临床分离铜绿假单胞菌喹诺酮耐药株gyrA基因突变及其对β-内酰胺类药物敏感性研究

利用PCR、限制性片段多态性分析（RFLP）、DNA单链构像多态性分析（SSCP）和DNA测序等方法，对10株成都地区临床分离耐喹诺酮类药物铜绿假单孢菌gyrA基因进行研究。结果表明，成都地区耐喹诺酮类药物铜绿假单胞菌gyrA的喹诺酮耐药决定区编码等83位氨基酸密码子表现出高频单点突变（10株中有8株），其突变方式为ACC→ATC。gyrA的PCR扩增产物SacII酶切片段与测序结果一致。SSCP的带谱与测序结果比较，除1株（PSA2）的SSCP带谱与标准株相同，但测序结果有点突变外，其余菌株与测序结果一致。因此，利用PCR－SSCP－RFLP系统，可快速、准确的检测耐喹诺酮类药物的铜绿假单胞菌gyrA中至少一个碱基的差异。用二倍稀释法测定喹诺酮和β－内酰胺类药物对耐药突变株体外抗菌活性，结果表明，铜绿假单胞菌gyrA基因突变株对诺氟沙星、环丙沙星、左氧氟沙星、头孢他啶、亚胺培南和哌拉西林表现出不同的敏感性。试验所用8株突变株对诺氟沙星和环丙沙星表现出高水平抗性；3株对左氧氟沙星敏感；3株对头孢他啶和哌拉西林表现出耐药，2株对亚胺培南耐药。     

耐氟喹诺酮类铜绿假单胞菌的gyrA基因突变研究

目的：研究临床分离的耐氟喹诺酮类药物铜绿假单胞菌gyrA基因突变情况。方法：测定临床分离的55株铜绿假单胞菌的MIC值，从中筛选出1株敏感菌和8株耐药菌。以标准敏感菌株ATCC27853作为质控菌株，用聚合酶链反应（PCR）扩增gyrA基因的喹诺酮耐药决定区（QRDR），扩增产物片段长度为350bp。用限制性内酶SacⅡ消化PCR产物，同时对上述10株菌的gyrA基因的喹诺酮决定区（QRDR）进行PCR－DNA直接测序分析。结果：临床分离铜绿假单胞菌敏感菌株的gyrA基因QRDR经限制性内切酶SacⅡ消化后出现118bp,232bp两条片段，表明该酶切位点未发生突变，此结果经DNA序列析证实。而耐药菌在酶切后均为一条片段，表明该酶切位点消失，经DNA序列分析发现，8株耐药株在83位（ACC→ATC）均有突变，该单位突变引起氨基酸由Thr→Ile的改变；其中有3株高度耐药菌同时发现在87位（GAC→GGC）有突变，该单位点突变引起氨基酸由Asp→Gly的改变，但没有发现87位点突变单独存在，且双位点突变菌株的MIC值与单一位点突变的MIC值相比，有显著的升高，MIC增加4－32倍，除此之外，有6株耐药菌株在132位有一静止突变（CAC→CAT），该 突变未引起氨基酸的改变。结论：gyrA基因突变是铜绿假单胞菌对氟喹酮类药物产生耐药的主要机制之一，铜绿假单胞菌gyrA上83位和87位突变最为常见。     

耐氟喹诺酮类铜绿假单胞菌gyrA及parC基因突变研究

目的　研究临床分离的耐氟喹诺酮类铜绿假单胞菌gyrA及parC基因突变情况。方法　测定临床分离的 5 5株铜绿假单胞菌MIC值 ,从中筛选出 1株敏感菌和 8株耐药菌 ,以标准敏感菌株ATCC2 785 3作为质控菌株。用聚合酶链反应 (PCR)扩增gyrA及parC基因的喹诺酮耐药决定区 (QR DR) ,扩增产物片段长度分别为 35 1bp、397bp。用限制性内切酶SacⅡ消化gyrAPCR产物 ,同时对上述 10株菌的gyrA及parC基因的喹诺酮决定区 (QRDR)进行PCR DNA直接测序分析。结果　有 8株耐菌株的gyrA基因在 83位 (ACC→ATC)有突变 ,导致氨基酸Thr→Ile的改变 ;有 3株高度耐药菌gyrA基因同时在 87位 (GAC→GGC)有突变 ,导致氨基酸Asp→Gly的改变 ;有 4株耐药菌株的parC基因在 87位有TCG→TTG突变 ,导致氨基酸由Ser→Leu的改变。同时具gy rA和parC突变MIC值是仅具gyrA突变菌株MIC值的 2～ 16倍。未发现parC突变单独存在。另外 ,有 6株耐药菌gyrA的 132位有CAC→CAT的突变 ;所有耐药菌株parC基因 115位有GCT→GCG的突变 ,该突变未引起氨基酸的改变。结论　gyrA83、87位突变及parC基因 87位突变都可引起铜绿假单胞菌对氟喹诺酮类药物产生耐药 ,但以gyrA基因 83位突变为主 ,合并gyrA基因 87位及parC基因 87位突变可增加耐药程度。     

铜绿假单胞菌环丙沙星耐药株Ⅱ类拓扑异构酶基因突变的研究

用PCR方法，扩增26株临床分离铜绿假单力环丙沙星耐药株、4株环丙沙星敏感菌株和PA01菌株的Ⅱ类拓扑异构酶gyrA、gyrB、parC和parE基因的喹诺酮类药物耐药决定区（QRDR）基因片断；并对铜绿假单胞菌Ⅱ类拓扑异构酶基因突变与耐药性关系进行研究。结果表明90％以上的铜绿假单胞菌耐药株表现出Ⅱ类拓扑异构酶基因突变（26株中有24株）。突变主要发生在gyrA基因（22株）和parC基因（14株）上，其中gyrA基因的第83位氨基酸密码子表现出高频突变（22株中有21株，ACC→ATC，占90％），parC基因第80位氨基酸密码子也表现出较高的突变率（14株中有12株，TCG→TTG，占60％），4株耐药株的gyrB和3株耐药株的parE基因发生单点突变，2株耐药株Ⅱ类拓扑异构酶基因朱检测到突变。用二倍稀释法，测定部分喹诺酮和β→内酰胺类药物对耐药突变株的体外抗菌活性。表明铜绿假单胞菌Ⅱ类拓扑异构酶基因突变株对诺氟沙星、左氧氟沙星、哌拉西林、头孢他啶和亚胺培南表现出不同的敏感性。试验所用24株突变株对诺氟沙星呈现100％的耐药；对左氧氟沙星65％的耐药；40％的菌株对哌拉西林表现出耐药；30％的菌株对头孢他啶耐药；75％的菌株对亚胺培南敏感。     

铜绿假单胞菌对喹诺酮类药物耐药作用的研究

目的研究铜绿假单胞茵对喹诺酮类药物的耐药性及其机制。方法收集、分离及鉴定从四川大学华西医院分离的27株铜绿假单胞茵,测定5种喹诺酮类药物的最低抑茵浓度（MIC）及碳酰氰基-对-氯苯腙（CCCP）对喹诺酮类药物的体外抗茵活性。采用RT—PCR方法扩增铜绿假单胞茵外膜蛋白基因oprM,PCR方法扩增外排蛋白阻遏基因mexR、质粒qnr基因。结果5种喹诺酮类药物中没有一种对27株铜绿假单胞菌完全敏感,均出现了比较高的耐药水平,且具有交叉耐药性。CCCP能使部分耐药 细菌的MIC逆转。耐药菌株oprM基因在RNA水平上表达增加,mexR经DNA测序表明临床分离铜绿假单胞菌主动外排耐药株基因在编码66住氨基酸处发生了碱基突变oqnr基因经PCR扩增后电泳无条带显示,表明没有质粒介导的耐药产生。结论成都地区铜绿假单胞菌临床分离株对5种喹诺酮类药物呈现交叉耐药。主动外排是导致铜绿假单胞菌耐药性的重要因素。     

结核分支杆菌耐喹诺酮分子机制的研究

目的了解结核分支杆菌耐喹诺酮药物的分子机制，建立快速的药敏的方法。方法通过16S rRNA聚合酶链反应-单链构象多态性(PCR—SSCP)技术分析77株分支杆菌临床分离株；通过PCR—SSCP和直接测序技术(PCR—DS)分析结核分支杆菌的gyrA基因突变的情况。结果75株为结核分支杆菌复合群。以结核分支杆菌标准菌株H37Rv和卡介苗为对照．30株喹诺酮敏感株的gyrA基因的SSCP图谱均泳动正常，测序分析与对照株相同。45株耐喹诺酮的菌株中，34株(75．6％)gyrA基因SSCP图谱泳动异常；测序证实10株为90位密码子的突变，24株为94位密码子突变，所有gyrA突变株的氧氟沙星4μg／ml≤MICs≤32vg／ml，左氧氟沙星2μg／ml≤MICs≤16vg／ml。结论gyrA基因突变，尤其90位和94位密码子突变是结核分支杆菌耐喹诺酮的主要分子机制，PCR—SSCP可能成为测定部分结核分支杆菌喹诺酮耐药基因型的简便、快速的方法，并有望直接用于临床标本喹诺酮敏感性试验。     

铜绿假单胞菌耐亚胺培南相关基因OprD2研究

目的研究铜绿假单胞菌对亚胺培南的耐药机制。 方法取临床分离的铜绿假单胞菌18株，经K-B法检测耐药性，根据耐药性分为耐亚胺培南菌组12株和敏感菌组6株。另设1标准菌株ATCC27853。采用PCR法检测各菌株的OprD2基因，探讨OprD2基因突变与细菌对亚胺培南耐药之间的关系。结果12株耐药菌经OprD2基因扩增，8株阴性，4株阳性；6株敏感菌株及标准菌株扩增全部为阳性。统计学检验结果表明，对亚胺培南耐药菌组和敏感菌组OprD2基因阳性率的差异有极显著性（P＜0.01），且在耐药菌中发现了新的OprD2基因突变方式。 结论OprD2突变是铜绿假单胞菌对亚胺培南耐药的重要机制，突变的方式有缺失突变与插入失活。     

肺炎克雷伯茵耐氟喹诺酮机理研究

目的：探讨肺炎克雷伯菌耐氟喹诺酮类药物的机理。方法：采用NCCLS推荐的K—B法测定50株肺炎克雷伯菌对氟喹诺酮类药物的耐药性并初步筛选出耐环丙沙星菌株。试管稀释法测定耐环丙沙星菌株对环丙沙星和左氧氟沙星的最低抑菌浓度（MIC）。对此23株菌GyrA的基因（gyrA）进行PCR扩增和基因序列的分析比较。结果：23株耐环丙沙星肺炎克雷伯菌中,20株存在gyrA变异：Ser83（TCC）→Phe（TTC）、Tyr（TAC）,Asp87（GAC）→Asn（AAC）、Ala（GCC）。结论：肺炎克雷伯菌对氟喹诺酮类药物耐药机理主要与药物作用靶位gyrA基因的突变有关,并且gyrA基因突变位点越多其耐药程度越高。     

突变选择窗内筛选的大肠埃希菌耐药突变体靶位变异位点的研究

目的研究不同药物浓度、不同化学结构的氟喹诺酮类药物对突变选择窗（MSW）内筛选的大肠埃希菌耐药突变体的靶位基因的影响。方法应用5种氟喹诺酮类药物在突变选择窗内接种约1×10^10菌量的ATCC25922筛选耐药突变体；用琼脂平板二倍稀释法测定ATCC25922和耐药突变体的MIC；用PCR及DNA测序方法确定ATCC25922和耐药突变体耐药决定簇（QRDRs）的gyrA、parC的突变位点和相应的氨基酸变化。结果在ATCC25922的MSW中，筛选53株耐药突变体，所有突变体均为gyrA位点突变，无parC位点突变。其中有79％（42株）为Set-83→Leu位点突变，19％（10株）为Asp-87→Asn，2％（1株）为Gly81→cys位点突变；83位和87位为大肠埃希菌的常见突变位点。氟喹诺酮对Set-83→h突变体的MIC。较Gly81→Cys突变体的MIC90高2～8倍，较Asp-87→Asn突变体的MIC90高1～2倍；Ser-83→Leu是所有的突变位点中对耐药影响最重要的位点。结论氟喹诺酮对大肠埃希菌的主要靶位是GyrA；83位和87位突变为大肠埃希菌最常见突变位点。     

两种氟喹诺酮类药物对肺炎克雷伯菌防耐药突变浓度及耐药突变体耐药性的研究

目的测定两种氟喹诺酮类药物（FQNs）对肺炎克雷伯菌（KP）的防耐药突变浓度（MPC），比较其防耐药突变能力，了解突变耐药菌对FQNs的耐药性。方法肉汤法富集10^10CFU／ml的菌液接种于不同浓度环丙沙星及加替沙星琼脂平皿上，采用琼脂二倍稀释法测定环丙沙星、加替沙星对临床分离肺炎克雷伯菌、ATCC700603及其耐药突变体的最低抑菌浓度（MIC）、防耐药突变浓度（MPC）。对不同药物浓度筛选出的耐药突变株进行编码拓扑异构酶VIC亚单位基因parC和回旋酶A亚单位基因gyrA喹诺酮耐药决定区的PCR扩增和测序，并测定外排泵抑制剂羟基氰氯苯腙（Carbonyl Cyanide m-chlorophenylhydrazone，CCCP）对环丙沙星和加替沙星MIC的影响。结果环丙沙星、加替沙星对ATCC700603的MPC分别为4、1.6mg／L，细菌耐药选择指数分别为16、4。两种药物的耐药突变体均出现了gyrA的第83位（TCC→ATC／TTG）均出现了突变，引起了相应氨基酸的改变（Ser-83→Ile／Leu）。其中有一株同时也出现了第87位点（GAC→AAC）的改变，氨基酸也由Asp→Asn。除第二步突变体parC第80位点突变（AGC→ATC），引起氨基酸由Ser→Ile的改变外，其余几株均没有发生parC的突变。MIC测定结果显示，单用两种FQNs及合用CCCP的结果一样。结论加替沙星限制KP耐药突变株选择的能力强于环丙沙星，KP的耐药突变株对FQNs耐药的主要原因是gyrA和parC基因突变。     

Detection of novel gyrA mutations in nalidixic acid-resistant isolates of Salmonella enterica from patients with diarrhoea

The aim of the current study was to detect mutations in the gyrA gene of quinolone-resistant Salmonella spp. isolates recovered in Tehran, Iran. Between April 2008 and September 2009, 174 Salmonella spp. were collected and assayed for quinolone resistance and detection of gyrA mutations. Isolates identified as Salmonella enterica were tested for susceptibility by the disk diffusion method. Polymerase chain reaction (PCR) amplification and sequencing of the gyrA gene segment encoding the quinolone resistance-determining region (QRDR) were performed for the nalidixic acid-resistant isolates. Amongst the 174 recovered Salmonella spp. isolates, 89 were resistant to nalidixic acid, of which 9 were resistant to enrofloxacin; 10 isolates had reduced susceptibility to nalidixic acid. None of the isolates were resistant to ciprofloxacin, but a single isolate showed reduced susceptibility. Twelve types of amino acid replacement were found in the QRDR region of GyrA, namely the previously described substitutions in positions 83 and 87 as well as five new substitutions Leu41-Pro, Arg47-Ser, Ser111-Thr, Ala118-Thr and Asp147-Gly. Double substitutions in both positions 83 and 87 were not identified. A Gly133-Glu substitution was identified in a single S. enterica serotype Typhi isolate.     

大肠埃希菌gyrA、parC和marOR基因突变与喹诺酮耐药的相关性

目的:研究大肠埃希菌gyrA、parC和marOR基因突变与喹诺酮类耐药的相关性。方法:采用微量稀释法进行常规药敏试验,筛选3株萘啶酸敏感大肠埃希菌和37株萘啶酸耐药大肠埃希菌株;PCR扩增大肠埃希菌喹诺酮耐药决定区(QRDR)相关gyrA、parC基因,进行聚合酶链反应-单链构象多态性(PCR-SSCP)分析,同时PCR扩增marOR基因;在耐药株选取部分菌株对gyrA、parC及marOR基因进行测序,检测其突变情况,其结果与体外药敏试验结果进行比较,研究其相关性。结果:37株耐药株均出现gyrA基因突变,但对环丙沙星低耐株最低抑菌浓度(MIC)=2mg/L只出现gyrA单位点突变,而parC基因未发生突变;环丙沙星高耐株(MIC=64mg/L)gyrA基因出现3个位点突变,parC基因出现单位点突变;在环丙沙星高耐株(MIC=256mg/L),并伴有其他类抗菌药物的多重耐药时,除了出现gyrA和parC基因双位点突变,同时检测到marOR基因的多位点突变。结论:gyrA和parC基因突变在大肠埃希菌对喹诺酮耐药中起着重要作用,gyrA和parC基因突变的程度与大肠埃希菌耐药水平有关,marOR基因多位点突变在多重耐药机制中具有一定的作用。     

碳青霉烯类异质性耐药铜绿假单胞菌的耐药机制研究

摘要：目的 筛选铜绿假单胞菌临床分离株的碳青霉烯类异质性耐药菌株,并分析相关机制。方法 采用E-test纸条法、 群体分析法(PAP)法对30株铜绿假单胞菌临床分离株进行亚胺培南异质性耐药的初筛和复筛、肉汤微量稀释法测定异质性耐药菌 株对亚胺培南及其他药物的MIC值、全基因组测序分析碳青霉烯类异质性耐药菌株的可能耐药机制。结果 通过E-test纸条法和 PAP法鉴别筛选出8株亚胺培南异质性耐药铜绿假单胞菌,检测发现异质性耐药菌株对多黏菌素B、头孢他啶、美罗培南耐药率 较高,对环丙沙星等更敏感。全基因组测序分析发现碳青霉烯类异质性耐药铜绿假单胞菌无可确证的基因突变,但可能与延伸 因子P基因有关。     

Comparison of gyrA gene mutations between laboratory-selected ofloxacin-resistant Mycobacterium tuberculosis strains and clinical isolates

To understand the relationship between mutations in the quinolone resistance-determining region (QRDR) of the gyrA gene and drug resistance to ofloxacin, 85 laboratory-selected ofloxacin-resistant Mycobacterium tuberculosis mutant strains and 110 M. tuberculosis clinical isolates, screened by denaturing high-performance liquid chromatography to contain mutations, were analysed for their mutation patterns by sequencing as well as their ofloxacin minimal inhibitory concentrations (MICs). All mutations detected occurred at the codons Ala74, Ala90, Ser91 and Asp94 in all strains. One of the five different forms of missense mutation in Asp94 occurred in 60% of the laboratory-selected strains and 78% of the clinical isolates. However, 53 clinical isolates (48%) and only 2 laboratory-selected strains (2.4%) harboured double point mutations. The mutation Ala74Ser occurred only in the clinical isolates and only in combination with the Asp94Gly mutation. The ofloxacin MIC for the clinical isolates ranged from 0.5microg/mL to 20microg/mL, whilst the MICs for the laboratory-selected strains were > or =10microg/mL. The differences in gyrA gene mutation patterns and MICs between the laboratory-selected resistant strains and clinically isolated resistant strains identified here might help to understand the mechanisms involved in fluoroquinolone resistance.     

Contribution of mutations in DNA gyrase and topoisomerase IV genes to ciprofloxacin resistance in Escherichia coli clinical isolates

DNA gyrase (GyrA and GyrB) and topoisomerase IV (ParC and ParE) are the two essential type II topoisomerases in Escherichia coli. These enzymes act via inhibition of DNA replication. Mutations in the quinolone resistance-determining region (QRDR) of the gyrA, gyrB, parC and parE genes from clinical isolates of E. coli were determined by DNA sequencing of 54 ciprofloxacin-resistant clinical isolates from a hospital in Delhi, India. The majority of the E. coli isolates were shown to carry mutations in gyrA, parC and parE. Ciprofloxacin resistance due to accumulation of such a high number of mutations in the QRDR regions of gyrA at positions Ser83 and Asp87 and parC at position Ser80 as well as outside of the QRDR region of parE at Ser458 and Glu460 confers high-level resistance of ciprofloxacin in clinical isolates. The high frequency of occurrence of mutations in the parE gene (44.4% strains) is alarming, as topoisomerase IV is a secondary target of quinolones.     

Analysis of topoisomerase mutations in fluoroquinolone-resistant and -susceptible Campylobacter jejuni strains isolated in Senegal

In this study, topoisomerase mutations in ciprofloxacin-resistant and -susceptible Campylobacter jejuni were analysed by DNA sequencing. In certain ciprofloxacin-resistant C. jejuni, the mechanism of resistance was complex. The Thr86-Ala substitution in the GyrA protein appears to play a role in increasing the minimum inhibitory concentration of nalidixic acid only. In addition, isolates with this amino acid change and those resistant to quinolones but lacking a mutation in the GyrA quinolone resistance-determining region could be derived from two different clones. Based on gyrA and gyrB polymorphisms, C. jejuni isolates from the Dakar region of Senegal appeared to be less diverse than those from other countries. Moreover, C. jejuni isolates in Senegal appeared to differ from European isolates by lack of a silent mutation at codon 120 of the gyrA gene.     

淋病奈瑟菌的耐药性与喹诺酮耐药基因突变的相关性分析

目的 分析本地区淋病奈瑟菌临床菌株gyrA基因突变与喹诺酮类药物耐药的相关性.方法 采用琼脂稀释法检测37株淋病奈瑟菌临床菌株对6种抗生素的最小抑菌浓度(MIC),PCR法扩增淋病奈瑟菌临床菌株gyrA基因片段并测序.结果37株淋病奈瑟菌临床菌株对大砚霉素、头孢曲松、四环素、青霉素、氧氟沙星和环丙沙星的耐药率分别为0、...     

Update on fluoroquinolone resistance in Helicobacter pylori: new mutations leading to resistance and first description of a gyrA polymorphism associated with hypersusceptibility

Helicobacter pylori eradication by standard therapy is decreasing due to clarithromycin and metronidazole resistance. Fluoroquinolones are valuable drugs for alternative therapy, but their activity needs to be updated. We determined minimum inhibitory concentrations (MICs) of the newly marketed fluoroquinolones (levofloxacin, moxifloxacin and gatifloxacin) and assessed the prevalence of resistance in 128 H. pylori strains isolated in 2004-2005. The quinolone resistance-determining region (QRDR) of gyrA was sequenced for all strains. Gatifloxacin MICs (MIC(50) = 0.25 mg/L) were two- to four-fold lower than those of the other fluoroquinolones. The prevalence of resistance (ciprofloxacin MIC > 1 mg/L) was 17.2% (22 strains). All resistant strains harboured one gyrA mutation at codons 86, 87 or 91, including three new mutations (Asp86Asn, Thr87Ile and Asn87Tyr). Ciprofloxacin-susceptible strains were devoid of such gyrA mutations, but harboured a polymorphism at codon 87 that distinguished 18 isolates (17%) with a Thr87 like the reference strain J99 from 88 strains with Asn87 like the reference strain 26695. Strains with Thr87 were four-fold more susceptible to nalidixic acid, pefloxacin, ciprofloxacin and levofloxacin and were equally susceptible to moxifloxacin and gatifloxacin. The high rate of quinolone resistance in H. pylori requires the use/implication of a 'test and treat' strategy that can confidently rely on QRDR gyrA sequencing.     

Type II topoisomerase mutations in clinical isolates of Enterobacter cloacae and other enterobacterial species harbouring the qnrA gene

The qnr genes are transferable genes that confer low-level quinolone resistance by protection of topoisomerase. The occurrence of mutations in DNA gyrase (gyrA, gyrB) and topoisomerase IV (parC, parE) genes in strains harbouring qnr was investigated in 28 qnrA-positive clinical isolates, among which 7 strains also harboured qnrS. Topoisomerase mutations were found in 25 (89%) of the 28 strains, with at least two mutations (gyrA and parC) in 13 strains and one mutation in 12 strains. Isolates of the Enterobacter cloacae complex were compared with reference strains of the new Enterobacter species. gyrA mutations were found at position 83 (Ser or Thr for Ile, Tyr, Leu or Phe depending on the species), and new gyrB mutations were described (S463A, S464F). qnrA had an additive effect of a 10-fold increase in the minimum inhibitory concentration (MIC) whatever the number of topoisomerase mutations, and qnrS was additive to qnrA with a further 2- to 10-fold increase in the MIC. Comparison of MICs with susceptibility breakpoints showed that strains combining qnrA and topoisomerase mutations were resistant to fluoroquinolones, but the three strains lacking a topoisomerase mutation were susceptible using ciprofloxacin and levofloxacin but not using nalidixic acid or moxifloxacin testing.