养殖动物及人分离大肠埃希菌染色体和质粒介导氟喹诺酮耐药机制的研究

目的 探讨从养殖动物及周围人群分离的大肠埃希菌染色体和质粒介导氟喹诺酮耐药机制. 方法 纸片扩散法和肉汤稀释法检测氟喹诺酮抗菌药物及其他抗生素的耐药性表型.PCR扩增DNA解旋酶(gyrA和gyrB)和拓扑异构酶IV(parC和parE)基因的喹诺酮耐药决定区、导致喹诺酮类抗生素耐药质粒的部分基因(qnr)以及氨基糖苷类抗生素乙酰转移酶Ib亚型cr变异体编码基因[aac(6')-I b-or],PCR产物进行直接测序.接合试验确定aac(6')-I b-cr酶的可转移性以及在氟喹诺酮耐药中的作用. 结果 鸡来源的大肠埃希菌对常用抗生素的耐药率明显高于猪和周围人群来源菌株.在PCR检测的64株大肠埃希菌中,环丙沙星MIC值大于1μg/ml以上的53株均存在gyrA和/或/parC基因上出现两个位点突变和氨基酸替代,环丙沙星的MIC>16μg/ml的菌株parE基因也发生了点突变及相应氨基酸替代.未发现gyrB亚单位有氨基酸替代.鸡来源28株菌和猪来源9株菌中分别有7株(25.O%)和1株(11.1%)携带有aac(6')-I b-cr基因;aac(6')-I b-cr基因可使环丙沙星、诺氟沙星乙酰化而降低药物抗菌活性. 结论 gyrA、parC和parE碱基突变导致氨基酸置换的数量与菌株对氟喹诺酮类耐药水平呈正相关,携带aac(6')-I b-cr基因的质粒在细菌氟喹诺酮耐药上也具有一定作用.     

大肠埃希菌尿液分离株中检出gyrA基因新变异株

目的 调查大肠埃希菌尿液分离株中喹诺酮类耐药相关基因的存在与变化状况.方法 收集宁波市第一医院2008年10月到2009年3月患者尿液标本中分离的大肠埃希菌共28株,采用聚合酶链反应(PCR)及序列分析的方法分析1种染色体介导的喹诺酮类耐药相关基因(gyrA基因)和5种质粒介导的喹诺酮类耐药相关基因[qnrA、qnrB、qnrS、aac(6＇)-Ⅰb、qepA].结果 28株大肠埃希菌检测到1株aac(6＇)-Ⅰb-Cr基因阳性株(经测序比对证实),qnrA、qnrB、qnrS、qepA基因均未检出.gyrA基因83位密码子28株菌都有突变(100.0%),其突变方式为TCG-83→HTG,导致氨基酸从丝氨酸(S)-83→亮氨酸(L);87位密码子22株菌(78.6%)有突变,可分为两种突变方式:21株(75.0%)突变方式为GAC-87→AAC,导致氨基酸从天冬氨酸(D)-87→天冬酰胺(N);5号株gyrA基因(3.6%)为新亚型,其突变方式为GAC-87→TAC,导致氨基酸从天冬氨酸(D)-87→脯氨酸(Y),另6株菌87位密码子无突变.结论 本组大肠埃希菌gyrA基因突变率为100.0%,是喹诺酮类耐药的主要原因.其他耐药相关基因阳性率很低.     

环丙沙星耐药肠杆菌科细菌临床株qnr和aac(6')-Ⅰ b-cr基因的检测

目的 了解温州地区环丙沙星耐药肠杆菌科细菌临床株的qnr、aac(6')-Ⅰ b-cr基因的分布情况.方法 收集2005年8月-2008年4月间温州医学院附属第一医院环丙沙星耐药的肠杆菌科细菌共461株,其中大肠埃希菌370株,阴沟肠杆菌39株,克雷伯菌属细菌52株.应用PCR方法 检测qnr和aac(6')-Ⅰ b幕因,DNA测序检测qnrA、qnrB、qnrS和aac(6')-Ⅰ b-cr基因;接合传递试验方法 探讨细菌质粒介导的耐药性传递情况.结果 461株环丙沙星耐药的肠杆菌科细菌临床株中检出含qnr基因阳性菌株15株(3.25%),包括qnrA基因阳性株5株(4株阴沟肠杆菌和1株解鸟氨酸克雷伯菌)、qnrB基因阳性株4株(2株肺炎克雷伯菌和2株大肠埃希菌)、qnrS基因阳性株6株(2株肺炎克雷伯菌和4株大肠埃希菌);检出52株细菌(包括42株大肠埃希菌、4株阴沟肠杆菌和6株克雷伯菌属细菌)携带aac(6')-Ⅰ b-cr.15株qnr基因阳性的菌株同时携带aac(6')-Ⅰ b-cr,药敏结果 显示对业胺培南敏感但对多种抗生素耐药.15株qnr基因阳性的菌株中7株质粒接合传递试验成功,临床株对喹诺酮类和氨基糖苷类的耐药性部分传递给了受体株.结论 qnr基因在温州地区环丙沙星耐药的肠杆菌科细菌临床株中较少见,而aac(6')-Ⅰ b-cr基因存在较普遍.     

产超广谱β-内酰胺酶大肠埃希菌和肺炎克雷伯菌遗传标记和耐药基因的研究

目的了解产超广谱β-内酰胺酶（extended spectrum beta-lactamases,ESBLs）大肠埃希菌和肺炎克雷伯菌的遗传标记基因（整合子qacE△1、转座子tnpU）、氨基糖苷类修饰酶编码aac（6′）-Ib基因和喹诺酮类药物耐药qnrA基因分布状况,为临床使用药物治疗感染和消毒灭菌工作提供参考。方法收集本院的产ESBLs大肠埃希菌和肺炎克雷伯菌共60株,采用PCR方法检测这些菌株中qacE△1、tnpU、aac（6′）-Ib和qnrA基因,MIC法药物敏感试验分析菌株的耐药性。结果 60株产ESBLs的大肠埃希菌和肺炎克雷伯菌中qacEΔ1基因检出率61.7%,tnpU基因检出率7.6%,aac（6′）-Ib检出率为11.7%,qnrA基因检出率3.3%。60株菌中有38株含1种或以上基因,其药敏敏感度最高的是亚胺培南、厄它培南、哌拉西林/他唑巴坦,敏感率均为97.4%,而对氨苄西林和头孢唑啉完全耐药。结论产ESBLs大肠埃希菌和肺炎克雷伯菌的耐药与耐药基因传递机制整合子和转座子系统密切相关,分离株携带qacE△1、tnpU、aac（6′）-Ib和qnrA基因是细菌呈多重耐药的主要原因,提示临床应慎重用药。     

常见肠杆菌科细菌临床株aac(6＇)-Ib-cr基因的流行现状和耐药特征

目的 研究氨基糖苷乙酰胺转移酶基因的突变形式aac(6’) -Ib -cr在肺炎克雷伯菌和大肠埃希菌临床株中的分布状况;分析携带该基因菌株对环丙沙星、氨基糖苷类和头孢菌素类的耐药特征.方法 利用多聚酶链式反应检测肺炎克雷伯菌和大肠埃希菌临床分离株中aac(6’)-Ib基因携带情况,采用BtsC I酶切消化,经DNA测序确认aac(6 ’)-Ib-cr基因;统计分析aac(6’) -Ib-cr基因与环丙沙星、头孢菌素类和氨基糖苷类耐药性关系.结果 aac(6’)-Ib-cr基因在肺炎克雷伯菌和大肠埃希菌的检出率分别为12.6％ (30/238)和18.8％ (13/69);aac(6 ’)-Ib-cr阳性株与阴性株对环丙沙星的耐药率分别为72.1％ (31/43)和48.1％(127/264),差异有统计学意义(X2=8.52,P＜0.05).肺炎克雷伯菌aac(6’)-Ib-cr基因阳性株对阿米卡星的耐药率高于aac(6’) -Ib-cr基因阴性株(X2=4.25,P＜0.05).aac(6’)- Ib-cr基因阳性株和阴性株对庆大霉素的耐药率、常用的头孢菌素类的耐药率和多重耐药率均较高,无统计学差异.结论 本地区肺炎克雷伯菌和大肠埃希菌临床分离株中,aac(6’) -Ib-cr基因均有检出;aac(6’)-Ib-cr基因阳性株对环丙沙星耐药率明显高于阴性株,肺炎克雷伯菌中aac(6’)-Ib-cr基因阳性株对阿米卡星的耐药率明显高于阴性株,具有统计学意义.aac(6’) - Ib-cr基因阳性株和阴性株对常用头孢菌素类的耐药率和多重耐药率均较高,无统计学意义.     

常见肠杆菌科细菌临床株aac（6′）-Ib-cr基因的流行现状和耐药特征

目的研究氨基糖苷乙酰胺转移酶基因的突变形式aac（6′）-Ib-cr在肺炎克雷伯菌和大肠埃希菌临床株中的分布状况;分析携带该基因菌株对环丙沙星、氨基糖苷类和头孢菌素类的耐药特征。方法利用多聚酶链式反应检测肺炎克雷伯菌和大肠埃希菌临床分离株中aac（6′）-Ib-cr基因携带情况,采用BtsCI酶切消化,经DNA测序确认aac（6′）-Ib-cr基因;统计分析aac（6′）-Ib-cr基因与环丙沙星、头孢菌素类和氨基糖苷类耐药性关系。结果aac（6′）-Ib-cr基因在肺炎克雷伯菌和大肠埃希菌的检出率分别为12．6％（30／238）和18．8％（13／69）;aac（6′）-Ib-cr阳性株与阴性株对环丙沙星的耐药率分别为72．1％（31／43）和48．1％（127／264）,差异有统计学意义（x2=8．52,P〈0．05）。肺炎克雷伯菌aac（6′）-Ib-cr基因阳性株对阿米卡星的耐药率高于aac（6′）-Ib-cr基因阴性株（x2=4．25,P〈0．05）。aac（6′）-Ib-cr基因阳性株和阴性株对庆大霉素的耐药率、常用的头孢菌素类的耐药率和多重耐药率均较高,无统计学差异。结论本地区肺炎克雷伯菌和大肠埃希菌临床分离株中,aac（6′）-Ib-cr基因均有检出;aac（6′）-Ib-cr基因阳性株对环丙沙星耐药率明显高于阴性株,肺炎克雷伯菌中aac（6′）-Ib-cr基因阳性株对阿米卡星的耐药率明显高于阴性株,具有统计学意义。aac（6′）-Ib-cr基因阳性株和阴性株对常用头孢菌素类的耐药率和多重耐药率均较高,无统计学意义。     

产AmpC酶大肠埃希菌和肺炎克雷伯菌中整合子与多重耐药的研究

目的探讨整合子介导的耐药机制在产AmpC酶大肠埃希菌和肺炎克雷伯菌多重耐药中的作用.方法5株产AmpC酶大肠埃希菌和肺炎克雷伯菌分离自2002年1月-2004年5月间我院呼吸科住院的患者,采用E-test试验条进行药敏试验、电转化试验,筛选、分离耐药质粒.PCR扩增Ⅰ型整合子基因盒插入序列,分子克隆和序列分析.结果所有产酶菌株通过电转化试验可将头孢西丁耐药性传递给受体菌,5个产AmpC酶耐药质粒中,有4个检出整合酶序列,其中3个携带2种抗药性基因盒,包括氨基糖苷乙酰转移酶基因aacA4;氨基糖苷腺苷转移酶基因aadA5;二氢叶酸还原酶基因dfrA17;氯霉素外排蛋白编码基因cmL44.结论整合子介导的抗药性基因盒参与了产质粒AmpC酶大肠埃希菌和肺炎克雷伯菌多重耐药的形成,应引起高度重视.     

大肠埃希菌中氟喹诺酮耐药机制功能分析

目的 分析拓扑异构酶的突变和外排泵系统在大肠埃希菌(Escherichia coli)氟喹诺酮类药物耐药机制中的作用.方法 本研究通过基因重组技术对大肠埃希菌中拓扑异构酶不同点突变的功能进行了准确测定,同时也对大肠埃希菌中不同外排泵及膜蛋白的功能进行了分析.结果 在不同的菌株中,acrAB或tolC的切除所引起细菌耐药性的变化不同.对拓扑异构酶点突变的功能分析显示,gyrA中的点突变(S83和D87)在喹诺酮耐药机制中起主要作用,没有gyrA上的点突变,parC上的点突变(S80和A108)对细菌的耐药性不产生影响,但单独gyrA上的点突变(S83和D87)也仅导致敏感菌株对萘啶酸耐药,而对其他氟喹诺酮类药物仍表现为敏感.当对喹诺酮敏感的大肠埃希菌K-12同时具备gyrA(S83L和D87N)和parC(S801和A108V)上的点突变后,重组菌株对氟喹诺酮会自然产生耐药性,而并不需要过度表达的外排泵.结论 拓扑异构酶的突变在大肠埃希菌氟喹诺酮药物的耐药机制中起主要作用,对氟喹诺酮药物耐药的菌株通常应同时具备gyrA和parC上的点突变.     

大肠埃希氏菌的qnrB基因检测与耐药机制研究

目的探索本地区医院大肠埃希氏菌喹诺酮耐药基因的分布及耐药机制。方法采用PCR扩增与测序、基因初步定位、质粒接合转移实验等方法确定喹诺酮耐药的大肠埃希氏菌qnr的基因类型,以分析研究有关的耐药特点与机制。结果各大肠埃希氏菌株中仅qnrB基因阳性,qnrA、qnrS、qnrC、qnrD、qepA、aac（6’）-Ib-cr基因均阴性;并且qnrB基因包括qnrB2、qnrB5、qnrB9、qnrB16、qnrB18、qnrB19和qnrB31等位基因;在这些qnrB等位基因中,qnrB31与qnrB16、qnrB2与qnrB9同源性较高;各qnrB等位基因分别位于约21.0 kb至28.0 kb长的质粒上。结论在本地区医院存在不同的qnrB等位基因流行;实验菌株的喹诺酮耐药与qnrB等位基因结构中LexA-蛋白结合位点共有序列缺失有关。     

含qnrA基因质粒介导不同水平环丙沙星耐药性的机制研究

目的 研究4个含qnrA基因质粒在大肠埃希菌接合子中介导环丙沙星耐药水平不同的发生机制.方法 以大肠埃希菌J53作为受体菌,通过接合试验从4株qnrA阳性的临床菌株中获得4株接合子.采用E test方法测定环丙沙星最低抑菌浓度(MIC);以PCR法检测aac(6')-Ib-cr基因,采用实时RT-PCR法测定qnrA的mRNA表达水平,通过启动子探针载体pKK232-8测定qnrA启动子强度,测定、比较启动子周边序列.结果 环丙沙星对仅含qnrA质粒pHS4及pUS5的接合子的MIC为0.094μg/ml及0.125μg/ml,同时携带qnrA及aac(6')-Ib-cr质粒pUS3及pUS6的接合子的环丙沙星MIC为0.25μg/ml及1.00μg/ml.含pUS6接合子qnrA相对表达水平较其他接合子高13～32.5倍,pUS6的qnrA上游序列启动子活性最强,比另3个质粒高12倍,序列分析发现与pUS3比较,pUS6中qnrA转录启始位点与启始密码之间有7 bp(GTYAGCA)的缺失.结论 1个质粒同时携带qnrA和aac(6')-Ib-cr 2个耐药基因及qnrA高表达是导致接合子对环丙沙星的耐药性不同的原因.     

Fecal colonization of Enterobacteriaceae carrying plasmid-mediated quinolone resistance determinants in Korea

The aims of the current study were to investigate the prevalence and molecular characteristics of plasmid-mediated quinolone resistance (PMQR) genes from colonizing fecal organisms and to compare the incidence and subtype of these genes according to bacterial species and hospital at five tertiary-care hospitals in Korea. A total of 500 nonduplicated clinical isolates of Enterobacteriaceae were obtained from fecal specimens at five tertiary-care hospitals between March and May 2008. The PMQR genes (qnrA, qnrB, qnrS, aac(6')-Ib-cr, and qepA) were amplified by PCR and confirmed by direct sequencing of the PCR products. A total of 83 (16.6%) qnr-positive isolates were detected. The prevalence rates of qnrA, qnrB, and qnrS were 1.4%, 13.6%, and 1.6%, respectively. The species distributions of qnrB-positive isolates were Klebsiella pneumoniae (37/109; 33.9%), Citrobacter freundii (10/34; 29.4%), Citrobacter braakii (8/13; 61.5%), and Escherichia coli (8/275; 2.9%). Sixteen subtypes of qnrB were detected, including seven novel variants. The prevalences of aac(6')-Ib-cr and qepA were 15.6% (n=78) and 0.6% (n=3), respectively. The aac(6')-Ib-cr gene was detected in 39 (47.0%) of 83 qnr-positive isolates and 39 (9.4%) of 417 qnr-negative isolates There was one qepA variant containing a novel mutation (Ala231Val). The prevalence of PMQR genes was high in Enterobacteriaceae from stool specimens in Korea, and there was a close relation between qnr and aac(6')-Ib-cr.     

Plasmid mediated quinolone resistance determinants qnr,aac(6′)-Ib-cr,and qep in ESBL-producing Escherichia coli clinical isolates from Egypt

Purpose: To characterize the prevalence of plasmid-mediated quinolone resistance determinants qnr, aac(6′)-Ib-cr and qep in extended-spectrum β-lactamase (ESBL) -producing E. coli and to determine the association of these determinants with CTX-M group in Cairo, Egypt. Materials and Methods: MICs of 15 antimicrobial agents against 70 E. coli clinical isolates were determined using agar dilution technique according to CLSI. Screening for the qnrA, qnrB, qnrS, aac(6′)-Ib, qep and CTX-M genes was carried out by PCR amplification and DNA sequencing. Curing was used to confirm whether qnr, aac(6′)-Ib, qep or ESBL-encoding genes were located on plasmids. Results: Out of 70 E. coli clinical isolates, 61 were resistant to at least one antibiotic, 16 (22.8%) were multidrug resistant and 30 (42%) were ESBL producers. Out of 30 ESBL producers E. coli isolates, 8 (26.6%) were positive for qnr genes, and the qnrA1-, qnrB1-and qnrS1-type genes were detected alone or in combination in 5 (16.6%), 7 (23.3%) and 5 (16.6%) isolates, respectively. Seven (23.3%) isolates were positive for aac(6′)-Ib-cr and only two (6.6%) isolates were positive for qepA4. Loss of all plasmids upon curing suggested that qnr, aac(6′)-Ib-cr, qep A4 and ESBL-encoding genes were always plasmid mediated. Out of 8 Qnr positive isolates 5 were associated with both CTX-M-1 and CTX-M-9 while 2 from 6 aac(6′)-Ib-cr positive isolates were associated with both CTX-M-1 and CTX-M-9. Conclusions: This study highlights the prevalence of quinolone resistance determinants qnr, aac(6′)-Ib-cr, qep A4 associated with CTX-M positive E. coli isolates from Egypt. This is the first report of the plasmid mediated fluoroquinolone efflux pump, Qep A from Egypt.     

Investigation of plasmid-mediated quinolone resistance in Pseudomonas aeruginosa clinical isolates

Aims: To investigate plasmid-mediated quinolone resistance in clinical isolates of Pseudomonas aeruginosa with the polymerase chain reaction (PCR). The plasmid-mediated quinolone resistance genes have been identified in many bacteria within the Enterobactericeae family, they have not been detected in P. aeruginosa isolates. Subjects and Methods: Identification of the isolates and testing of antibiotic susceptibility was performed in Vitek2 Compact (Biomeriux, France) and Phoinex (BD, USA) automated systems. Screening for the qnrA, qnrB, qnrS, qnrC, aac (6′)-Ib-cr and qepA genes was carried out by PCR amplification and aac (6′)-Ib-cr DNA sequencing. Results: The qnr and the qepA genes were not detected in any of P. aeruginosa isolates. The aac (6’)-Ib gene was detected in six of the isolates and positive isolates for aac (6’)-Ib were sequenced for detection of the aac (6’)-Ib-cr variant but aac (6’)-Ib-cr was not detected in any isolates. Conclusions: Plasmid-mediated quinolone resistance genes have so far not been identified in P. aeruginosa isolates. However, qnrB have detected in P. florescens and P. putida isolates. This is the first study conducted on the qnrA, qnrB, qnrS and qnrC genes as well as the qepA and aac (6’)-Ib-cr genes in P. aeruginosa clinical isolates.     

Prevalence and characterization of plasmid-mediated quinolone resistance genes in extended-spectrum β-lactamase-producing Enterobacteriaceae isolates in Mexico

The objectives of this study were to investigate the prevalence of plasmid-mediated quinolone resistance genes in a collection of 226 extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae isolates and characterize the qnr-positive isolates. The rate of qnr-positive isolates was 21.6% (49/226), 49.5% for aac(6')-Ib-cr (112/226), and 1.7% for qepA1 (4/226). Those isolates carried qnr genes corresponding to types qnrB (71.4%), qnrS1 (24.4%), and qnrA1 (18.3%). The distribution among bacterial species was as follows: 55.8% (19/34) to Enterobacter cloacae, 50% (28/56) to Klebsiella pneumoniae, and 1.4% (2/136) to Escherichia coli. The characterization of qnr-positive isolates indicated the ESBL SHV-types as the most prevalent (81.6%), including the ESBLs SHV-12, SHV-5, and SHV-2a, followed by CTX-M-15 (44.9%) and TLA-1 (8.1%). In addition, for qnr-positive isolates, the prevalence of aac(6')-Ib-cr was 55.1%, but qepA was not identified. Alterations at codons Ser-83 and Asp-87 in GyrA and at codons Ser-80 in ParC were observed in 69% and 80% of the qnr-positive isolates, respectively. The analysis of the transconjugants revealed a cotransmission of bla(CTX-M-15) with qepA1 or aac(6')-Ib-cr and/or qnrA1 and bla(SHV-type) with qnrB5 and qnrB6 genes. To conclude, these findings indicate a high prevalence of qnr and aac(6')-Ib-cr among ESBL-producing isolates from Mexican hospitals and point to the wide spread of qnr-like determinants associated to ESBLs SHV- and CTX-M-type in Mexican clinical isolates.     

Detection of drug resistance-associated genes of multidrug-resistant Acinetobacter baumannii

The beta-lactamase (BLA) genes, the genes for aminoglycosides-modifying enzymes (AMEs), disinfectant-sulfanilamide resistance (qacEDelta1-sul1) genes, class 1 integrase (intl1) gene, and the qnr gene associated with plasmid-mediated quinolone resistance were analyzed using PCR and verified by DNA sequencing for 31 clinical isolates of multidrug-resistant Acinetobacter baumannii (MDRAB). The organism typing was performed by pulsed-field gel electrophoresis (PFGE). The positive rate of ADC, TEM, PER, and DHA of BLA genes were 100%, 61.3%, 19.4%, and 3.2%, respectively; however, the genes of SHV, OXA-23 group, OXA-24 group, GES, VIM, IMP, and qnr gene were negative. The positive rate of the genes of AMEs for aac (3)-I, aac (6')-I, ant (3")-I, ant (2")-I, aac (3)-II, and aac (6')-II were 67.7%, 45.2%, 29.0%, 22.6%, 12.9%, and 3.2%, respectively. The positive rate of qacEDelta1-sul1 and intl1 were 80.6% and 58.1%, respectively. Six different PFGE clones were found, of which two dominated. The findings show that clinical isolates of MDRAB harbor various kinds of resistance genes.     

Prevalence of the plasmid-mediated quinolone resistance genes, aac(6')-Ib-cr, qepA, and oqxAB in clinical isolates of extended-spectrum β-lactamase (ESBL)-producing Escherichia coli and Klebsiella pneumoniae in Korea

In this study, we sought to determine the prevalence of the plasmid-mediated quinolone resistance (PMQR) genes aac(6')-Ib-cr, qepA, and oqxAB in extended-spectrum β-lactamase (ESBL)-producing Escherichia coli and Klebsiella pneumoniae clinical isolates in South Korea. In total, 104 isolates (63 E. coli and 41 K. pneumoniae) were collected. We found that 23 of the 63 (36.5%) E. coli and nine of the 41 (22.0%) K. pneumoniae isolates were positive for aac(6')-Ib-cr. No isolate was positive for qepA, while transferable oqxAB was detected only in 10 (24.4%) K. pneumoniae isolates. Among the 32 aac(6')-Ib-cr-positive isolates, 30 (93.8%) were positive for both aac(6')-Ib-cr and bla(CTX-M) (CTX-M-15, -14, and -57). Our results suggest that PMQR determinants are highly prevalent in ESBL-producing E. coli and K. pneumoniae isolates in Korea.     

Molecular epidemiology of ciprofloxacin-resistant extended-spectrum β-lactamase-producing Klebsiella pneumoniae in Taiwan

Fluoroquinolone resistance in extended-spectrum β-lactamases (ESBL)-producing isolates results in very few antimicrobial treatment options. In Taiwan's Surveillance of Antimicrobial Resistance (TSAR) III program, 124 (52.8%) cases of ESBL-producing Klebsiella pneumoniae (ESBL-KP) were resistant to ciprofloxacin. The prevalence of plasmid-mediated quinolone resistance (PMQR) determinants and chromosomal quinolone resistance-determining regions (QRDR) of gyrA and parC genes among ESBL-KP isolates was assessed via PCR sequencing. Chromosomal QRDR mutations were present in most of the 123 (96.8%) cases of ciprofloxacin-resistant ESBL-KP isolates. Sixty-six (53.2%) isolates had at least one PMQR gene. qnrB2, qnrB4, and qnrS1 were detected in 26, 19, and 13 isolates, respectively, whereas qnrA, qnrC, and qnrD were not detected. ESBL genes were transferable via conjugation with either aac(6')Ib-cr or qnrB in 63.6% of the isolates carrying PMQR genes. QnrB was associated with either CTX-M-15 or SHV-12, and aac(6')Ib-cr was linked to CTX-M-3 or CTX-M-14 in plasmids. qnrS did not co-transfer with ESBL genes. Clonal spread of PMQR genes harboring ESBL-KP isolates was observed in three hospitals. QnrA, which is common in Asia, was unexpectedly absent in ESBL-KP in Taiwan. Aside from transmission via clonal spread for ciprofloxacin-resistant ESBL-KP, concomitant transference of PMQR genes with either bla(CTX-M) or bla(SHV) via plasmid was common.     

Diverse phenotypic and genotypic characterization among clinical Klebsiella pneumoniae and Escherichia coli isolates carrying plasmid-mediated quinolone resistance determinants

A total of 59 and 74 nonduplicate plasmid-mediated quinolone resistance (PMQR) genes-carrying Klebsiella pneumoniae and Escherichia coli isolates were collected. All strains were assayed for fluoroquinolone susceptibility and the prevalence of quinolone resistance-determining regions (QRDRs) mutation. The association between PMQR determinants and common β-lactamase genes was also analyzed. Genetic relatedness of the isolates was analyzed by pulsed-field gel electrophoresis (PFGE). The PMQR genes-carrying K. pneumoniae and E. coli isolates exhibited high fluoroquinolone resistance rates, indicating that PMQR determinants play an essential role in the development of fluoroquinolone resistance. Remarkably, most qnr-carrying strains had only a single or no QRDR mutation in GyrA or ParC, and most exhibited decreased ciprofloxacin (CIP) susceptibility or low-level CIP resistance. However, 71.4% and 98.4% of qnr-negative K. pneumoniae and E. coli contained double QRDR mutations, and most presented high-level CIP resistance. Additionally, K. pneumoniae presented a lower CIP resistance rate than E. coli (59.3% vs. 91.9%) and low carriage of double QRDR mutations (38.9% vs. 89.9%). Also, most (88.1%) K. pneumoniae examined in this study carried qnr and only 14.9% of E. coli were qnr positive. Thus, the high fluoroquinolone susceptibility of K. pneumoniae isolates is primarily due to fewer QRDR substitutions as a result of high prevalence of qnr alleles in the host. Our findings support the hypothesis that chromosomal resistance mutations could be affected by the presence of Qnr, in other words, Qnr may protect the QRDR domains in the gyrase and topoisomerase IV from mutations under the inhibition of fluoroquinolones. Another remarkable finding was that the PMQR genes-carrying K. pneumoniae exhibited much higher proportions of extended-spectrum β-lactamases (ESBLs)-positive phenotype than E. coli (73.5% vs. 59.5%). This is due to not only the high prevalence of SHV-type ESBL-conferring enzymes in K. pneumoniae but also the interference of DHA-producing K. pneumoniae as a result of the strong association between qnrB and bla(DHA).