质粒介导的喹诺酮耐药基因在中国大陆尿液分离的大肠埃希菌中的广泛分布

目的分析中国大陆20家三甲医院尿来源大肠埃希菌的耐药特点并调查质粒介导的喹诺酮类耐药基因的分布情况和流行特点。方法收集卫生部全国耐药监测网2007年1月至2008年3月非重复298株尿液分离大肠埃希菌;琼脂稀释法测定其对20种抗菌药物的敏感性,多聚酶链反应和DNA测序分析qn-rA,qnrB,qnrS,aac(6’)-ib和qepA基因的流行性;接合实验分析质粒的转移性;Eric-PCR分析喹诺酮基因阳性菌株之间的遗传相关性;卡方检验用于分析耐药基因与氟喹诺酮耐药之间的相关性。结果 298株大肠埃希菌对20种抗菌药物耐药现象严重,其中对环丙沙星和左氧氟沙星有很高的耐药性,耐药率高达78.5%和74.2%。经基因比对分析,62株(20.8%)细菌携带aac(6’)-Ib基因;45株(15.1%)细菌携带喹诺酮耐药基因,1株(0.3%)检测出qnrA基因,3株(11.4%)检出qnrB基因,5株(1.7%)检出qnrS基因,25株(8.4%)确定为aac(6’)-Ib-cr基因,12株(4.7%)检出qepA基因;此外,有3株细菌分别发现aac(6’)-Ib-cr和qepA1基因aac(6’)-Ib-cr和qnrB1基因,qepA和qnrS1基因共存。45株喹诺酮基因阳性菌株之间具有很大的遗传差异,并且其中有16株细菌携带的基因具有可转移性。aac(6’)-Ib的流行性与细菌的环丙沙星和左氧氟沙星不敏感性相关(P<0.05);喹诺酮耐药基因的流行性与细菌的氟喹诺酮不敏感性相关(P<0.05)。结论尿液分离的大肠埃希菌耐药严重,质粒介导的喹诺酮耐药基因主要以aac(6’)-ib-cr为主,qepA1次之,这些潜在播散的喹诺酮耐药基因对于临床尿路感染的治疗有很大的挑战。     

大肠埃希菌耐喹诺酮类抗菌药的相关耐药基因检测及耐药机制分析

目的 探讨大肠埃希菌耐喹诺酮类抗菌药的相关耐药基因检测及耐药机制.方法 选取我院尿液等排泄物和分泌物标本中分离出的150株大肠埃希菌株,应用纸片扩散法进行药敏试验,采用PCR技术检测qnr以及aac(6')-Ib-cr质粒基因的表达,并对PCR产物进行DNA测序分析.结果 150株大肠埃希菌对喹诺酮类抗菌药物的耐药率均超过50％,20株菌检出阳性基因qnrB,qnrS以及aac(6')-Ib-cr的阳性率分别为12.20％、9.76％、13.41％,共有8株菌同时携带超过2种质粒基因,其对喹诺酮类药物均耐药,同时合并有其他类型抗菌药物的耐药情况,12株菌检出单个质粒基因,其中4株对喹诺酮类敏感.结论 大肠埃希菌的耐药情况十分严重,存在qnrB、qnrS、aac(6')-1b-cr流行,并存在两种及多种耐药基因共存于同一细菌中的现象,质粒介导的喹诺酮耐药基因数量与耐药种类数量呈正相关.     

市售整鸡中环丙沙星耐药大肠埃希氏菌的分布及耐药机制研究

目的 本研究旨在阐明市售整鸡中环丙沙星耐药大肠埃希氏菌的分布和耐药机制特点.方法 对广东省3个不同地区市售整鸡中的环丙沙星耐药大肠埃希氏菌进行分离和鉴定,并对分离株进行种系分群、药敏试验和喹诺酮耐药机制研究.结果与结论 从58份市售整鸡中分离出41株环丙沙星耐药大肠埃希氏菌,其中27株属于A群.分离株耐药谱主要有两种:AMP-CAZ-CIP-CTX-Cl-SXT-TET和AMP-CAZ-CIP-CTX-Cl-GEN-SXT-TET.喹诺酮耐药机制研究显示,拓扑异构酶编码基因gyrA和parC均有点突变出现,其中,26株分离株携带质粒介导的喹诺酮耐药基因,包括oqxAB、qnrS、aac(6')-Ib-cr,而qnrA、qnrB、qnrC、qnrD和qepA基因则均未检出.耐药菌株在市售整鸡中的广泛分布和复杂的喹诺酮耐药机制,应引起相关部门的重视.     

天津地区大肠埃希菌临床株质粒介导的喹诺酮类耐药机制的研究

目的 了解天津地区临床分离的大肠埃希菌中qnr,aac(6')-Ib-cr及gepA基因的流行情况,分析阳性菌株感染的微生物学及临床特征.方法 从天津某三甲医院收集环丙沙星耐药(CPLX,MIC 4gg/mL)的大肠埃希菌共75株,采用PCR法检测gnrA,gnrB,gnrS,aac(6')-Ib及gepA基因,对aac(6'),-Ib基因阳性菌株用Fok I酶切确认aac(6')-Ib-cr基因,接合试验验证qnr的转移性.所有阳性菌株用PCR法检测p-内酰胺酶基因,并收集阳性菌株感染患者临床资料进行分析.结果 75株环丙沙星耐药的大肠埃希菌中共有18株(24%)携带qnr基因和(或)aac(6)-Ib-cr基因,其中gnrB,gnrS和aac(6')-Ib-cr检出率分别为5.3%,4%和21.3%,未检出gnrA和gepA o 5株qnr阳性菌株均接合传递成功.18株gnr1aac(6')-1b-cr阳性的大肠埃希菌中均检测出R-内酸胺酶基因.阳性菌株对喹诺酮类和头孢菌素类药物高度耐药且主要来源于泌尿系感染,感染患者均为中老年人.结论 天津地区临床存在qnr和aac(6')-Ib-cr阳性菌株的流行,这是首次在天津发现qnr介导的喹诺酮类耐药.     

碳青霉烯类耐药肺炎克雷伯菌临床分离株中质粒介导的喹诺酮类耐药基因检测

目的：探讨质粒介导的喹诺酮类耐药（PMQR）基因在碳青霉烯类耐药肺炎克雷伯菌（CRKP）临床分离株中的分布情况。方法：收集CRKP29株,聚合酶链反应（PCR）扩增并确定产物基因型。结果：CRKP中PMQR基因检出率为48．3％（14／29）,其中qnrB5株,包括qnrB21株、qnrB43株、qnrB101株;qnrS 5株,均为qnrS1;1株同时携带qnrS1和qnrB4;有3株携带aac（6′）-Ib-cr基因。所有PMQR基因阳性菌株均同时携带β-内酰胺酶基因,其中8株同时携带碳青霉烯酶基因,占57．1％（8／14）,以blaKPC-2（4／14）及blaNDM-1（3／14）为主。结论：PMQR基因在临床分离的CRKP中较为普遍,以qnr基因为主,且qnr阳性菌株碳青霉烯酶基因携带率较高,均为多重耐药株。     

尿源产超广谱β-内酰胺酶大肠埃希菌质粒介导氟喹诺酮类耐药性研究

目的 研究尿源性产CTX-M型超广谱β-内酰胺酶(ESBLs)菌株中qnrA、qnrB以及qnrS的流行情况和耐药特征,为控制产ESBL细菌感染提供实验依据.方法 琼脂稀释法测定喹诺酮类及氨基糖苷类对46株尿源产ESBL细菌的最低抑菌浓度.PCR法检测qnrA、qnrB以及qnrS的存在情况.结果 46株产CTX-M型大肠埃希菌对环丙沙星、左氧氟沙星、莫西沙星、庆大霉素和阿米卡星的耐药率分别为26.1％、19.6％、13.0％、60.9％和43.5％.46株大肠埃希菌中检出2株含qnrB(4.3％),未检出qnrA和qnrS.结论 尿源性产CTX-M型超广谱β-内酰胺酶大肠埃希菌中有qnrB的存在,且对喹诺酮类以及氨基糖苷类药物耐药性严重,需参考药敏结果,不宜经验性选择喹诺酮类药物及氨基糖苷类药物治疗产ESBL且qnr基因阳性的大肠埃希菌尿路感染及尿源性血流感染.     

肠杆菌科细菌喹诺酮耐药基因qnrB的研究

目的 对2005年10月～2006年4月临床分离的120株肠杆菌科细菌检测qnrB的分布,同时研究qnr阳性菌株与blaSHV、bhtCTX-M或质粒型AmpC β-内酰胺酶基因的关系,为进一步研究质粒介导的喹诺酮类耐药机制(PMQR)提供实验依据.方法 采用碱裂解变性法提纯质粒DNA,PCR法检测qnrA和qnrB的存在,接合实验验证qnr的转移性.扩增qnrB全序列、测序.对所有qnr阳性菌株,采用nitrocefin法检测qnr阳性菌产β-内酰胺酶情况,多重PCR法检测blaSHV、btaCTX-M及多种质粒型AmpC β-内酰胺酶基因.结果 120株肠杆菌科细菌中,qnrA和qnrB的检出率分别为30.8%与27.5%,其中13株细菌同时检出qnrA和qnrB;肠杆菌属、克雷伯菌属细菌及大肠埃希菌qnr的检出率分别为52.9%、59.3%和50.0%.17株细菌可扩增出681bp的全序列片段.测序结果显示,部分菌株包含qnrBl;另有4株细菌的qnrB序列相同,但与qnrB3相比存在3个位点核苷酸序列突变(201位A→T,271位T→G,498位G→A)而导致编码蛋白的氨基酸存在2个位点差异,为新的qnrB亚型(qnrB6),GenBank注册号为:EF520349.所有qnr阳性菌均可使nitrocefin纸片变红色.qnr阳性菌中,49株(89.1%)包含blaSHV或blaCTX-M;约70.0%可检测到质粒型AmpC相关的β-内酰胺酶基因,分别有18和16株细菌可扩增到blaDHA及blaEBC.结论 我院临床存在qnr阳性菌株的流行,发现一种新的qnrB亚型(qnrB6).     

志贺菌临床株耐药性分析及对氟喹诺酮类抗生素耐药机制的研究

目的:了解本地区志贺菌抗生素耐药情况,探讨志贺菌对氟喹诺酮类药物的耐药机制和耐药基因的突变及流行情况。方法:K-B纸片扩散法测定2009—2010年天津地区临床分离的119株志贺菌的药敏情况,对氟喹诺酮耐药志贺菌的gyrA、parC基因及质粒介导的喹诺酮耐药(PMQR)基因qnrA、qnrB、qnrS、aac(6′)-ib-cr、qepA进行扩增、测序,将PMQR阳性菌株与大肠埃希菌J53ARZ进行质粒接合试验,比较接合前后受体菌对抗菌药物的敏感性变化。结果:119株志贺菌对萘啶酸敏感率最低(1.72%),其次为氨苄西林(2.52%)及复方磺胺甲口恶唑(3.36%)。福氏志贺菌与宋内氏志贺菌对氟喹诺酮耐药性差别较大,5株志贺菌对环丙沙星等氟喹诺酮耐药,均为福氏志贺菌,其中2株对左氧氟沙星耐药。4株同时存在gyrA83、87位点及parC80位点突变,1株缺乏gyrA87位点突变。PMQR基因阳性菌3株,包括1株qnrS及2株aac(6′)-ib-cr阳性菌,接合菌对多种抗生素的敏感性降低。结论:本地区志贺菌临床株对多种抗生素的多重耐药率较高,对氟喹诺酮耐药率低于5%。喹诺酮耐药机制既有染色体介导靶位酶突变,也存在质粒介导喹诺酮耐药。     

雏鸭腹泻大肠埃希菌耐药性和氨基糖苷类抗生素耐药基因检测与分析

目的 研究雏鸭腹泻大肠埃希菌的耐药性和氨基糖苷类抗生素耐药基因的存在情况。方法 采取山东济宁某养鸭场腹泻雏鸭粪便病料，通过实验室分离培养，对雏鸭腹泻大肠埃希菌进行药敏试验，比较雏鸭腹泻大肠埃希菌分离株对抗菌药物的敏感性，并采用PCR法对氨基糖苷类抗生素耐药菌株进行氨基糖苷类抗生素耐药基因aac(3)-Ⅰ、aac(3)-Ⅱ、aac(6’)-Ⅰb、aac(6’)-Ⅱ、ant(3”)-Ⅰ和aph(3’)-Ⅱ的检测。结果 在24种药物敏感性试验中，雏鸭腹泻大肠埃希菌对头孢他啶、头孢哌酮、头孢吡肟和阿米卡星4种药物敏感，对氧氟沙星、头孢曲松、头孢噻肟和大观霉素4种药物中度敏感；对青霉素、链霉素、庆大霉素和四环素等16种药物耐药。在20株菌株中，检测的氨基糖苷类抗生素耐药基因中5株细菌检测出aac(3)-Ⅰ，10株细菌检测出aac(3)-Ⅱ，5株细菌检测出aac(6’)-Ⅰb,6株细菌检测出aac(6’)-Ⅱ，8株细菌检测出ant(3”)-Ⅰ，17株细菌检测出aph(3’)-Ⅱ。结论此次试验分离培养出的雏鸭腹泻大肠埃希菌呈现普遍耐药性，普遍检测出氨基糖苷类抗生素耐药基因aac(3)-Ⅰ、aac(3...     

PFGE和质粒图谱分析人和猪来源德尔卑沙门菌多重耐药株的同源性

目的 分析人和猪来源的德尔卑沙门菌的分子同源性,为研究多重耐药性在人畜之间传播提供分子生物学技术依据.方法 药敏试验检测人和猪来源的德尔卑沙门菌多重耐药株对10种抗菌药物的敏感性;用脉冲凝胶电泳方法(PFGE)检测菌株间的分子同源性;结合实验或电转移试验获得目标质粒,质粒酶切图谱检测质粒的同源性.PCR分析染色体介导喹诺酮耐药基因gyrA和arC,质粒可介导的喹诺酮耐药基因qnrA/qnrB/qnrC/qnrS、aac(6)-Ⅰ b-cr、qepA和oqxAB.结果 共收集48株德尔卑沙门菌,经药敏试验筛选获得11株多重耐药株(MDR),人源性8株,猪源性3株.11株菌除对氨苄青霉素、氯霉素、链霉素、磺胺和四环素(R型:ACSSuT)耐药外,所有菌株也对萘啶酸和环丙沙星耐药,6株对左氧氟沙星耐药,5株对头孢曲松耐药.PFGE分析,11株菌共分为6个克隆,3株动物源性菌株与8株人源性菌株无克隆相关性.喹诺酮耐药基因分析显示,有5株和3株动物源株检测到gyrA的Ser83Leu或/和Asp87Asn突变,6株人源株和3株动物源株检测到parC的Ser80Ile或/和Thr57Ser突变.质粒电泳分析显示,所有菌株都存在1-4条质粒条带,大小介于2～180kb,其中3株人源性菌株和1株动物源性株都存在4kb左右大小的质粒;2株动物源性菌株仅存在20 kb大小质粒.用结合实验和电转移方法,分析4kb质粒结构,证实4个质粒的酶切图谱完全一致,质粒中均存在介导外排基因oqxAB和喹诺酮耐药基因aac(6')-Ⅰ b-cr.结论 人和猪来源的德尔卑沙门菌多重耐药株中质粒介导的外排基因oqxAB和喹诺酮耐药基因aac(6')-Ⅰ b-c是喹诺酮重要的耐药机制,可能在沙门菌间传递,导致对喹诺酮类高耐药性.通过人和猪的接触或间接途径,沙门菌有传播喹诺酮耐药性的风险,同一种质粒可在人和动物中转移而传播该耐药机制.     

Prevalence of plasmid-mediated quinolone resistance determinants qnr and aac(6')-Ib-cr in Escherichia coli and Klebsiella pneumoniae producing extended-spectrum β-lactamases in Spain

The presence of the plasmid-mediated quinolone resistance determinants qnrA, qnrB, qnrS and aac(6')-Ib-cr was evaluated in a collection of 382 isolates of extended-spectrum β-lactamase (ESBL)-producing Escherichia coli and Klebsiella pneumoniae collected between February and March 2006 for the nationwide Spanish GEIH-ESBL 2006 project. In total, 14 isolates (3.7%) were positive for qnr genes (3 qnrA1, 5 qnrB-like and 6 qnrS1) and 62 isolates (16.2%) were positive for the mutant variant of aac(6')-Ib-cr. The Aac(6')-Ib-cr enzyme was the most prevalent plasmid-mediated mechanism of quinolone resistance in Spain. Most of the Aac(6')-Ib-cr-producing E. coli isolates (94.2%) carried two mutations in gyrA and two in parC, whilst only 57.2% of K. pneumoniae harbouring this enzyme were gyrA and/or parC mutants. Most qnr plasmids were transferable, but only four were conjugative. Plasmid incompatibility groups were identified for only four plasmids, belonging to FIA, HI2 and I1γ. The most prevalent ESBLs associated with qnr plasmids belonged to the SHV and CTX-M families. The present study highlights the broad geographical spread of qnr-like determinants in Spain and their association with the SHV-12 and CTX-M-9 ESBLs in human clinical isolates.     

肺炎克雷伯菌质粒介导的喹诺酮类抗生素耐药机制的研究

目的 研究质粒介导的喹诺酮类耐药机制(PMQR)在肺炎克雷伯菌临床分离株上的分布情况,并对阳性菌株上染色体介导的喹诺酮类耐药机制进行分析.方法 细菌的鉴定和药敏采用Vitek-2 compact系统;采用PCR法检测质粒介导的喹诺酮类耐药基因qnrA、qnrB、qnrS、aac-(6')-Ib-cr和qepA的分布情况.对包含PMQR的细菌,采用E-试验测定环丙沙星的MIC大小,同时扩增测序分析染色体基因gyrA、gyrB、parC、parE的突变情况.结果 临床分离的67株肺炎克雷伯菌中,qnrS、qnrB、aac-(6')-Ib-cr、qepA的检出率分别为14.93%、2.99%、2.99%和16.42%.8株细菌同时包含qnr和qepA基因,其中2株qnr、qepA和aac-(6')-Ib-cr同时阳性.PMQR阳性菌株对环丙沙星的MIC值不定(0.032～≥64μg/mL),其中8株(占61.54%)对环丙沙星高水平耐药(≥64μg/mL).比对结果显示,环丙沙星MIC≤0.5μg/mL的3株细菌几乎未见染色体的氨基酸序列改变;而环丙沙星MIC≥8μg/mL的菌株全部存在gyrA和parC编码氨基酸序列改变,且突变主要集中在gyrA 83位、87位和parC 80位上.所有PMQR阳性的肺炎克雷伯菌的gyrB和parE均未发现任何氨基酸序列突变.结论 临床分离的肺炎克雷伯菌上检测到qnr、aac-(6')-Ib-cr、qepA的分布与共存.PMQR阳性菌株对环丙沙星的MIC值不定,但染色体机制仍是肺炎克雷伯菌对喹诺酮类抗生素耐药的主要机制,突变主要见于gyrA的83位、87位及parC的80位上.     

Prevalence of β-lactamases and 16S rRNA methylase genes amongst clinical Klebsiella pneumoniae isolates carrying plasmid-mediated quinolone resistance determinants

To characterise the prevalence of β-lactamases and 16S rRNA methylase genes amongst clinical Klebsiella pneumoniae isolates carrying plasmid-mediated quinolone resistance (PMQR) determinants in China, 59 non-duplicate K. pneumoniae isolates harbouring at least one PMQR gene were screened for common β-lactamases and 16S rRNA methylases genes. The genetic relatedness of the isolates was analysed by pulsed-field gel electrophoresis (PFGE). Most of PMQR gene-positive isolates carried no substitutions within the quinolone resistance-determining regions (QRDRs) or single point mutation in GyrA or ParC. Over one-half (52.5%) of the isolates exhibited decreased susceptibility to ciprofloxacin [minimum inhibitory concentration (MIC)=0.5-2 μg/mL] or low-level resistance to ciprofloxacin (MIC=4-8 μg/mL). qnr, aac(6')-Ib-cr and qepA were positive in 52 (88.1%), 16 (27.1%) and 3 (5.1%) isolates, respectively. The identified genes for β-lactamases were distributed as follows: bla(TEM), 50.8%; bla(SHV), 91.5%; bla(CTX-M), 55.9%; bla(DHA), 59.3%; and bla(OXA-1), 22.1%. armA and rmtB were detected in 16.9% and 3.4% of isolates, respectively. All qnrB were detected in DHA-producing K. pneumoniae. Approximately 81.3%, 68.8% and 43.8% of aac(6')-Ib-cr carrying isolates produced OXA-1, DHA and ArmA, respectively. In conclusion, owing to few QRDR substitutions, most of the PMQR gene-carrying K. pneumoniae isolates exhibited low-level resistance to fluoroquinolones. qnr appears to be the predominant PMQR gene and it presented a significant correlation with bla(SHV), bla(CTX-M) and bla(DHA), whereas aac(6')-Ib-cr exhibited a close relationship with bla(OXA-1), bla(DHA) and armA. qepA was rarely detected in this study.     

High levels of multidrug resistance in clinical isolates of Gram-negative pathogens from Nigeria

In Nigeria, quinolones and β-lactam antibiotics are widely used to treat bacterial infections. This study aimed to identify the prevalence of resistance to these drugs and to determine the mechanisms of resistance to these agents. In total, 134 non-duplicate, Gram-negative enteric isolates of 13 species from different hospitals were investigated for susceptibility to a panel of antibiotics, carriage of plasmid-mediated quinolone and β-lactam resistance genes, production of extended-spectrum β-lactamases (ESBLs), and mutations within topoisomerase genes. The level of resistance to all antibiotics tested was extremely high, with minimum inhibitory concentrations for 90% of the organisms (MIC(90) values) of ≥ 256 μg/mL for all drugs. Of the 134 isolates, 92 had mutations within the quinolone resistance-determining region (QRDR) of gyrA or within gyrA and parC. In addition, the plasmid-mediated quinolone resistance genes qnrA, qnrB, aac(6')-Ib-cr and qepA were identified. The qnrD allele, which has previously only been found in Salmonella isolates from China, was identified in two Proteus isolates and one Pseudomonas isolate. Of the 134 isolates, 23 (17.2%) carried aac(6')-Ib-cr, 11 (8.2%) carried a qnr variant and 5 (3.7%) were positive for qepA. Twenty-eight isolates (20.9%) produced ESBL variants, with a CTX-M variant being carried by 25 isolates (18.7%). In addition, six isolates (4.5%) carried ampC variants [ACT-1 (1 isolate), DHA-1 (4 isolates) and CMY-2 (1 isolate)]. This study demonstrates a very high level of multidrug resistance amongst Gram-negative enteric bacilli isolated from different sites from patients in Nigerian hospitals as well as the presence of a variety of plasmid-associated resistance genes, including some identified from Africa for the first time.     

临床分离鲍曼不动杆菌耐药性及qnr基因检测

目的了解临床分离鲍曼不动杆菌中qnr基因和ESBLs基因的分布及其耐药特征。方法采用PCR法对115株鲍曼不动杆菌进行qnrA、qnrB、qnrS基因筛查,并用PCR法检测qnr阳性菌株SHV、TEM、CTX-M-14及CTX-M-3型ESBLs基因;用琼脂对倍稀释法测定15种抗菌药物对qnr阳性株的MIC值。结果115株鲍曼不动杆菌中,2株(1.74%)细菌检出qnrB基因;qnr阳性菌株同时检出SHV、CTX-M-14、TEM、CTX-M-3型ESBLs基因。1株qnr阳性菌株对4种喹诺酮类耐药,1株对左氧氟沙星及莫西沙星中介,对环丙沙星和洛美沙星耐药;2株阳性菌除对亚胺培南和头孢哌酮/舒巴坦敏感外,对其他的β-内酰胺类抗菌药物均耐药。结论临床分离鲍曼不动杆菌中存在qnrB基因,qnr阳性株同时含有ESBLs基因,且呈多重耐药,临床应加强监测。     

Role of IncHI2 plasmids harbouring blaVIM-1, blaCTX-M-9, aac(6')-Ib and qnrA genes in the spread of multiresistant Enterobacter cloacae and Klebsiella pneumoniae strains in different units at Hospital Vall d'Hebron, Barcelona, Spain

Seven Enterobacter cloacae isolates and seven Klebsiella pneumoniae isolates harbouring a phenotype compatible with the production of a metallo-β-lactamase were recovered between 2009 and 2011 in three Intensive Care Units of Hospital Vall d'Hebron (Barcelona, Spain). The presence of bla(VIM), bla(IMP), bla(NDM), bla(CTX-M), aac(6')-Ib, qnrA, qnrB and qnrS genes was screened by polymerase chain reaction (PCR) and sequencing. Clonal relatedness of the isolates was assessed by pulsed-field gel electrophoresis (PFGE) and, in the case of K. pneumoniae isolates, by multilocus sequence typing (MLST). PCR-based replicon typing, Southern hybridisation, plasmid double-locus sequence typing and MOB relaxase classification methods were used to identify and characterise the plasmids carrying the resistance genes. Transferability of the identified plasmids was tested by conjugation assays. All 14 isolates were found to carry bla(VIM-1), bla(CTX-M-9) (except one isolate), aac(6')-Ib and qnrA genes. Clonality assessment demonstrated that E. cloacae isolates were distributed in three clonal clusters, whereas all of the K. pneumoniae isolates belonged to one unique clone, identified as sequence type ST252. All studied isolates harboured a large conjugative IncHI2 MOB(H11) plasmid carrying all of the detected resistance genes. Plasmid DNA analysis showed that all of them belonged to the ST1 IncHI2 plasmid cluster and shared the same relaxase partial sequence. In conclusion, the present study describes the dissemination within a hospital of multiresistant E. cloacae and K. pneumoniae isolates producing VIM-1. A complex clonal epidemiology of the E. cloacae isolates was observed and plasmid DNA analysis strongly supports horizontal exchanges of the same IncHI2 plasmid between different strains and species.     

Detection and genetic characterisation of qnrB in hospital isolates of Klebsiella pneumoniae in Singapore

Polymerase chain reaction (PCR) screening of 116 ciprofloxacin-resistant Klebsiella pneumoniae hospital isolates for the presence of qnr genes that mediate plasmid quinolone resistance revealed that none were positive for qnrA or qnrS. However, qnrB was detected in ca. 5.2% of the isolates. Southern hybridisation demonstrated that the qnrB-hybridising plasmids were large (>70kb) and capable of transferring quinolone resistance by conjugation. Sequence analysis of the qnrB genes detected in this study showed that they were identical to previously identified qnrB1, qnrB4 and qnrB6 genes, although a novel variant designated qnrB20 was also identified. Analysis of the genetic environment around the cloned qnrB genes showed that they were present in diverse plasmid backbones, sometimes within novel genetic contexts, but always associated with mobile or transposable elements.