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。     

临床分离肺炎克雷伯菌Ⅰ整合子分布及其与qnr基因关系研究

目的 了解临床分离肺炎克雷伯菌中qnr基因和Ⅰ类整合子基因的分布及其耐药特征.方法 采用PCR法对45株耐环丙沙星肺炎克雷伯菌进行qnrA、qnrB、qnrS基因筛查并测序,用PCR法检测qnr阳性菌株Ⅰ类整合子基因,并采用SPSS 13.0和Whonet 5.4软件分析药敏结果及比较.结果 45株肺炎克雷伯菌中,24株(51.1％)细菌检出qnrS基因,未检出qnrA和qnrB基因.20株qnr阳性菌株同时携带Ⅰ类整合子基因.qnr阳性菌株Ⅰ类整合子基因携带率显著高于阴性菌株,qnr阳性菌株对阿米卡星、妥布霉素、亚胺培南、哌拉西林/他唑巴坦及头孢哌酮舒巴坦的敏感性较高.结论 肺炎克雷伯菌对氟喹诺酮类抗菌药物耐药主要由qnrS引起,qnr阳性株同时携带Ⅰ类整合子,导致呈现多重耐药性,加强临床耐药监测对控制多重耐药传播有着重要的意义.     

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。     

质粒介导的耐氟喹诺酮类药物基因的研究

目的 了解南昌大学第二附属医院质粒介导的耐氟喹诺酮类药物大肠埃希菌的流行情况和耐药机制.方法 收集该院2009年1月至2011年12月常规培养的耐左氧氟沙星的大肠埃希菌株100株,提取DNA后PCR扩增qnrA、qnrB、qnrC、qnrD、qnrS、aac(6’)-Ib和qepA基因,并对aac(6’)-Ib阳性产物测序比对.结果 (1)7株细菌检测出qnr基因,其中qnrA2株,qnrS 5株,1株qnrS和qnrA同时阳性,qnrB、qnrC和qnrD均未检出.(2)5株检测出aac(6’)-Ib基因,测序结果经BLAST比对均为野生型,未发现aac(6’)-Ib-cr基因.(3)所有菌株均未检测出qepA基因.结论 该院的氟喹诺酮类抗菌药的耐药机制主要还是靶位突变和细胞膜通透性改变导致的,但7％的质粒介导的耐药基因的检出率也提醒我们要密切关注质粒介导的耐药情况.     

鸡肉源沙门氏菌对喹诺酮和氟喹诺酮类抗生素耐药状况及相关基因

【目的】研究分离于陕西、河南、四川和北京四省(市)鸡肉源沙门氏菌对喹诺酮和部分氟喹诺酮类抗生素的药敏性及相关耐药基因,更好地了解耐药性的产生和传播途径,确保食品安全。【方法】用琼脂稀释法测定沙门氏菌的药敏性,用PCR和基因序列测定法确定耐药沙门氏菌中与(氟)喹诺酮类抗生素耐药相关的喹诺酮类抗性决定区基因突变及质粒携带的耐药基因。【结果】390株沙门氏菌中,63.59%的菌株对萘啶酮酸产生抗性,21.28%、16.67%和14.62%的菌株分别对环丙沙星、左氧氟沙星和加替沙星产生抗性。248株萘啶酮酸抗性菌中,aac(6’)-Ib-cr、qnrA、qnrB和qnrS基因的检出率分别为20.16%、10.89%、10.08%和1.61%。83株耐环丙沙星的菌株中,gyrA和parC基因的点突变共199个;其中gyrA基因中以Ser83Phe和Asp87Gly双突变最为常见,其次分别为Ser83Phe和Asp87Asn双突变、Ser83Tyr、Ser83Phe、Asp87Gly;parC基因的65个点突变均为Ser80Arg突变。【结论】四省市中鸡肉源沙门氏菌耐药状况严重,其解旋酶和拓扑异构酶基因突变及质粒携带的耐药基因是导致沙门氏菌耐药的重要机制。     

PCR-RFLP检测猪源致病性沙门氏菌gyrA基因点突变

四川农业大学谭炳乾、王红宁和叶如俊等先生利用PCR-RFLP法检测猪源致病性沙门氏菌gyrA基因点的突变。他们测定了16株猪源致病性沙门氏菌对5种常用氟喹诺酮类药物的最低抑菌浓度（MIC）。结果表明对此5种药物的耐药率在31．2％～56．2％。分别以质粒和染色体为模板,应用PCR扩增16株沙门氏菌的gyrA基因,以后所有菌株均获得以染色体为模板的扩增产物,还从1株鼠伤寒沙门氏菌获得了以质粒为模板的扩增产物。     

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。     

质粒介导的大肠埃希菌对喹诺酮类抗菌药物耐药机制的研究

目的了解临床分离的耐环丙沙星的大肠埃希菌质粒介导的喹诺酮类耐药基因的携带情况,并进行相关耐药机制的分析。方法采用VITEK-2全自动微生物检测系统鉴定细菌,用K-B法检测细菌对16种常用抗生素的敏感性,采用聚合酶链反应检测喹诺酮类耐药基因qnrS、qnrA、qnrB、qepA和aac（6′）-Ib,并对阳性的aac（6′）-Ib结果进行测序分析。结果 30株耐环丙沙星的大肠埃希菌中,2株（6.67%）检出qepA基因,8株（26.67%）检出aac（6′）-Ib基因,经测序证实其中6株为aac（6）′-Ib-cr（20.0%）。未检出qnrS、qnrA和qnrB基因。结论对环丙沙星耐药的大肠埃希菌携带aac（6′）-Ib-cr和qepA基因,引起质粒介导的对喹诺酮类抗菌药物的低水平耐药。     

临床分离纹带棒状杆菌喹诺酮类耐药株gyrA和parC基因突变的研究

目的 探讨gyrA 、parC基因的改变与纹带棒状杆菌耐喹诺酮类抗生素的关系.方法 采用微量稀释法测试纹带棒状杆菌对环丙沙星、左旋氧氟沙星、万古霉素的敏感性.PCR扩增检测gyrA和parC基因喹诺酮类耐药决定区相关片段并测序,在GenBank中进行Blast及BlastX分析并观察氨基酸突变位点,用NcoI酶进行PCR-RFLP.结果 37株纹带棒状杆菌对环丙沙星和左旋氧氟沙星的耐药率为94.6％,30株菌发生双突变(87位Ser突变为phe,91位Asp突变为Ala),5株菌单点突变(87位Ser突变为phe).PCR-RFLP经NcoI酶切后显示,敏感株和耐药株均产生3个条带.结论 纹带棒状杆菌对环丙沙星、左旋氧氟沙星的耐药率较高,对万古霉素全部敏感.纹带棒状杆菌对喹诺酮类抗生素的耐药机制主要是gyrA突变引起的,尤以双突变占优势.纹带棒状杆菌不携带parC基因,不编码拓扑异构酶Ⅳ.     

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。     

CIP耐药的铜绿假单胞菌两种分子耐药机制关系的研究

目的探讨环丙沙星（CIP）耐药的铜绿假单胞菌临床分离株主动外排药物与gyrA、parC基因突变的关系。方法联合碳酰氰基-对-氯苯腙（CCCP）和CIP对CIP耐药的铜绿假单胞菌株进行主动外排阳性株和阴性株的筛选,并对这些菌株的gyrA,parC基因进行聚合酶链式反应-限制性片段长度多态性分析（PCR—RFLP）。结果57％（55／97）的CIP耐药菌株最小抑菌浓度（MIC）可被逆转,gyrA单基因突变率为65％,gyrA和pa-C双基因突变率为35％,未发现parC单基因突变的菌株。主动外排阳性组与阴性组gyrA、parC基因突变情况差异无显著性。结论在本地区铜绿假单胞菌对CIP的耐药机制中,主动外排系统表达上调与抗菌药物作用靶位的改变均占有重要的地位,两者可能是并存的两种相对独立的机制。     

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).     

Occurrence of qnr-positive clinical isolates in Klebsiella pneumoniae producing ESBL or AmpC-type beta-lactamase from five pediatric hospitals in China.

The plasmid-mediated quinolone resistance qnr genes in clinical isolates in adults have been described in different countries; however, the frequency of their occurrence has not been detected in pediatric patients. A total of 410 clinical isolates of Klebsiella pneumoniae, identified as producers of an extended-spectrum beta-lactamase (ESBL), or AmpC beta-lactamase, were collected from five children's hospitals in China during 2005-2006. The isolates were screened for the presence of the qnrA, qnrB, and qnrS genes, and then the harboring qnr gene isolates were detected for a bla gene coding for the TEM, SHV, CTX-M, and plasmid-mediated ampC gene by a PCR experiment. Ninety-two isolates (22.7%) were positive for the qnr gene, including 10 of qnrA (2.4%), 25 of qnrB (6.1%), and 62 of qnrS (15.1%). Eighty-one of the 92 (88.0%) qnr-positive isolates carried at least one bla gene for TEM, SHV, CTX-M, or DHA-1. The ciprofloxacin resistance increased 16-256-fold and oflaxacin resistance increased 2-32-fold in transconjugants, respectively. These results indicated that the plasmid-mediated qnr quinolone resistance gene was qnrS, followed by qnrB and qnrA. Most of the isolates also carried a bla gene coding ESBL or ampC gene coding DHA-1 among Klebsiella pneumoniae isolated from Chinese pediatric patients.     

一株猪源鼠伤寒沙门氏菌的耐药性鉴定及其消除

猪源鼠伤寒沙门氏菌临床分离株17Y,检测其对19种抗生素的耐药性,结果耐14种抗生素。用高温及高浓度SDS处理后,获得对11种抗生素的敏感性,该菌株命名为17S1。PCR检测证明,大部分耐药基因存在于质粒上,包括I型整合子携带耐药基因,且随着质粒的消除而被消除。所鉴定的耐药基因有blaTEM、blaOXA-1、cat1、tet(B)、aacC2、aadA8b、dhfrXⅡ和sul1等。喹诺酮类药物的靶基因gyrA与parC位于染色体上。GyrA在耐药决定区第87位氨基酸突变(N78D),导致了喹诺酮类药物的耐药性逆转。敏感菌中扩增不到质粒毒力基因spv与rck。耐药性消除后的菌株17S1对小鼠的毒力降低(LD50增加10倍),在小鼠体内的增长与散速度也显著降低(P<0.05)。以上证据表明,鼠伤寒沙门氏菌的多重耐药性主要由质粒决定,研究开发新型质粒消除剂将对克服鼠伤寒沙门氏菌多重耐药性具有重要意义。     

Development of fluoroquinolone (ciprofloxacin) resistance in Mycoplasma hominis in the presence of Hela cells

The effect of cocultivation of eukaryotic HeLa cells and Mycoplasma hominis mycoplasma on the resistance of the latter to fluoroquinolones (ciprofloxacin) was examined. It was shown that cocultivation of the M. homonis and HeLa cells during 24 h with subsequent addition of ciprofloxacin resulted in an increase of the mircoplasma resistance to this antimicrobial agent. In the M. hominis cells cultivated in the presence of HeLa cells and the increasing concentration of ciprofloxacin mutations in the parC gene were observed only at low concentrations of the antimicrobial agent, while mutations in the gyrA gene were never detected. A gradual elevation of ciprofloxacin concentration up to 10 micrograms/ml resulted in the reversion of the parC mutations in mycoplasmas. Mycoplasma cells resistant to high flouroquinolone concentrations and isolated after cocultivation with the HeLa cells were characterized by the wild-type genotype in respect of the gyrA and parC genes. It was shown for the first time that infection of HeLa cells resulted in the appearance of genome rearrangements in M. hominis cells.     

Rapid detection of gyrA and parC mutations in fluoroquinolone-resistant Neisseria gonorrhoeae by denaturing high-performance liquid chromatography

The detection of DNA sequence variation is fundamental to the identification of the genomic basis of phenotypic variability. Denaturing high-performance liquid chromatography (DHPLC) is a novel technique that is used to detect mutations in human DNA. This is the first report that this technique is used as a tool to detect mutations in genes encoding fluoroquinolone resistance in Neisseria gonorrhoeae. Eighty-one strains of N. gonorrhoeae were used in this study. Genomic DNA from each strain was subjected to PCR amplification of 225 bp in gyrA and 166 bp in parC spanning the fluoroquinolone-resistance determining regions (QRDRs). After we performed DNA sequencing of these amplicons and identification of mutations in the QRDRs, DHPLC was undertaken to investigate whether its results correlate the distinctive chromatogram with their DNA mutations pattern. The profilings detected by DHPLC completely corresponded to the results of the DNA sequencing in mutation patters in gyrA and parC genes. They resulted in the following amino acid substitutions: Ser-91Phe, Asp-95Gly, and Asp-95Asn in gyrA; and Gly-85Asp, Asp-86Asn, Ser-87Arg, and Ser-88Pro in parC, respectively. These mutations existed alone or as combinations, and we identified five mutations patterns in gyrA and six in parC including wild-type. These mutations and their patterns could be rapidly and reproducibly identified from the PCR products using DHPLC, producing specific peak patterns that correlate with genotypes. This novel detection system facilitates the detection of resistance alleles, providing a rapid (5 min per sample), economic (96 sample per run), and reliable technique for characterizing fluoroquinolone resistance in N. gonorrhoeae.