【作者】 邵宜波；

【导师】 李旭；

【作者基本信息】 安徽医科大学 ， 内科学， 2012， 博士

【摘要】 目的了解枸橼酸杆菌中质粒介导喹诺酮耐药基因（PMQR）阳性率，以期发现新型PMQR基因。掌握临床分离的PMQR阳性枸橼酸杆菌对临床常见药物的敏感性及耐药性。了解PMQR基因有无与ESBL及整合子共同传播，为临床合理使用喹诺酮类药物提供依据。了解新型质粒介导喹诺酮耐药基因（qnrB24）的耐药特性和旁侧序列。材料与方法菌株来源收集安徽医科大学第一附属医院检验科2009年临床分离的枸橼酸杆菌，采用全自动微生物分析仪对标本进行重新鉴定。方法用煮沸法提取细菌总DNA，聚合酶链反应（PCR）方法扩增qnr，aac(6，)-Ib-cr，qepA基因。PCR产物纯化测序法明确具体基因型别，测序结果在Genbank上比对。应用PFGE、ERIC-PCR技术分析PMQR阳性菌株克隆流行传播情况。对PMQR阳性的菌株进行转移接合实验。对收集的PMQR阳性枸橼酸杆菌及其接合子，根据2010年CLSI的标准，采用琼脂对倍稀释法进行临床常用抗菌药物的药物敏感实验。对PMQR阳性菌株进行整合子及ESBL的基因分型。对新发现的质粒介导喹诺酮耐药基因qnrB24基因阳性菌株克隆测序，同时进行转移接合实验，了解qnrB24对喹诺酮类药物的耐药性，碱裂解法提取接合子质粒，Southern blot明确质粒大小。采用热不对称交错PCR技术研究qnrB24基因5’端以及3’端未知侧翼碱基序列，结果通过GenBank比对进行DNA序列分析。在研究该耐药基因旁侧序列的基础上，初步预测该基因是否可能导致快速的水平或垂直传播。结果2009年安医大一附院收集的枸橼酸杆菌共31株，用特异性PMQR引物扩增后，8株弗劳地枸橼酸杆菌扩增结果阳性，经测序均为qnr基因型，qnr的阳性率是25.8%（8/31），qnrA1，qnrB1, qnrB2，qnrB4, qnrB10和qnrB24的阳性率分别是6.5%（2/31），3.2%（1/31），6.5%（2/31），3.2%（1/31），3.2%（1/31），3.2%（1/31），没有发现aac(6，)-Ib-cr，qepA基因。8株PMQR阳性菌株中的7株细菌对环丙沙星的MIC值在8-64μg/ml，其中一株细菌对环丙沙星耐药表型为敏感，MIC值为1μg/ml。qnr阳性菌株对喹诺酮类药物的耐药率在87.5%，对头孢噻肟、阿米卡星、头孢他啶、头孢吡肟和庆大霉素的耐药率分别为75.0%，37.5%，62.5%，37.5%和87.5%，所有PMQR阳性菌株对亚胺培南耐药表型为敏感。PFGE和ERIC-PCR结果显示菌株间没有一致条带，不属于同一血清型。4株qnr阳性的菌株的qnr基因（3，4，13，23号）成功地进行了转移接合，qnrA基因转移接合率是100%（2/2），qnrB基因转接合率33.3%（2/6）。与受体菌E.coilJ53相比较，qnr阳性的接合子对喹诺酮类药物的敏感性下降，环丙沙星的MIC值是0.125-1.0μg/ml，左氧氟沙星的MIC值是0.19-0.75μg/ml，qnr阳性接合子对喹诺酮药物的MIC值较受体菌升高了10-23倍，部分接合子也呈现出对其他抗菌药物的协同耐药性。3株qnr阳性枸橼酸杆菌携带ESBL基因，分别是CTX-M-3型、SHV-12型、CTX-M-27型。其中4号菌株同时携带有CTX-M-3和SHV-12型。qnr阳性菌株中I类整合酶阳性率是75.0%（6/8），其中3株qnr阳性菌株的I类整合子可变区扩增结果阳性，耐药基因盒排列为dfrA12-aadA2。测序分析显示一株qnr阳性的弗劳地枸橼酸杆菌有3个碱基位点发生有义突变，3个突变位点分别为139位C→A，257位C→T，670位A→G，相应的氨基酸发生变化，分别为47位亮氨酸→蛋氨酸，86位丙氨酸→缬氨酸，224位异亮氨酸→缬氨酸。这个突变基因命名为qnrB24（Genbank登录号：HM192542）。药敏结果显示携带qnrB24基因克隆表达株对环丙沙星，左氧氟沙星的MIC值均为0.25μg/mL。接合子对环丙沙星的MIC值为0.125μg/ml，左氧氟沙星MIC值为0.19μg/ml，表达株对常见氟喹诺酮类抗菌药物的敏感性较E.coil JM109受体菌下降约8倍,接合子对常见氟喹诺酮类抗菌药物的敏感性较E.coil J53受体菌下降约8-11倍，表达株及接合子耐药水平低于临床分离菌株。Southern blot显示该基因位于约60kb质粒上。热不对称交错PCR结果显示qnrB24基因5，端为假定的转座酶。结论本地区枸橼酸杆菌中qnr基因型的检出率较高，以qnrB基因型为主，没有发现aac(6，)-Ib-cr，qepA基因型。2009年安徽医科大学第一附属医院来源的的PMQR阳性枸橼酸杆菌流行病学上没有克隆相关性。qnr阳性菌株中部分携带ESBL基因，同时大都携带I类整合酶基因。qnr阳性枸橼酸杆菌对喹诺酮、头孢类、氨基糖甙类等常用抗菌药物耐药性较高，应加强耐药性监测和防治。在qnr阳性的枸橼酸杆菌中进行测序后发现一种新型质粒介导喹诺酮耐药基因，qnrB24（Genbank号：HM192542）。qnrB24基因通过质粒介导引起细菌对喹诺酮类药物的耐药性上升。qnr阳性菌株的喹诺酮耐药性可以通过质粒在不同菌株间传递。qnr基因的单独存在可以使细菌对喹诺酮药物的敏感性降低，但单独作用对喹诺酮类抗菌药物靶位点的保护作用相对比较低，临床菌株的耐药性可能是qnr基因与其他因素共同作用的结果。更多还原

【Abstract】 ObjectivesThis study was conducted to detect and analyse the presence of plasmid-mediatedquinolone resistance (PMQR) determinants [qnr, aac(6′)-Ib-cr and qepA] among clinicalisolates of Citrobacter freundii strains isolated from patients in Anhui, China. Thegenotyping of them was investigated. The I、II、III Class integron and ESBL gene wereinvestigated.Materials and MethodsIsolatesIn2009,31Citrobacter strains were collected from the first affiliated hospital of Anhuimedical university.MethodPCR was used to detect PMQR genes. The class I, II and III integrons as well asExtended-spectrum β-lactamase were also detected by PCR, and their contribution toresistance of antimicrobial agents were analysed.Conjugation experiments were conducted to determine whether the qnr-carrying plasmidswere self-transferable. The relatedness of PMQR positive isolates was analysis by PFGE, ERIC-PCR.The susceptibility of the positive isolates and transconjugants were tested by agar dilutionmethod according to CLSI guidelines. The minimum inhibitory concentrations (MICs) ofciprofloxacin and levofloxacin were determined by Etest strips according to themanufacturer’s instructions.The qnrB24PCR product was purified by double-enzyme cleavage method, cloned intoPHSG398, and expressed in E. coli JM109competent cells. The plasmid DNA oftransformant was extracted, then the recombinant plasmid was comfirmed by restrictionenzyme analysis and sequenced.Southern blotting was used to reveal that the novel gene located on a plasmid.The TAIL-PCR was used to understand the genetic environment of the qnrB24gene.ResultThe qnr genes were detected in eight isolates, whereas aac(6′)-Ib-cr and qepA were notidentified in these isolates. qnrA1, qnrB1, qnrB2, qnrB4, qnrB10and qnrB24werepresent in6.5%,3.2%,6.5%,3.2%,3.2%and3.2%of Citrobacter freundii isolatesrespectively. Sequence analysis identified one novel qnrB variant (qnrB24).The results of PFGE, ERIC-PCR showed that PMQR positive isolates were not clonerelated.87.5%qnr-positive clinical isolates were resistant to quinolone resistance. Most of themwere also resistant to various antibiotics, including aminoglycosides, β-lactams, et al. The qnr genes were transferred from four clinical isolates to their transconjugants. All MICs oftransconjugants showed reduced susceptibility to fluoroquinolones.The ESBL gene and I class integron were found in almost qnr positive isolates.Sequence analysis revealed that the qnrB24gene had a maximal identity of98.7%to theqnrB10gene. Three nucleotide changes were observed. The C→A at nucleotide position139resulted in Leu→Met, the C→T at position257produced a Ala→Val change, and theA→G at position670produced a Ile→Val change.Susceptibility results showed that the MIC of transformant carrying qnrB24gene againstciprofloxacin, levofloxacin was both0.25μg/ml. The transconjugant of carrying qnrB24gene against ciprofloxacin, levofloxacin was0.125μg/ml,0.19μg/ml respectively. Thesensitivity of the transformant and transconjugant was lower than the recipient starin, butthe drug resistance was lower than the clinical isolate.Southern hybridization of plasmid DNA from transconjugant of qnrB24revealed thatthe gene was located on about60kb plasmid.The quinolone resistance of qnrB24could be transferred by conjugation.There was putative transposase adjust to5’flank of qnrB24gene.ConclusionOur study shows that qnr gene has occurred in Citrobacter freundii isolates from AnhuiProvince, China. qnr gene was therefore present in both quinolone-resistant and-susceptible isolates andsome of them could be transferred by conjugation experiments.qnr positive isolates strains showed multi-resistance and no clone was spread found inthese isolates.Class I integrons and ESBL genes were prevalence among PMQR positive isolates.Meanwhile, The qnrB24gene was discovered firstly. qnrB24gene could increase the drugresistance to fluoroquinolones slightly. The qnrB24could be transferred by plasmid.PMQR provide the low-level quinolone resistance shown in vitro to facilitate theemergence of higher-level resistance in the presence of quinolones at therapeutic levels.Our study suggests that surveillance for PMQR determinants should be undertaken on aregular basis.更多还原