5株第一类整合子阳性铜绿假单胞菌的耐药机制分析

目的了解5株分离于临床的第一类整合子阳性铜绿假单胞菌分子生物学特点。方法应用PCR的方法确定5株铜绿假单胞菌含有第一类整合子并且对其整合耐药基因盒进行扩增和测序,通过PFGE分析该5株菌的同源性。结果5株铜绿假单胞菌均为第一类整合子阳性菌株并且都携带2360bp的耐药基因盒,包括aacA4和含有终止突变的cmlA1。PFGE结果显示5株铜绿假单胞菌来自同一克隆。结论整合子在临床致病菌的多重耐药性中起着重要作用,本研究提示应加强细菌耐药性的监测以及防止相关病菌在院内的传播。     

铜绿假单胞菌Ⅰ类整合子相关基因的解析和定位

目的对4株铜绿假单胞菌临床菌株携带的Ⅰ类整合子相关基因进行解析和定位,探讨Ⅰ类整合子相关基因在细菌耐药性形成和耐药基因播散中的作用。方法采用PCR扩增、DNA测序、DNA序列比对的方法对4株铜绿假单胞菌临床菌株携带的Ⅰ类整合子相关基因进行解析,采用接合试验对该类整合子进行定位分析。结果4株铜绿假单胞菌I类整合子都含有耐药基因,该耐药基因在相应菌株中发挥耐药作用;该菌整合子位于质粒上。结论Ⅰ类整合子耐药基因在铜绿假单胞菌耐药性形成和耐药基因的播散中发挥作用。     

铜绿假单胞菌I类整合子相关基因的解析和定位

目的对4株铜绿假单胞菌临床菌株携带的I类整合子相关基因进行解析和定位，探讨I类整合子相关基因在细菌耐药性形成和耐药基因播散中的作用。方法采用PCR扩增、DNA测序、DNA序列比对的方法对4株铜绿假单胞菌临床菌株携带的I类整合子相关基因进行解析，采用接合试验对该类整合子进行定位分析。结果4株铜绿假单胞菌I类整合子都含有耐药基因．该耐药基因在相应菌株中发挥耐药作用；该菌整合子位于质粒上。结论I类整合子耐药基因在铜绿假单胞菌耐药性形成和耐药基因的播散中发挥作用。     

多重耐药铜绿假单胞菌的耐药性及耐药基因检测

目的:了解铜绿假单胞菌耐药性及β-内酰胺酶基因类型,指导临床合理用药。方法:琼脂稀释法测定12种抗生素对临床分离70株铜绿假单胞菌的最低抑菌浓度;筛选出20株多重耐药菌,三维试验检测产ESBLs和AmpC酶的情况;用聚合酶链反应(PCR)法检测β-内酰胺酶基因并进行测序分析。结果:铜绿假单胞菌对亚胺培南、阿米卡星和环丙沙星的敏感率较高;6株菌产AmpC酶,2株菌产ESBLs;PCR检测出4株菌产TEM酶,经测序2株产TEM-1型,一株产TEM-29型,另一株产酶与TEM-29比较有1处氨基酸的改变,命名为TEM-147;未检出SHV、OXA、PER和VEB型β-内酰胺酶基因。结论:必须重视铜绿假单胞菌ESBLs的检测,对于该菌引起的感染采用碳青霉烯类、阿米卡星或环丙沙星治疗是较好的选择。     

多重耐药铜绿假单胞菌判定方法探讨

目的探讨多重耐药铜绿假单胞菌（MDR—PA）的监测方法,分析MDR—PA的耐药情况。方法收集2009年1月至2011年12月我院分离的铜绿假单胞菌（PA）591株,将11种抗菌药物分为6大类,筛查MDR—PA,分析MDR—PA的耐药性。细菌鉴定和药敏试验采用VITEK2Compact全自动细菌培养鉴定仪。结果在6大类抗菌药物中,耐药率最高的为氨基糖苷类（34．2％、163／477）,最低的为碳青霉烯类（12．9％、76／591）。除碳青霉烯类一氨基糖苷类-力口酶抑制剂复合类外（18例）,其他含有碳青霉烯类的9组,MDR—PA监测结果均≤9例,而非含有碳青霉烯类的10组,MDR—PA监测结果均≥22例。氨苄西林、氨苄西林／舒巴坦、头孢唑啉、呋喃妥因、头孢呋辛酯、复方新诺明在6类抗菌药物中耐药率均〉95．4％;左旋氧氟沙星、头孢他啶、氨曲南、环丙沙星、头孢吡肟和哌拉西彬他唑巴坦对耐碳青霉烯类的PA抗菌活性尚可（耐药率27．6％～35．1％）,对其他5类非耐碳青霉烯类的PA抗菌活性最好的是亚胺培南（耐药率13．7％-24．0％）;对其他5类非耐氟喹诺酮类的PA抗菌活性中,左旋氧氟沙星、环丙沙星抗菌活性尚可（耐药率27．6％～49．0％）。结论筛查MDR—PA时,应优先选择耐头孢菌素类、加酶抑制剂复合类、氨基糖苷类、单环β-内酰胺类,少选择耐碳青霉烯类,以便提高MDR—PA的检出率。     

铜绿假单胞菌中整合子的分布及其可转移耐药性研究

目的 研究淮北地区Ⅰ类、Ⅱ类整合子在多重耐药铜绿假单胞菌(PA)中分布及其可转移耐药情况.方法 用聚合酶链反应(PCR)技术榆测多重耐药菌中整合子基因,并对整合酶基因阳性菌株进行转移接合试验;采用K-B法对接合转移成功菌株及接合子进行13种药物敏感试验.结果 多重耐药铜绿假单胞菌中Ⅰ类整合子检出率为86.1%,未检出Ⅱ类整合子,含整合酶基因的接合子在转移成功接合子中所占比率为35.7%.结论 淮北地区铜绿假单胞菌中整合子的分布以Ⅰ类整合子为主,且水平传播率达到35.7%,应引起高度重视.     

不同地区铜绿假单胞菌第一类整合子和整合子相关基因盒的分布

目的对石家庄、昆明两地分离的铜绿假单胞菌临床菌株进行第一类整合子的分布及整合子相关基因盒携带率的比较分析，探讨第一类整合子在两地菌株分布的差别。方法采用PCR方法筛选检测64株石家庄、昆明两地临床分离的铜绿假单胞菌第一类整合子和及其所携带的整合子相关基因盒。结果64株铜绿假单胞菌临床菌中共有43株为第一类整合子阳性菌（阳性率67．2％）；其中石家庄菌株和昆明菌株阳性率分别为71．4％和64．3％，两地铜绿假单胞菌第一类整合子阳性率无显著性差异。第一类整合子阳性铜绿假单胞菌中，石家庄菌株和昆明菌株携带一类整合子相关基因盒的阳性率分别为80．0％和38．％，两地第一类整合子阳性菌中整合子相关基因盒的携带率具有显著性差异（P〈0．01）；两地菌株携带的基因盒大小不同。结论第一类整合子在铜绿假单胞菌临床菌株中广泛分布，环境的选择压力影响细菌整合子相关基因盒的分布。     

多重耐药铜绿假单胞菌β-内酰胺类相关耐药基因研究

目的 调查我院2005年临床分离多重耐药铜绿假单胞菌（PA）的耐药性分析并探讨其耐药基因的存在状况。方法对31株临床分离的铜绿假单胞菌用纸片扩散法检测其药敏表型,采用聚合酶链反应（PCR）法检测β-内酰胺酶编码基因和外膜通道蛋白OprD2基因,并与相关药敏表型比较。结果31株铜绿假单胞菌对16种抗菌药物的耐药率为48．4％～100％;β-内酰胺酶编码基因PER、VIM、CARB、IMP、TEM、OXA-10的检出率分别为6．4％、3．2％、3．2％、3．2％、3．2％、3．2％,未检出GES、SHV、VER基因,31株外膜通道蛋白OprD2基因缺失,检出率为100％。结论本院多重耐药铜绿假单胞菌对β-内酰胺酶类抗生素的耐药主要与外膜通道蛋白OprD2基因缺失有关。     

亚胺培南耐药铜绿假单胞菌的金属β-内酰胺酶检测

目的:了解金属β-内酰胺酶在广州地区铜绿假单胞菌中的流行状况及其流行亚型,完善广州地区金属β-内酰胺酶的分子流行病学资料.方法:采用双纸片协同法和双纸片增效法对164株亚胺培南耐药的铜绿假单胞菌筛选金属β-内酰胺酶表型阳性的菌株,采用PCR检测5类金属β-内酰胺酶的编码基因(IMP家族、VIM家族、GIM-1、SPM-1和SIM-1),利用生物信息学的方法对检测到的金属β-内酰胺酶亚型进行比对分析并制作分子进化树.结果:164株亚胺培南耐药的铜绿假单胞菌中筛选出22株金属β-内酰胺酶表型阳性的菌株,IMP阳性15株(9.1%),其中11株为IMP-9亚型,另外4株携带一个新的亚型,命名为IMP.25,VIMI阳性5株(3.0%),均为VIM-2亚型,未检测到GIM-1、SPM-1和SIM-1型金属β-内酰胺酶.结论:广州地区铜绿假单胞菌产金属β-内酰胺酶的基因型已经出现多型化的特征,同时存在IMP型和VIM型金属β-内酰胺酶的流行,本次研究发现了一个新的金属β-内酰胺酶亚型(IMP-25),为IMP家族金属p.内酰胺酶的多样性及其分子流行病学资料提供了新的信息.     

耐碳青霉烯类铜绿假单胞菌金属β-内酰胺酶基因检测及其与整合子的关系研究

目的 了解临床分离铜绿假单胞菌对常用抗生素的耐药特征;调查金属β-内酰胺酶在铜绿假单胞菌的流行状况和基因型以及与整合子之间的关系.方法 收集广东省4家三甲医院临床分离的铜绿假单胞菌,采用K-B纸片扩散法进行药敏试验;双纸片协同法和增效法筛选金属β-内酰胺酶表型阳性菌株;设计合成金属β-内酰胺酶和整合子的引物序列进行PCR扩增和测序分析,检测金属β-内酰胺酶基因型并分析其与整合子之间的关系.结果 287株铜绿假单胞菌对11种抗生素的耐药率分别为:亚胺培南(33.80％),美罗培南(33.45％)、头孢他啶(27.87％)、头孢吡肟(36.24％)、庆大霉素(45.30％)、阿米卡星(36.94％)、环丙沙星(33.10％)、左氧氟沙星(37.63％)、氨曲南(47.39％)、哌拉西林(35.19％)、哌拉西林/三唑巴坦(28.57％);多重耐药菌占34.84％(100/287),泛耐药菌株占9.41％(27/287);共有9株铜绿假单胞菌呈金属β-内酰胺酶表型阳性,其中2株产IMP-9型MBL,1株产IMP-25型MBL,4株产VIM-2型MBL;有8株金属β-内酰胺酶表型阳性菌株携带了Ⅰ类整合子,但只有2株成功扩出可变区,其中1株携带了IMP-9基因.结论 铜绿假单胞菌耐药情况严重,本地区铜绿假单胞菌中MBLs的基因型以IMP-9、IMP-25和VIM-2为主;MBLs的基因环境较为复杂,部分MBLs为整合子所携带.     

耐亚胺培南铜绿假单胞菌β-内酰胺酶相关基因及Ⅰ类整合子的检测

目的 研究耐亚胺培南铜绿假单胞菌(PA)β-内酰胺酶相关基因及Ⅰ类整合子的携带情况.方法 采用K-B法对临床分离的PA进行药敏试验,选出26株耐亚胺培南PA,用PCR法检测其β-内酰胺酶相关基因和Ⅰ类整合子(int Ⅰ1和qacE△1-sull基因).结果 26株耐亚胺培南PA体外对15种抗菌药物的药敏结果显示对多黏菌素B的敏感率为100%,对头孢哌酮/舒巴坦、哌拉西林、哌拉西林/他唑巴坦、头孢他啶、阿米卡星的敏感率分别为61.54%、61.54%、65.39%、30.77%、50.00%.对复方新诺明的耐药率为100%.16种基因检出率由高到低依次为qacE△1-sull(92.31%),OprD2缺失(46.15%),TEM(42.31%),OXA-10(26.92%),VIM(11.76%),SPM(7.69%),VEB(7.69%),SHY(7.69%),未检出IMP、GIM、PER、GES、CARB、DHA、CTX-M、intⅠ 1基因.结论 耐亚胺培南PA的耐药主要与Ⅰ类整合子、OprD2缺失有关.     

Complete sequence of pBM413, a novel multidrug resistance megaplasmid carrying qnrVC6 and blaIMP-45 from pseudomonas aeruginosa

This study aimed to characterise a novel multidrug resistance megaplasmid carrying qnrVC6 and bla_IMP-45 from Pseudomonas aeruginosa strain Guangzhou-Pae617 isolated from a patient hospitalised in Guangzhou, China, in 2012. The plasmid pBM413 has a length of 423 017?bp and an average G?+?C content of 56.41%. A qnrVC6 gene flanked by two copies of insertion sequence (IS) elements ISCR1, a multiresistance class 1 integron In786 containing aacA4–bla_IMP-45–bla_OXA-1–catB3 cassettes, an armA gene, and an aphA7 gene flanked by two copies of IS26 were identified. To our knowledge, this is the first identification of a qnrVC6 gene in P. aeruginosa.     

Identification of an atypical integron carrying an IS26-disrupted aadA1 gene cassette in Acinetobacter baumannii

An unusual class 1 integron was identified that carries an IS26-disrupted aadA1 gene cassette (designated as 'integron-IS26') in an imipenem-resistant Acinetobacter baumannii (IRAB) outbreak strain. DNA sequencing revealed that integron-IS26 contained two gene cassettes, the aac(6')-Im cassette and a peculiar aadA1 cassette that was disrupted by IS26 (disrupted aadA1 cassette). Southern blotting localised integron-IS26 to the chromosome. Nested polymerase chain reaction (PCR) was performed to define the frequency of integron-IS26 in five groups of bacteria. Nested PCR identified integron-IS26 in 19 (73.1%) of 26 clinical outbreak strains of IRAB, 10 (100%) of 10 IRAB isolated from environmental cultures, 3 (13.0%) of 23 imipenem-susceptible A. baumannii (ISAB) non-outbreak strains, 1 (3.6%) of 28 netilmicin- and tobramycin-resistant A. baumannii and none of the netilmicin- and tobramycin-resistant Pseudomonas aeruginosa. In conclusion, we have identified a novel class I integron that carries the aac(6')-Im cassette and an IS26-disrupted copy of aadA1 (integron-IS26) in most IRAB outbreak strains and in a few ISAB non-outbreak control strains. Integron-IS26 is located chromosomally.     

定量分析铜绿假单胞菌第一类整合酶基因的表达

目的整合于是具有整合和表达外来耐药性基因盒能力的基因元件。在细菌耐药性基因的积累和传递中起重要作用。本研究通过定量分析第一类整合子整合酶基因（intI1）在细菌不同生存状态的表达水平，探讨整合子的作用机制。方法培养和提取两株第一类整合子阳性铜绿假单胞菌的液相培养菌、生物被膜菌、被膜脱落菌等三种生长状态的总RNA，采用竞争RT-PCR方法定量检测菌体在以上三种状态下intI1 mRNA的表达水平。结果铜绿假单胞菌的液相培养菌、生物被膜菌和被膜脱落菌都有intI1 mRNA表达；两株菌在三种状态下intI1 mRNA的表达量不同，其中生物被膜菌表达最高，被膜脱落菌次之，液相培养菌最低；生物被膜菌intI1 mRNA的量较液相培养物高约15倍，较被膜脱落菌高约4倍。结论铜绿假单胞菌intI1在生物被膜中mRNA表达上调，随着菌体从被膜脱落以及菌体持续的液相培养intI1表达下调。提示整合子在生物被膜菌中可进行活跃的基因盒的捕获、移动和积累。有利于细菌耐药性的提高及扩散。     

Emergence of multidrug-resistant Salmonella enterica serovar Typhi associated with a class 1 integron carrying the dfrA7 gene cassette in Nepal

A total of 121 Salmonella enterica serovars Typhi and Paratyphi A isolated from enteric fever patients at a university hospital in Nepal between February 2004 and January 2006 were tested for their antimicrobial susceptibility. The occurrence and cassette content of integrons as well as the molecular mechanisms of resistance among the multidrug-resistant (MDR) S. Typhi were evaluated. Thirty-nine percent of the isolates were susceptible to all the antimicrobial agents tested. Seven of the S. Typhi strains were MDR. None of the 121 S. enterica isolates were resistant to ciprofloxacin, cefazolin, rifampicin or kanamycin. All MDR S. Typhi isolates contained a class 1 integron with a single cassette, dfrA7, conferring resistance to trimethoprim. Pulsed-field gel electrophoresis (PFGE) of XbaI-generated genomic restriction fragments yielded 12 different patterns. Five of the seven MDR isolates containing class 1 integrons had an identical PFGE pattern. Resistance to sulfamethoxazole, streptomycin, ampicillin, tetracycline and chloramphenicol was mediated by sul1, strA-strB, blaTEM-like, tetB and catA genes, respectively. To the best of our knowledge, this is the first report of integron-associated multidrug resistance as well as the first molecular characterisation of the mechanism of resistance of S. Typhi isolated from Nepal. This study indicates the spread of integron-associated multidrug resistance in S. Typhi in Nepal.     

Characterization of a novel gene related to antibiotic susceptibility in Pseudomonas aeruginosa

Pseudomonas aeruginosa is a human pathogen with increased intrinsic resistance to a large number of antibiotics used in clinical therapy. Therefore, understanding the mechanisms of resistance and developing therapy alternatives for P. aeruginosa are of profound importance. Previous work from our laboratory demonstrated that several mutants have isolated with altered expression of the phzA1B1C1D1E1F1G1 (phzA1) operon in the presence of sub-inhibitory concentrations (SICs) of tetracycline (TET). The present study investigates the roles of the PA0011 gene in mediating phzA1 expression at SIC of TET. The PA0011 gene encodes 2-OH-lauroytransferase by controlling the synthesis of the cell envelope and the outer membrane. We found that the PA0011 mutant strain was susceptible to several different antibiotics and environmental stresses. Complementation in the PA0011 mutant restored these phenotypes to wild-type levels. In addition, expression of the PA0011 gene, as monitored through a luciferase reporter, is increased at SICs of antibiotics. Indeed, the expression of the PA0011 gene increased about threefold in pqsR and pqsH mutants compared with the wild-type PAO1. However, the PA0011 gene negatively regulates the quorum sensing (QS) system. Taken together, these data suggest that PA0011 is involved in susceptibility to antimicrobial agents in P. aeruginosa, and that its susceptibility effect maybe partly dependent on increased QS expression.     

Survey of extended-spectrum beta-lactamases in Escherichia coli isolates from a Portuguese hospital and characterisation of a novel class 1 integron (In60A) carrying the blaCTX-M-9 gene

Between November 2001 and November 2004, 231 Escherichia coli isolates resistant to beta-lactam antibiotics were identified. In 14 isolates, bla(TEM-24) (2 isolates), bla(TEM-52) (5 isolates) and bla(TEM-26) (7 isolates) were identified. In 145 E. coli isolates with the same M13 fingerprinting profile and the same resistance phenotype, the bla(CTX-M-15) gene was found in association with an insertion sequence ISEcp1. The bla(CTX-M-2) gene was identified in one E. coli isolate (290HSM), and in other E. coli isolate (246HSM) the bla(CTX-M-9) gene was contained in a new complex sul1-type class 1 integron (named In60A). This is the first report of three cefotaximases (CTX-M-15, CTX-M-2 and CTX-M-9) in E. coli isolates from a Portuguese hospital.