多重耐药菌中质粒介导喹诺酮耐药基因的检测和分析

目的分析广州医学院第一附属医院临床分离的60株多重耐药大肠埃希菌和肺炎克雷伯菌质粒介导的喹诺酮耐药基因存在状况。方法采用VITEK-2全自动细菌鉴定仪检测大肠埃希菌和肺炎克雷伯菌对抗生素的药物敏感性,选择多重耐药的大肠埃希菌和肺炎克雷伯菌作为研究对象,用多重PCR法进行质粒介导的喹诺酮耐药基因的检测和分析。结果检测出耐药基因qnrA型2株,qnrB型耐药基因15株,aac（6’）-Ib基因9株,其中aac（6’）-Ib—cr型耐药基因5株。其中有1株肺炎克雷伯菌检测到同时含有qnrA和aac（6’）-Ib—cr基因;还有3株肺炎克雷伯菌检测到同时含有qnrB和aac（6’）-Ib—cr基因。未检测到qnrC、qnrD、qnrS、qepA型的耐药基因。结论临床分离的多重耐药大肠埃希菌和肺炎克雷伯菌含有多种质粒介导的喹诺酮耐药基因,应引起临床的重视。     

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.     

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.     

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。     

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.     

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。     

肠杆菌科细菌质粒介导的喹诺酮耐药基因研究进展

继首个质粒介导的喹诺酮耐药基因qnrAl之后,qnrB,qnrS,qnrC和qnrD等其他一些类似基因也相继被发现.另 外,两种质粒介导的喹诺酮耐药机制,即外排泵QepA和OqxAB以及氨基糖苷甲基转移酶Aac(6’)-Ib-cr陆续被报道.本文综述肠杆菌科细菌质粒介导的喹诺酮耐药基因研究进展.     

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...     

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.     

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.     

High prevalence of plasmid-mediated quinolone resistance determinant aac(6')-Ib-cr amongst Salmonella enterica serotype Typhimurium isolates from hospitalised paediatric patients with diarrhoea in China

In this study, the antimicrobial susceptibilities and prevalence of plasmid-mediated quinolone resistance determinants amongst Salmonella enterica serotype Typhimurium isolates from hospitalised paediatric patients with diarrhoea in China were investigated. In total, 40 (64.5%) of 62 S. Typhimurium isolates were resistant to ciprofloxacin (minimum inhibitory concentration ≥0.5 μg/mL), comprising 28 isolates with low-level resistance and 12 isolates with high-level resistance. All ciprofloxacin-resistant isolates were multiresistant to other antimicrobial agents. Four pulsed-field gel electrophoresis (PFGE) clusters were found amongst the 40 ciprofloxacin-resistant isolates, amongst which PFGE clusters A, B, E and D accounted for 7, 4, 1 and 28 isolates, respectively. Two isolates with high-level ciprofloxacin resistance had two mutations in the quinolone resistance-determining regions (QRDRs) of gyrA and parC. The remaining ciprofloxacin-resistant isolates had only one mutation in the QRDR of gyrA. All 62 S. Typhimurium isolates were negative for qnr genes and qepA and 23 (37.1%) of the isolates were positive for aac(6')-Ib-cr. Nineteen isolates harbouring aac(6')-Ib-cr belonged to PFGE cluster D. A high prevalence of ciprofloxacin resistance and aac(6')-Ib-cr was found amongst S. Typhimurium isolates in China from hospitalised paediatric patients with diarrhoea not receiving quinolones. A single mutation in the QRDR of gyrA as well as production of AAC(6')-Ib-cr contributed to ciprofloxacin resistance. Clonal spread was responsible for the dissemination of aac(6')-Ib-cr amongst S. Typhimurium isolates.     

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.     

Analysis of plasmid-mediated multidrug resistance in Escherichia coli and Klebsiella oxytoca isolates from clinical specimens in Japan

This study investigated the relationship of plasmid-mediated quinolone resistance (PMQR) and aminoglycoside resistance among oxyimino-cephalosporin-resistant Escherichia coli (n = 46) and Klebsiella oxytoca (n = 28) clinical isolates in Japan. Seventy-three isolates appeared to produce an extended-spectrum β-lactamase (ESBL) and one K. oxytoca isolate produced IMP-1 metallo-β-lactamase (MBL). Polymerase chain reaction (PCR) and sequencing confirmed that eight CTX-M-9/SHV-12-producing isolates, one IMP-1-producing K. oxytoca isolate, and six ESBL-positive E. coli isolates respectively possessed PMQR genes qnrA1, qnrB6, and aac(6′)-Ib-cr. All qnr-positive isolates also carried either aac(6′)-Ib or aac(6′)-IIc aminoglycoside acetyltransferase genes. Resistance determinants to β-lactams, quinolones and aminoglycosides were co-transferred with a plasmid of ca. 140 kb. The qnrA1 gene was located downstream of insertion sequence ISCR1 in complex class 1 integrons. A novel qnrA1-carrying class 1 integron with the cassette arrangement aac(6′)-IIc–aadA2 as well as a unique class 1 integron with bla_IMP-1–aac(6′)-IIc cassettes on the plasmid carrying qnrB6 were found in K. oxytoca isolates. We describe the identification of qnrB6 and aac(6′)-Ib-cr and the close association of qnr with aac(6′)-Ib and aac(6′)-IIc for the first time in clinical isolates producing ESBL or MBL in Japan.