gyrA、gyrB和外排系统共同介导铜绿假单胞菌对喹诺酮类耐药机制研究

目的探讨临床分离的对环丙沙星和左氧氟沙星均耐药的铜绿假单胞菌耐药机制。方法收集临床分离经VITEK-2（C0mpact细菌鉴定仪检测环丙沙星和左氧氟沙星均耐药的铜绿假单胞菌,琼脂稀释法测定环丙沙星和左氧氟沙星的MIC值,PCR扩增DNA促旋酶基因gyrA和gyrB以及DNA拓扑异构酶Ⅳ的parC和parE基因,实时-RT-PCR分析细菌外排系统表达情况。结果琼脂稀释法检测结果与VITEK-2 Compact细菌鉴定仪检测结果相符。PCR扩增测序发现以DNA促旋酶基因gyrA（在位点941处插入碱基C）和gyrB（3株在位点1588处缺失碱基A,其他菌株在位点1543处插入碱基T）基因缺失或者插入导致移码突变为主,parE基因有3株在位点1895处插入碱基C。实时定量PCR检测发现以mexA和mexC表达增加为主。结论检出的耐环丙沙星和左氧氟沙星铜绿假单胞菌是由于DNA促旋酶基因gyrA和gyrB基因的突变和mexAB-OprM和mexCD-OprJ表达增加共同作用的结果。     

鲍曼不动杆菌对喹诺酮类药物的耐药机制研究

目的探讨鲍曼不动杆菌对喹诺酮类药物的耐药机制。方法收集我院2006年5月—2007年2月不同患者痰培养标本中分离的鲍曼不动杆菌80株,剔除重复菌株用肉汤稀释法测定菌株在环丙沙星、含有不同浓度羟基氰氯苯胺(CCCP)的环丙沙星、亚胺培南-西司他丁的MIC。PCR扩增gyrA基因和parC基因并进行酶切分析,选取部分PCR扩增产物进行测序鉴定。荧光定量PCR方法检测环丙沙星敏感株与耐药株的adeBmRNA表达的相对定量。结果鲍曼不动杆菌对环丙沙星的敏感率为15%,随加入CCCP的浓度增高而增加,并接近亚胺培南的敏感率。PCR限制性片段长度多态性(RFLP)分析显示,58株耐药菌中39株gyrA基因不能被Hinf I酶切,23株praC基因不能被Hinf I酶切,敏感株均能被Hinf I酶切,耐药株中的gyrA和parC基因存在突变。环丙沙星耐药菌株的adeBmRNA表达量明显高于敏感菌株。结论鲍曼不动杆菌对喹诺酮类药物耐药除了与gyrA、praC基因突变有关外,还与主动外排泵基因adeBmRNA过表达有关。     

铜绿假单胞菌耐药性及氟喹诺酮类耐药相关基因的研究

目的 了解医院临床分离的铜绿假单胞菌耐药情况及氟喹诺酮类相关耐药基因存在状况.方法 用BD Phoenix100全自动微生物分析仪进行细菌鉴定和药物敏感试验,用聚合酶链反应（PCR）检测parC、gyrA两种氟喹诺酮类耐药相关基因.结果 70株铜绿假单胞菌呈现多重耐药,对环丙沙星、左旋氧氟沙星的耐药率分别为51.4%、50.0%,其余11种的耐药率在10.0%～100.0%.在70株铜绿假单胞菌中,39（55.7%）株分离菌检出parC基因,18（25.7%）株分离菌检出gyrA基因.结论 临床分离的铜绿假单胞菌多重耐药严重,携带parC和gyrA基因是本组试验菌株对氟喹诺酮类抗生素耐药的重要机制.     

西安地区志贺氏菌属耐氟喹诺酮类药物基因突变研究

目的 调查西安地区志贺氏菌属对氟喹诺酮类药物的耐药情况,并研究其基因突变.方法 对收集西安地区2004年1月～2009年11月三所三级甲等医院粪便分离的98株志贺氏菌属,经法国生物-梅里埃公司VITEK-32细菌鉴定系统鏊定,Kirby-Bauer法测定菌株对左氧氟沙星、环丙沙星2种喹诺酮类药物药敏试验,PCR扩增gyrA基因和parC基因并进行酶切分析,选取部分PCR扩增产物进行测序.结果 志贺氏菌属对左氧氟沙星耐药率为26.5%(26/98);对环丙沙星耐药率为43.9%(43/98),对2种氟喹诺酮类药物均耐药的有12株,占12.2%.对gyrA基因.对药株有93%(40/43)不能被Hinf I酶切,敏感株100%(55/55)被Hinf I酶切;对parC基因,耐药株69.8%(30/43)不能被Hinf I酶切,敏感株有69.2%(9/13)被Hinf Ⅰ酶切.测序结果 :耐药株中的gyrA和parC基因存在突变,敏感株中则不存在突变.结论 西安地区志贺氏菌属对氟喹诺酮类药物耐药情况严重,其耐药与gyrA和parC基因突变有关.     

运用PCR-RFLP技术研究耐喹诺酮类铜绿假单胞菌gyrA基因的突变

目的 研究临床分离的耐喹诺酮类铜绿假单胞菌(Pseudomonas aeruginosa.PA)gyrA基因突变情况.方法 以铜绿假单胞菌gyrA基因序列为靶序列,用PCR-RFLP、DNA测序等方法,对铜绿假单胞菌gyrA基因突变进行研究.结果 在57株耐喹诺酮类铜绿假单胞菌中,有35株(61.4%)菌gyrA基因的83位出现突变,其突变方式为Thr83(ACC)→Ile(ATC),其余22株(38.6%)耐喹诺酮类铜绿假单胞菌未发现基因位点突变.33株喹诺酮类敏感临床分离铜绿假单胞菌菌株未发现上述突变.gyrA基因的PCR扩增产物Sac Ⅱ酶切片段与它们测序结果一致.结论 gyrA基因83位氨基酸密码子突变(Thr-83→Ile)是临床分离铜绿假单胞菌耐喹诺酮类药物的主要机制之一.     

铜绿假单胞菌整合子Ⅰ和ISCR1分布及ERIC-PCR分型

目的研究临床分离铜绿假单胞菌的整合子Ⅰ和ISCR1的分布情况,并对其进行基因分型。方法分离临床234株铜绿假单胞菌,用WHONET5.4分析菌株药敏情况,PCR检测整合酶Ⅰ、整合子Ⅰ、ISCR1以及ISCR1携带的耐药基因。ERIC-PCR进行基因分型。结果铜绿假单胞菌对阿莫西林/克拉维酸、氨苄西林、氯霉素、头孢唑啉、米诺环素、氨苄西林/舒巴坦高度耐药,对环丙沙星、头孢他啶、头孢哌酮/舒巴坦、阿米卡星、亚胺培南、美洛培南较敏感,118株整合酶Ⅰ阳性,95株Ⅰ类整合子可变区阳性,3株ISCR1和ISCRI携带的耐药基因阳性。118株整合酶Ⅰ阳性铜绿假单胞菌分为89个基因型。结论Ⅰ类整合子广泛存在于铜绿假单胞菌中,ISCRI携带率较低,ERIC-PCR可用于临床分离铜绿假单胞菌的基因分型。     

南京地区铜绿假单胞菌的整合子流行性调查

目的调查南京地区铜绿假单胞菌的整合子流行情况,并分析整合子与铜绿假单胞菌耐药的相关性。方法收集98株南京地区临床分离的铜绿假单胞菌,K-B法测定其药敏情况,简并引物PCR法扩增整合子的整合酶基因,对阳性PCR产物采用HinfⅠ内切酶作限制片段长度多态性(RFLP)分析进行整合子分类。结果98株铜绿假单胞菌对哌拉西林等14种抗菌药的耐药率从14.3%到82.7%。40.8%(40/98)的铜绿假单胞菌中检出整合子;PCR-RFLP结果显示均为Ⅰ类整合子,未检出Ⅱ类和Ⅲ类整合子。结论Ⅰ类整合子较广泛地存在于南京地区铜绿假单胞菌中;整合子与铜绿假单胞菌的耐药和多重耐药具有相关性。     

铜绿假单胞菌的临床分布及对环丙沙星耐药机制的研究

目的 分析医院铜绿假单胞菌（PAE）的临床分布和耐药性,并了解其对环丙沙星的耐药机制.方法 我院2011年9月-2013年9月收集的287株铜绿假单胞菌.采用琼脂纸片扩散法（K-B法）进行药敏试验,微量肉汤稀释法测定环丙沙星对PAE的最低抑菌浓度（MIC）.应用PCR方法扩增PAE的gyrA、gyrB、parC及parE基因,并测序来确定基因的突变.结果 287株铜绿假单胞菌以痰液标本中分离最多（76.3％）;主要感染分布在重症监护室（29.3％）和呼吸内科（22.6％）;且对哌拉西林/他唑巴坦及亚胺培南敏感,敏感率均在70％以上;而对氨苄西林及环丙沙星耐药率高,分别达97.9％和44.9％.在45株（环丙沙星MICs≥32 μg/ml）铜绿假单胞菌中,有42株均存在gyrA基因突变,且28株存在着gyrA基因和parC基因双突变.结论 铜绿假单胞菌多重耐药现象严重,gyrA基因突变是PAE对环丙沙星耐药的主要机制之一.应加强对铜绿假单胞菌的临床分布及耐药性监测,更好地指导临床合理用药,控制医院感染.     

南京地区鲍曼不动杆菌喹诺酮类药物耐药基因突变的研究

目的 调查南京地区鲍曼不动杆菌对喹诺酮类药物的耐药情况,并研究其耐药机制。方法 在南京地区的医院随机收集了73株鲍曼不动杆菌,历时9个月。定量肉汤稀释法测定菌株对左氧氟沙星、环丙沙星和加替沙星3种喹诺酮类药物的MIC,PCR扩增gyrA基因和parC基因并进行酶切分析,选取部分PCR扩增产物进行测序。结果 鲍曼不动杆菌对左氧氟沙星耐药率为39．7％（29／73）;对环丙沙星耐药率为76.7％（56／73）;对加替沙星耐药率为32．9％（24／73）。对3种喹诺酮类药物均耐药的有16株,占21．9％。PCR—RFLP显示,对gyrA基因,耐药株有80．4％（45／56）不能被Hinf Ⅰ酶切,敏感株100％（17／17）被Hinf Ⅰ酶切;对parC基因,耐药株73．2％（41／56）不能被Hinf Ⅰ酶切,敏感株有88.2％（15／17）被Hinf Ⅰ酶切。测序结果：耐药株中的gyrA和parC基因存在突变,敏感株中则不存在突变,并在耐药株中发现gyrA和parC基因新的点突变,Genbank号分别为DQ270238和DQ270239。结论 南京地区鲍曼不动杆菌对喹诺酮类药物耐药情况严重,其耐药与gyrA和parC基因突变有关。     

铜绿假单胞菌整合子基因盒的相关研究

目的对北京大学人民医院不同年度临床分离的铜绿假单胞菌进行整合子基因盒检测,分析其变化趋势及其与细菌耐药性的相关性。方法应用PCR对2006—2008年临床分离的420株铜绿假单胞菌进行整合子检测,对阳性PCR产物采用HinfⅠ内切酶作限制片段多态性(RFLP)分析进行整合子分类,并对整合子阳性株进行耐药基因盒的扩增与测序。结果 420株铜绿假单胞菌中116株(27.6%)检出Ⅰ类整合子,未检出Ⅱ、Ⅲ类整合子。对2006年及2008年的整合子阳性菌株可变区基因盒扩增得到7种不同的基因盒图谱,片段大小在710～2526bp,基因盒为介导氨基糖苷类抗生素耐药的aadB、aadA族和介导甲氧苄啶耐药的dfrA1和dhfrXVB。结论该院铜绿假单胞菌中整合子检出率随年度呈上升趋势,携带的基因盒与其耐药表型有相关性。     

Broad-host-range gyrase A gene probe.

The Escherichia coli gyrase A gene was cloned in the broad-host-range cosmid vector pLA2917. The resulting plasmid, pNJR3-2, conferred quinolone susceptibility on a gyrA mutant of E. coli. To analyze the expression of this E. coli gene in Pseudomonas aeruginosa, pNJR3-2 or pLA2917 was mobilized via conjugation into P. aeruginosa PAO2 and several well-characterized quinolone-resistant mutants of this strain. The vector pLA2917 did not significantly affect the quinolone susceptibilities of any of the P. aeruginosa strains. However, pNJR3-2 conferred wild-type quinolone susceptibility on P. aeruginosa cfxA (gyrA) mutants and intermediate quinolone susceptibility on cfxA-cfxB double mutants of P. aeruginosa. The quinolone susceptibility of P. aeruginosa PAO2 gyrA+ was unaffected by pNJR3-2. Also, pNJR3-2 had no significant effect on P. aeruginosa cfxB (permeability) mutants. These results demonstrate that the DNA gyrase A gene from E. coli is expressed in P. aeruginosa and confers dominant susceptibility on gyrA mutants. Thus, pNJR3-2 can be used to detect the quinolone resistance mutations that occur in the gyrase A gene of this organism. pNJR3-2 also appears to discriminate between mutations in gyrA and mutations which alter permeability. This gyrase A probe was used successfully in the analysis of quinolone resistance in clinical isolates of P. aeruginosa.     

Use of a broad-host-range gyrA plasmid for genetic characterization of fluoroquinolone-resistant gram-negative bacteria.

The gyrA genotypes of ciprofloxacin-resistant clinical isolates of Escherichia coli (n = 3), Klebsiella pneumoniae (n = 4), Providencia stuartii (n = 2), Pseudomonas aeruginosa (n = 1), and Acinetobacter calcoaceticus (n = 1) were analyzed in a dominance test. This test is based on the dominance of a wild-type gyrA gene (gyrA+) over the quinolone resistance allele (gyrA) in a heterodiploid strain. Plasmid pBP515, developed to carry the gyrA+ gene of E. coli K-12 on a broad-host-range vector derived from pRSF1010, was used to obtain heterodiploid strains. Plasmid pBP515 encodes kanamycin and gentamicin resistance and is transferable via mobilization by a pRP1-derived helper plasmid (pRP1H) to strains of several gram-negative species. After the introduction of pBP515, single-cell MICs (as measured by reduction of the viable cell count) of ciprofloxacin and nalidixic acid decreased by 4- to greater than 8,000-fold for all strains tested, and 8 of the 11 strains regained ciprofloxacin susceptibilities similar to those of the respective wild types. The results indicate that (i) high-level fluoroquinolone resistance in clinical isolates of E. coli, K. pneumoniae, P. aeruginosa, and A. calcoaceticus can result from mutational alteration of the gyrA gene, and (ii) gyrA mutations are involved in high levels of fluoroquinolone resistance in P. stuartii. Additional mutations outside the gyrA locus may contribute to resistance in K. pneumoniae and P. stuartii.     

Cloning and nucleotide sequence of Pseudomonas aeruginosa DNA gyrase gyrA gene from strain PAO1 and quinolone-resistant clinical isolates.

The Pseudomonas aeruginosa DNA gyrase gyrA gene was cloned and sequenced from strain PAO1. An open reading frame of 2,769 bp was found; it coded for a protein of 923 amino acids with an estimated molecular mass of 103 kDa. The derived amino acid sequence shared 67% identity with Escherichia coli GyrA and 54% identity with Bacillus subtilis GyrA, although conserved regions were present throughout the sequences, particularly toward the N terminus. Complementation of an E. coli mutant with a temperature-sensitive gyrA gene with the PAO1 gyrA gene showed that the gene is expressed in E. coli and is able to functionally complement the E. coli DNA gyrase B subunit. Expression of PAO1 gyrA in E. coli or P. aeruginosa with mutationally altered gyrA genes caused a reversion to wild-type quinolone susceptibility, indicating that the intrinsic susceptibility of the PAO1 GyrA to quinolones is comparable to that of the E. coli enzyme. PCR was used to amplify 360 bp of P. aeruginosa gyrA encompassing the so-called quinolone resistance-determining region from ciprofloxacin-resistant clinical isolates from patients with cystic fibrosis. Mutations were found in three of nine isolates tested; these mutations caused the following alterations in the sequence of GyrA: Asp at position 87 (Asp-87) to Asn, Asp-87 to Tyr, and Thr-83 to Ile. The resistance mechanisms in the other six isolates are unknown. The results of the study suggested that mechanisms other than a mutational alteration in gyrA are the most common mechanism of ciprofloxacin resistance in P. aeruginosa from the lungs of patients with cystic fibrosis.     

Multidrug-resistant Pseudomonas aeruginosa strain that caused an outbreak in a neurosurgery ward and its aac(6')-Iae gene cassette encoding a novel aminoglycoside acetyltransferase

We characterized multidrug-resistant Pseudomonas aeruginosa strains isolated from patients involved in an outbreak of catheter-associated urinary tract infections that occurred in a neurosurgery ward of a hospital in Sendai, Japan. Pulsed-field gel electrophoresis of SpeI-, XbaI-, or HpaI-digested genomic DNAs from the isolates revealed that clonal expansion of a P. aeruginosa strain designated IMCJ2.S1 had occurred in the ward. This strain possessed broad-spectrum resistance to aminoglycosides, beta-lactams, fluoroquinolones, tetracyclines, sulfonamides, and chlorhexidine. Strain IMCJ2.S1 showed a level of resistance to some kinds of disinfectants similar to that of a control strain of P. aeruginosa, ATCC 27853. IMCJ2.S1 contained a novel class 1 integron, In113, in the chromosome but not on a plasmid. In113 contains an array of three gene cassettes of bla(IMP-1), a novel aminoglycoside resistance gene, and the aadA1 gene. The aminoglycoside resistance gene, designated aac(6')-Iae, encoded a 183-amino-acid protein that shared 57.1% identity with AAC(6')-Iq. Recombinant AAC(6')-Iae protein showed aminoglycoside 6'-N-acetyltransferase activity by thin-layer chromatography. Escherichia coli expressing exogenous aac(6')-Iae showed resistance to amikacin, dibekacin, isepamicin, kanamycin, netilmicin, sisomicin, and tobramycin but not to arbekacin, gentamicins, or streptomycin. Alterations of gyrA and parC at the amino acid sequence level were detected in IMCJ2.S1, suggesting that such mutations confer the resistance to fluoroquinolones observed for this strain. These results indicate that P. aeruginosa IMCJ2.S1 has developed multidrug resistance by acquiring resistance determinants, including a novel member of the aac(6')-I family and mutations in drug resistance genes.     

志贺菌gyrA、parC基因突变与喹诺酮类耐药

目的探讨DNA旋转酶A亚单位（gyrA）和拓扑异构酶Ⅳ C亚单位（parC）基因突变与志贺菌耐喹诺酮类药物的相关性。方法用聚合酶链反应（PCR）检测志贺菌喹诺酮耐药决定区（QRDR）相关gyrA、parC基因并挑选11株菌扩增片段进行DNA测序,分析突变位点与药敏结果的关系。结果11株扩增片段测序结果显示,9株耐萘啶酸菌均在gyrA83位发生有意义突变TCG（Ser）→TTG（Leu）,宋内志贺菌未发生parC基因突变,5株耐萘啶酸、诺氟沙星和／或环丙沙星中介敏感福氏志贺菌在parC80位发生有意义突变AGC（Ser）→ATc（Ile）。结论志贺菌对喹诺酮类药物耐药严重,福氏志贺菌比宋内志贺菌更耐此类药物,靶酶基因突变是其耐喹诺酮类药物的主要机制之一,gyrA Ser83→Leu突变是导致志贺菌临床株对萘啶酸耐药的关键突变。parC基因突变在gyrA基因突变的基础上才会发生,parC突变可能引起诺氟沙星和／或环丙沙星不敏感。     

亚胺培南耐药的铜绿假单胞菌中oprD基因突变研究

目的 研究耐哑胺培南(IMP)铜绿假单胞菌(Pa)外膜孔蛋白oprD基因突变方式和突变意义.方法 对来自临床样本的34株耐IMP的Pa用PCR方法直接扩增oprD基因,扩增产物纯化后进行DNA双向序列分析,测得序列在www.ncbi.nlm.nih.gov/BLAST中的BLASTn(序列号:X63152.1 Pseudomonas aeruginosa PA01)和BLASTx(序列号:NF249649.1 Pseudomonas aeruginosa PA01)进行DNA序列和氨基酸序列分析,并与标准菌株ATCC27853和2株临床分离IMP敏感Pa比较,分析突变类型和可能影响的oprD外膜蛋白功能域.结果 oprD基因突变率高达92.3%(12/13),突变方式多样(包括点突变、缺失突变、插入突变),导致外膜蛋白oprD L2、L3茎环结构上103、115、127、154、158、170、185、186、189位氨基酸改变和(或)移码突变.发现新的碱基变异位点(1 079、1114、1196、1206、1288、1300、1301 bp)和新的氨基酸突变位点(115、127、154、158、185、189位氨基酸).以上突变在标准菌株ATCC27853和2株临床IMP敏感的Pa中均未检测到.结论 oprD基因发生广泛有意突变,导致L2、L3茎环结构氨基酸改变和(或)移码突变,可能影响oprD与IMP结合,导致本组Pa对IMP耐药.     

Mutations in the gyrA and parC genes in fluoroquinolone-resistant clinical isolates of Pseudomonas aeruginosa.

We determined partial sequences of the gyrA and parC genes of the fluoroquinolone-susceptible strain ATCC 27853 and 22 clinical isolates of Pseudomonas aeruginosa. While a single amino acid change in GyrA with or without a change in ParC was found in 14 isolates with decreased susceptibility to fluoroquinolones, 3 higher-level fluoroquinolone-resistant isolates had a double amino acid change in GyrA and a single amino acid change in ParC.     

铜绿假单胞菌的基因芯片检测研究

目的用基因芯片对临床标本铜绿假单胞菌进行快速检测。方法结合临床检验经验,抽取98例临床标本,其中78例常规细菌培养鉴定疑似铜绿假单胞菌感染,20例非铜绿假单胞菌感染,铜绿假单胞菌质控菌株(ATCC27853),大肠埃希菌(ATCC25922),用研究制备出铜绿假单胞菌筛查的基因芯片进行测定,同时进行比对分析。结果铜绿假单胞菌检出符合率为100%,基因芯片检测的标本需要量少,检验时间短,敏感性、特异性、准确性高。结论针对细菌培养的临床标本特点,采用高效的基因芯片检测方法,具有现实的临床意义。     

Role of mutations in DNA gyrase genes in ciprofloxacin resistance of Pseudomonas aeruginosa susceptible or resistant to imipenem.

In Pseudomonas aeruginosa, resistance to imipenem is mainly related to a lack of protein OprD and resistance to fluoroquinolones is mainly related to alterations in DNA gyrase. However, strains cross resistant to fluoroquinolones and imipenem have been selected in vitro and in vivo with fluoroquinolones. We investigated the mechanisms of resistance to fluoroquinolones in 30 clinical strains of P. aeruginosa resistant to ciprofloxacin (mean MIC, >8 micrograms/ml), 20 of which were also resistant to imipenem (mean MIC, >16 micrograms/ml). By immunoblotting, OprD levels were markedly decreased in all of the imipenem-resistant strains. Plasmids carrying the wild-type gyrA gene (pPAW207) or gyrB gene (pPBW801) of Escherichia coli were introduced into each strain by transformation. MICs of imipenem did not change after transformation, whereas those of ciprofloxacin and sparfloxacin dramatically decreased (25- to 70-fold) for all of the strains. For 28 of them (8 susceptible and 20 resistant to imipenem), complementation was obtained with pPAW207 but not with pPBW801. After complementation, the geometric mean MICs of ciprofloxacin and sparfloxacin (MICs of 0.3 microgram/ml and 0.5 microgram/ml, respectively) were as low as those for wild-type strains. Complementation was obtained only with pPBW801 for one strain and with pPAW207 and pPBW801 for one strain highly resistant to fluoroquinolones. These results demonstrate that in clinical practice, gyrA mutations are the major mechanism of resistance to fluoroquinolones even in the strains of P. aeruginosa resistant to imipenem and lacking OprD, concomitant resistance to these drugs being the result of the addition of at least two independent mechanisms.