示,耐药株均有gyrA基因的突变,其中1株出现第83、87和27位氨基酸的改变,2株出现第83位氨基酸的改变,1株出现第47位点的改变;环丙沙星敏感株中未出现gyrA基因的突变。4株耐药株均有parC基因的突变,引起相应氨基酸Ser80→Arg的改变,2株环丙沙星敏感株也发生了同样的改变。结论哈尔滨地区肺炎克雷伯菌对环丙沙星和左氧氟沙星的耐药性显著,在喹诺酮耐药株中有gyrA和parC基因的同时突变,在敏感株中也发现了parC基因的突变。     

Mutations in the gyrA and parC genes in ciprofloxacin-resistant clinical isolates of Acinetobacter baumannii in Korea

Mutation with Ser-83-->Leu in gyrA gene was associated with the principal mutation for ciprofloxacin resistance in clinical isolates of Acinetobacter baumannii. Double mutation, Ser-83-->Leu in gyrA gene and Ser-80-->Leu in parC gene, was the most frequently detected among ciprofloxacin-resistant isolates. A novel mutation with Ser-80-->Trp in parC gene, in addition to mutation in gyrA gene, was associated with a high-level ciprofloxacin resistance. These results suggested that the presence of an additional mutation in the parC gene contributed to a higher-level of ciprofloxacin resistance than a single mutation in the gyrA gene (geometric mean MICs of ciprofloxacin, 44.1 versus 16 microg/ml, P < 0.05).     

Fluoroquinolone resistance and gyrA and parC mutations of Escherichia coli isolated from chicken

Escherichia coli is a common inhabitant of the intestinal tracts of animals and humans. The intestines of animals also represent an ideal environment for the selection and transfer of antimicrobial resistance genes. The aim of this study was to investigate the resistance of E. coli isolated from chicken fecal samples to fluoroquinolones and to analyze the characterization of mutations in its gyrA and parC gene related resistance. One hundred and twenty-eight E. coil isolates showed a high resistance to ciprofloxacin (CIP; 60.2%), enrofloxacin (ENO; 73.4%) and norfloxacin (NOR; 60.2%). Missense mutation in gyrA was only found in the amino acid codons of Ser-83 or Asp-87. A high percentage of isolates (60.2%) showed mutations at both amino acid codons. Missense mutation in parC was found in the amino acid codon of Ser-80 or Glu-84, and seven isolates showed mutations at both amino acid codons. Isolates with a single mutation in gyrA showed minimal inhibitory concentrations (MIC) for CIP (相似文献   

耐喹诺酮类铜绿假单胞菌的耐药性分析及血清学分型

目的了解广州地区喹诺酮类耐药铜绿假单胞菌的耐药性及泵抑制剂对其耐药水平降低的作用,并调查血清型分布情况。方法用法国生物梅里埃公司的微生物鉴定和药敏分析系统VITEK-2对127株铜绿假单胞菌进行鉴定和药敏检测,并采用羰酰氰基-对-氯苯胺(CCCP)与环丙沙星共同作用,以琼脂稀释法测定耐药菌的最低抑菌浓度(M IC)的变化,同时用玻片凝集法对耐药株进行血清学分型。结果环丙沙星耐药菌对哌拉西林/他唑巴坦(65.5%)的敏感率最高,只有阿米卡星(64.4%)、哌拉西林(51.7%)和妥布霉素(50.6%)的敏感率大于50.0%,而敏感菌对美罗培南(97.5%)及左氧氟沙星(97.5%)的敏感率最高,妥布霉素(95.0%)次之,对临床常用的13种抗生素,耐药菌较敏感菌的敏感性明显降低(P值均<0.001);耐药菌受泵抑制CCCP作用,M IC降低1~4个稀释度;血清分型率为93.1%,耐药菌的血清型以B型(20.7%)和L型(19.5%)为主。结论耐喹诺酮类铜绿假单胞菌对临床常用抗生素的敏感性降低,并呈多重耐药,使用抗生素 泵抑制剂可提高药物对铜绿假单胞菌的敏感性;血清学分型可以快速简单地监测铜绿假单胞菌在医院内的流行情况。     

Novel gyrase mutations and characterization of ciprofloxacin-resistant clinical strains of Enterococcus faecalis isolated in Poland

The activity of ciprofloxacin, sparfloxacin and moxifloxacin was determined for 205 Enterococcus faecalis isolates from patients of five hospitals (Warsaw, Poland; collected from 2000 to 2002). Ciprofloxacin resistant and intermediate isolates were numerous (53.7%). Among them, highly resistant (MIC > or = 16 mg/l) isolates predominated (98%). Isolates resistant to ciprofloxacin were also resistant to sparfloxacin and moxifloxacin. The parC and gyrA QRDRs (quinolone-resistance-determining region) of 11 isolates with ciprofloxacin MICs from 1 to 256 mg/l were analysed by DNA sequencing. In ParC one kind of amino acid substitution (of Ser-85 to Ile) in 9 E. faecalis strains with MICs from 16 to 256 mg/l was observed. In GyrA Ser-84 was changed to one of four different amino acids: Arg, Ile, Cys or Tyr, however no association between the amino acid type and MIC value was found. The last two substitutions have not been reported to date for E. faecalis. Moreover, our results may suggest that mutations within parC and gyrA are associated with development of a high-level of ciprofloxacin resistance.     

Real-time PCR detection of Pseudomonas aeruginosa in clinical and municipal wastewater and genotyping of the ciprofloxacin-resistant isolates

Real-time quantification of Pseudomonas aeruginosa was performed in various wastewater systems including clinical, municipal wastewaters and inflow from a wastewater treatment plant. The highest concentrations of P. aeruginosa-specific targets were detected in clinical wastewaters. Limitations of the detection system resulting from inhibition or cross-reaction were identified. Ciprofloxacin-resistant P. aeruginosa strains were isolated after specific enrichment from clinical and municipal wastewaters. In some cases they were also cultivated from effluent of a wastewater treatment plant, and from its downstream river water. A total of 119 isolates were phenotypically characterized as ciprofloxacin-resistant via antibiogram testing. Subsequently, the fluoroquinolone-resistance-mediating mutations in the genes gyrA codon positions 83 and 87, gyrB codon position 466 and parC codon positions 87 and 91 were determined by mini-sequencing. Ciprofloxacin resistance was mainly associated with mutations in gyrA codon position 83 and parC mutation in codon positions 87 or 91 of the bacterial gyrase and topoisomerase II genes. All ciprofloxacin-resistant P. aeruginosa strains were compared with genotypes from clinical data of fluoroquinolone-resistant P. aeruginosa infections. The results were in agreement with data from clinical analyses, with the exception that no gyrA 87 and no gyrB mutations were found in ciprofloxacin-resistant P. aeruginosa wastewater isolates.     

氟喹诺酮类药物体外诱导肺炎克雷伯菌耐药后GyrA和ParC的变异

目的了解3种氟喹诺酮类（FQS）体外诱导肺炎克雷伯菌（Klebsiella pneumoniae,Kpn）耐药性的差异,研究肺炎克雷伯菌DNA旋转酶A亚单位（GyrA）和拓扑异构酶ⅣC亚基（ParC）的变异与其耐喹诺酮类药物的关系。方法采用环丙沙星（CW）、左氧氟沙星（LEX）和加替沙星（GAT）对从临床分离8株Kpn进行体外分步诱导,采用琼脂平板二倍稀释法测定CIP、LVF及GAT对Kpn诱导前、后的最低抑菌浓度（MIC）,并对诱导成功的17株Kpn的GyrA的基因（gyrA）和ParC的基因（parC）进行PCR扩增,选取其中8株KpnDNA测序并进行序列分析比较。结果8株耐FQS菌株都存在GyrA变异,同FQS耐药性相关的变异有Ser83（TCC）→Phe（TTC）、Ile（ATC）和Tyr（TAC）,Asp87（GAC）→Ala（GCC）、ma（GCC）和Glu（GAA）,5株Kpn同时存在ParC的变异：丝氨酸Ser80（AGC）→Ile（ATC）。结论本研究体外实验证实了Kpn可在长期低剂量的接触抗菌药物后形成耐药菌株。在高度耐FQS的Kpn中同时存在GyrA和ParC变异。     

主动外排机制在鲍曼不动杆菌耐药性中的作用

目的探讨细菌主动外排机制在临床分离的鲍曼不动杆菌耐药性中的作用。方法琼脂稀释法检测临床分离的鲍曼不动杆菌对常用抗生素的耐药性,测定经外排泵抑制剂碳酰氰基-对-氯苯腙（CCCP）处理前后鲍曼不动杆菌对抗生素最小抑菌浓度（MIC）的变化,以聚合酶链反应（PCR）、逆转录-聚合酶链反应（RT-PCR）检测多重耐药主动外排基因以出及其表达水平。结果临床分离的鲍曼不动杆菌对常用抗生素耐药率高且具有多重耐药性,并存在药物的主动外排。所有临床分离的菌株均能检测到adeB基因,但多重耐药株表达水平明显高于敏感株（P〈0．01）。结论临床分离的鲍曼不动杆菌的耐药性尤其是多重耐药性与外排泵介导的耐药机制密切相关。     

Genetic basis of quinolone resistance and epidemiology of resistant and susceptible isolates of porcine Campylobacter coli strains

AIMS: The aims of this study were to investigate the epidemiology of quinolone-resistant and -susceptible porcine isolates of Campylobacter coli and to characterize the genetic basis of quinolone resistance. METHODS AND RESULTS: Penner serotyping and flagellin gene sequence polymorphisms were used to investigate the epidemiology of the C. coli isolates. A total of 55 isolates were included, of which 30 were paired resistant and susceptible isolates from 15 pigs. Amplification of gyrA, gyrB and parC, followed by direct sequencing of amplicons was used to identify mutations in the targets of quinolones. Overall, 31 of the isolates were resistant to ciprofloxacin (minimum inhibitory concentrations (MIC), 2- >or = 32 microg x ml(-1)). Thirteen DdeI-flaA profiles were observed and resistant and susceptible strains were identified for nine profiles. The majority of resistant strains exhibited either profile 1 or 6. While profile 1 comprised susceptible and resistant strains, all of the strains with profile 6 were resistant to ciprofloxacin. The serogroup (O:24) of the profile 6 strains was identical. The only other serogroup to be uniformly associated with quinolone resistance was O:5. Strains with this phenotype comprised a number of genotypes, including profile 1. Only four of the paired isolates from individual pigs had the same profile. The genetic basis of quinolone resistance was investigated in two strains with ciprofloxacin MICs of 2 and > or = 32 miccrog x ml(-1), respectively. The amino acid substitution of isoleucine for threonine at position 86 was identified in the GyrA proteins from both strains. No mutations were identified in the GyrB proteins. CONCLUSIONS: There was an association between two of the genotypes, serotypes 5 and 24, and quinolone resistance. The association between genotype, serotype and resistance in C. coli isolates has not been reported previously. Only the mutation in GyrA associated with quinolone resistance was identified. No mutations in GyrB were identified. Amplification products of parC were not obtained and it may be that this gene is not present in some Campylobacter spp. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides data on the distribution of ciprofloxacin resistance between subtypes of C. coli.     

铜绿假单胞菌氨基糖苷16SrRNA甲基化酶基因分析

目的调查215株湖州地区临床分离铜绿假单胞菌对氨基糖苷类抗生素的耐药性和16S rRNA甲基化酶基因分布情况。方法收集2011年1月至2012年12月湖州地区临床分离铜绿假单胞菌215株,琼脂稀释法测定5种氨基糖苷类抗菌药物（庆大霉素、阿米卡星、妥布霉素、伊帕米星、奈替米星）的MIC值;PCR检测armA、rmtA、rmtB、rmtC、rmtD和npmA六种氨基糖苷类16S rRN甲基化酶基因,序列分析明确基因型。测定产16S rRNA甲基化酶菌株对常见抗菌的敏感性,并检测碳青霉烯耐药株产碳青霉烯酶情况。结果铜绿假单胞菌对异帕米星敏感率最高为81.4%,对5种氨基糖苷类抗生素全部耐药的22株菌株中,17株检出armA基因;未发现其他16S rRNA甲基化酶基因阳性菌株。17株armA阳性菌株对碳青霉烯类抗生素耐药5株（耐药率为29.4%）,对头孢他啶、头孢吡肟、哌拉西林/他唑巴坦、环丙沙星耐药率均超过40%。5株碳青霉烯耐药菌株中检测到2株产VIM-2型金属碳青霉烯酶。结论铜绿假单胞菌对氨基糖苷类抗生素耐药率高,检测到16S rRNA甲基化酶基因armA。产16S rRNA甲基化酶铜绿假单胞菌耐药性强,部分菌株同时产金属碳青霉烯酶,给临床抗感染治疗及院内感染控制带来挑战。