一组泛耐药鲍曼不动杆菌中发现拓普异构酶Ⅳ编码基因新的变异型

目的 调查一组泛耐药鲍曼不动杆菌中喹诺酮类耐药相关基因的存在与变化状况.方法 收集南通大学附属医院2011年1月-2011年4月患者标本中分离的泛耐药鲍曼不动杆菌共20株,采用聚合酶链反应(PCR)及序列分析的方法分析喹诺酮类药物作用靶位基因gyrA与parC,以及可移动遗传元件可介导的喹诺酮类耐药基因[ qnrA、qnrB、qnrS、qepA、aac( 6')-Ⅰ b-cr].结果 本组20株鲍曼不动杆菌均检出gyrA基因第83位密码子TCA→TTA有义突变(氨基酸序列Ser→Leu).parC基因第80位密码子TCG→TTG有义突变(氨基酸序列Ser→Leu),并同时存在3个同义突变(第40位密码子CCC→CCT同义突变;第41位密码子GTA→GTT同义突变;第44位密码子CGT→CGC 同义突变),且本组parC基因序列为新的变异型.可移动遗传元件可介导的喹诺酮类耐药基因qnrA、qnrB、qnrS、qepA、aac(6’)-Ⅰ b-cr均没有检出.结论 本组鲍曼不动杆菌耐喹诺酮类药物可认为主要与喹诺酮耐药决定区(QRDR)突变相关,发现parC基因新的变异型国内未见报道.     

养殖动物分离的大肠埃希菌质粒介导喹诺酮耐药机制的研究

目的 调查养殖业动物大肠埃希菌对喹诺酮类抗生素的耐药情况,阐明耐药机制,为控制畜牧养殖业滥用抗生素提供依据.方法 从7省市9家养殖场采集鸡和猪的肛门拭子样本,分离并鉴定其中的大肠埃希菌,PCR扩增筛选其中的质粒上的喹诺酮耐药基因qnrA、qnrB、qnrS、qnrC、qnrD和aac(6')-I 6-cr,并进行测序和药敏试验,通过接合试验以确定耐药基因的可转移性以及在喹诺酮耐药中的作用.结果 在818株大肠埃希菌中检出38株qnr阳性菌株和75株aac阳性菌株,其中gyrA突变的有15株,parC突变的有13株.结论 养殖业动物分离大肠埃希菌对喹诺酮的耐药性较为普遍,质粒介导的喹诺酮耐药作为一个重要的机制参与其中,提示应加强食源性细菌耐药性的监测.     

环丙沙星药敏检测在大肠埃希菌graA基因突变关系研究

为了解3株3次环丙沙星药敏检测结果不一致的大肠埃希菌gyrA基因基因突变状况，本文对该3株菌和另3株3次环丙沙星药敏检测均为耐药的大肠埃希菌进行了gyrA基因基因喹诺酮耐药区（QRDR）PCR扩增和产物DNA测序。结果为3株3次环丙沙星药敏检测结果不一致的和另3株3次检测均为耐药的大肠埃希菌均存在gyrA基因基因第83和第87位氨基酸密码子的突变（TCG→TTG，GAC→AAC）。     

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.     

环丙沙星药敏检测与大肠埃希菌gyrA基因突变关系研究

为了解3株3次环丙沙星药敏检测结果不一致的大肠埃希菌gyrA基因突变状况,本文对该3株菌和另3株3次环丙沙星药敏检测均为耐药的大肠埃希菌进行了gyrA基因喹诺酮耐药决定区(QRDR)PCR扩增和产物DNA测序。结果为3株3次环丙沙星药敏检测结果不一致的和另3株3次检测均为耐药的大肠埃希菌均存在gyrA基因第83和第87位氨基酸密码子的突变(TCGTTG,GACAAC)。     

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。     

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.     

海藻希瓦菌中发现一种新的qnrA7基因亚型

目的 研究海藻希瓦菌中质粒介导的喹诺酮类耐药基因的分布和特性.方法 PCR检测qnr、qepA、aac(6')Ib-cr基因,阳性产物进行DNA测序以确定其基因型,接合转移试验探讨质粒介导的喹诺酮类耐药基因的体外转移性,E-test法测定菌株MIC,提取质粒对qnrA基因进行初步定位.结果 海藻希瓦菌中检出qnrA基因,为新发现的亚型,命名为qnrA7,GenBank登录号为GQ463707,未检出qnrB、qnrS、qnrC、qnrD、qepA、aac(6')-Ib-cr基因;qnrA7位于约33 kb的质粒上,但体外接合转移试验未成功;该菌对喹诺酮类药物敏感.结论 海藻希瓦菌质粒上检出新的qnrA基因亚型,其作为qnr基因的环境宿主值得关注.     

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.     

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')-Ⅰ b-cr的检测

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')-Ⅰ b-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')-Ⅰ b-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(Aeromonas punctata)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;aa47(6')一Ⅰ b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')-Ⅰ b-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.     

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.     

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.     

Prevalence of plasmid-mediated quinolone resistance genes in uropathogenic Escherichia coli isolated in a teaching hospital of northern Italy

A retrospective study was conducted to determine the prevalence of plasmid-mediated quinolone resistance (PMQR) determinants in uropathogenic Escherichia coli isolated from inpatients and outpatients in a teaching hospital of northern Italy. The presence of qnrA, qnrB, qnrS, aac(6')-Ib-cr, and qepA was evaluated in 76 and 72 nalidixic acid-resistant E. coli, isolated in 2004 and 2006, respectively. Positivity for the aac(6')-Ib-cr gene was demonstrated in 3 of 76 (3.9%) and 8 of 72 (11%) isolates, respectively; no other PMQR determinant was found. All aac(6')-Ib-cr-positive strains also showed two point mutations in the gyrA and parC genes. Most aac(6')-Ib-cr-positive isolates demonstrated the contemporary presence of bla(CTX-M-15), bla(OXA-1/30), and bla(TEM-1) genes and 4/11 harbored a class 1 integron with a dfrA17-aadA5 gene cassette arrangement. Interestingly, all aac(6')-Ib-cr-positive isolates belonged to B2 phylogenetic group, O25b antigen type, multi locus sequence type 131, and to a cluster of approximately 70% similarity level by pulsed-field gel electrophoresis (PFGE). These findings suggest the circulation of the previously described intercontinentally spreading E. coli O25:H4-ST131 clone in our geographical area since 2004. Hybridization studies of the PFGE profiles showed the aac(6')-Ib-cr gene to be associated with different molecular weight bands (40-350 kb) and interestingly aac(6')-Ib-cr chromosomal integration was demonstrated in one strain by I-Ceu I method. This represents the first report to investigate the presence and diffusion of PMQR determinants in northern Italy and to describe aac(6')-Ib-cr chromosomal integration in E. coli.     

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.     

High rates of plasmid-mediated quinolone resistance QnrB variants among ciprofloxacin-resistant Escherichia coli and Klebsiella pneumoniae from urinary tract infections in Korea

The aims of this study were to investigate the prevalence of qnrA, qnrB, and qnrS determinants and their molecular characteristics in ciprofloxacin-resistant isolates of Escherichia coli and Klebsiella pneumoniae from urinary tract infections (UTI) in Korea. A total of 202 nonduplicated clinical isolates of ciprofloxacin-resistant E. coli (n = 143) and K. pneumoniae (n = 59) were collected between July 2005 and August 2006. The qnr determinant screening was carried out by PCR amplification of qnrA, qnrB, and qnrS, and all positive results were confirmed by direct sequencing of the PCR products. For qnr-positive strains and their conjugants, antimicrobial susceptibility tests and pulsed-field gel electrophoresis (PFGE) were performed. The qnrB gene was detected in 41 of the 202 isolates. Among 33 of 59 (55.9%) K. pneumoniae isolates showing qnrB, 29 isolates contained the qnrB4 gene, 3 isolates had the qnrB2 gene, and 1 isolate had the qnrB6 gene. All 8 (5.6%) of the qnrB-positive isolates among the 143 E. coli strains possessed the qnrB4 gene. The minimum inhibitory concentrations (MICs) of ciprofloxacin for the transconjugants were 0.03-2 mug/ml, representing an increase of 4- to 256-fold relative to the recipient, E. coli J53Az(r). Resistances to various other antimicrobial agents also were transferred with the plasmid. The PFGE analysis revealed indistinguishable or closely related patterns in several strains and highly diverse patterns in general. QnrB variants, especially the qnrB4 subtype, are highly prevalent in ciprofloxacin-resistant E. coli and K. pneumoniae from UTI in Korea. The emergence of plasmid-mediated quinolone resistance may contribute by several means to the rapid increase in bacterial resistance to these drugs.