淋球菌gyrA和parC基因突变与氟喹诺酮类药物关系的研究

目的：探讨淋球菌gyrA和parC基因突变与淋球菌耐氟喹诺酮类药物之间的关系。方法：①纸片扩散法检测58株淋球菌对5种氟喹诺酮类药物的敏感性。②E测定法定量检测环丙沙星的最小抑菌浓度（MIC）。③PCR技术扩增gyrA和parC基因的喹诺酮耐药决定区（QRDR）相关序列并作测序分析。结果：①对环丙沙星、氧氟沙星、氟罗沙星、洛美沙星、依诺沙星同为敏感、中介、耐药者分别为2株、4株和39株。②环丙沙星MIC为敏感、中介、耐药分别为2株、17株和39株。③环丙沙星MIC为0．004－0．016μg/mL的2株淋球菌gyrA和parC基因均未发生突变；MIC为0．064－0．094μg/mL的菌株仅发生gyrA单位点突变；而MIC≥0.25μg/mL的菌株均发生gyrA双位点突变。MIC≤0．25μg/mL的菌株无parC基因突变，而MIC≥1.0μg/mL的菌株除出现gyrA双位点突变外均同时发生parC单位点突变。④在发生突变的16株菌中，Ser91(TCC)→Phe(TTC）突变为15株。结论：①gyrA基因突变介导淋球菌对氟喹诺酮类药物低和中水平耐药，而对氟喹诺酮类药物高水平耐药需要parC基因突变的共同参与。②gyrA基因Ser91→Phe的突变是导致淋球菌对氟喹诺酮类药物耐药的关键突变。     

淋病奈瑟菌分离株3种药物耐药基因检测

目的：从基因水平了解淋病奈瑟菌对青霉素类、四环素类、喹诺酮类3种抗菌药物的耐药状况。方法：采用聚合酶链反应(PCR)对常州地区的50株淋病奈瑟菌进行TEM-1基因、tetM基因检测并对gyrA基因的DNA进行测序分析。结果：50株淋病奈瑟菌中32株TEM-1基因阳性，24例tet基因阳性，经PCR扩增和DNA测序，gyrA基因全部存在突变，PPNG—TRNG—QRNG3种分子检测发现青霉素类、四环素类与喹诺酮类二重耐药分别占26％和10％，耐多种药物(MDR)菌株总数已达74％。结论：淋球菌对青霉素类、四环素类、喹诺酮类抗菌药物的耐药性已相当高，迫切需要寻找新的敏感抗菌药物。     

生殖支原体耐药机制及治疗方案的研究进展

生殖支原体是引起非淋菌性尿道炎、子宫颈炎和盆腔炎的病原体,近几年报道生殖支原体对大环内酯类和氟喹诺酮类抗生素的耐药呈逐渐增加的趋势。其对大环内酯类抗生素的耐药机制主要为23SrRNA基因突变,氟喹诺酮类耐药的机制主要为拓扑异构酶IV基因ParC以及DNA促旋酶基因GyrA的突变。随着全球范围内生殖支原体感染率的升高,如何控制生殖支原体耐药成为亟待解决的难题,新的治疗方案主要包括传统抗生素的使用及新型抗生素的开发。     

人型支原体氟喹诺酮耐药性的分子机制

DNA螺旋酶和拓扑异构酶Ⅳ是氟喹诺酮类药物作用的两个靶位，其分别由GyrA、GyrB和ParC、ParE两组基因编码。大量研究表明，这两组基因的变异与人型支原体氟喹诺酮的耐药密切相关。文中将近年来国外在人型支原体氟喹诺酮耐药性基因研究方面的进展作一综述。     

淋球菌对氟喹诺酮耐药性的研究进展

淋球菌对氟喹诺酮耐药性问题已引起人们的广泛关注。淋球菌外膜蛋白结构改变所致的膜通透性下降、细菌对药物的摄取减少,可能是导致淋球菌对氟喹诺酮耐药性的—种重要机制,而最新研究表明:CyrA和ParC基因的突变与淋球菌对氟喹诺酮耐药性之间密切相关。     

淋球菌rpsE基因突变与大观霉素耐药性的研究

目的 探讨淋球菌rpsE基因突变与淋球菌大观霉素耐药的相关性。 方法 对临床分离的4株大观霉素耐药的淋球菌株（MIC128 μg/ml、256 μg/ml）的rpsE基因进行PCR扩增测序分析,寻找可能的突变位点,通过DNA转化技术将含有突变基因的细菌基因组DNA转化入敏感的淋球菌株,检测转化成功的淋球菌的MIC并进行PCR扩增测序,分析发现的突变位点与淋球菌大观霉素耐药的相关性。 结果 4株大观霉素耐药的菌株均发现rpsE基因A70C（Thr24Pro）突变,而16S rRNA大观霉素耐药决定区（SRDR）未发现任何突变;大观霉素敏感菌株的16S rRNA和rpsE基因均未发现突变。转化后获得的大观霉素耐药淋球菌中亦发现了同样的rpsE基因突变。 结论 rpsE基因单点突变与淋球菌对大观霉素耐药相关。     

淋病奈瑟菌mtrR基因启动子区的IR区碱基缺失与其耐药性关系的研究

目的探索淋病奈瑟菌(NG,简称淋球菌)多传递耐药系统R基因(m trR)启动子区反向重复序列(IR)区基因缺失与淋病奈瑟菌染色体所介导的耐药性的关系。方法以琼脂稀释法做药物敏感试验,选择临床敏感株采用自身配对法以次抑菌浓度法诱导筛选出对不同抗生素耐药的淋球菌株,以聚合酶链反应(PCR)扩增包含IR区在内的目的基因后,对扩增产物测序,并将其IR区基因突变与临床分离自然耐药株比较。结果8株敏感株及24株单独耐1种药物菌株及1株诱导的多重耐药株无IR区基因突变,7株诱导多重耐药菌株IR区有碱基A/T缺失,与自然多重耐药株两者相比较碱基缺失无差异。结论淋球菌染色体IR区基因缺失与单独耐一种药物菌株产生无关,IR区基因缺失参与了淋球菌多重耐药株的产生,人工诱导的多重耐药株与自然多重耐药株IR区碱基突变无差异。     

体外诱导解脲脲原体对喹诺酮类药物交叉耐药的研究

目的 探讨经次抑菌浓度喹诺酮类药物多代培养后,解脲脲原体（Uu）敏感株对喹诺酮类药物敏感性的变化及其机制.方法 将3株Uu临床分离株及标准株Uu3在含有次抑菌浓度诺氟沙星、氟罗沙星、环丙沙星或左氧氟沙星的液体培养基中传代培养10-12代后,检测其对4种药物的MIC值.提取标准株及耐药株的DNA,PCR扩增喹诺酮耐药决定区的gyrA和parC基因,测序分析耐药株的基因突变情况.结果 经次抑菌浓度喹诺酮类药物多代培养诱导后,3株临床分离敏感株与Uu3均出现对诱导药物的耐药及交叉耐药,共诱导出16株药物诱导耐药株.在它们及9株临床分离耐药株中,parC基因未检出错义突变,但gyrA基因检出2种错义突变,即：V175A（n=8）和N165D（n=1）.结论 次抑菌浓度喹诺酮类药物可诱导解脲脲原体出现交叉耐药,其产生可能与gyrA基因突变有关.     

淋球菌IR区基因突变及mtrF表达水平与多重耐药的相关性研究

目的探讨淋球菌多传递耐药系统回文序列(IR)区基因突变及mtrF表达水平与多重耐药的相关性。方法采用纸片扩散法测定菌株的耐药性,琼脂稀释法检测菌株的耐药水平,PCR法扩增mtrR编码基因和IR区基因并进行测序分析,同时用RT-PCR测定mtrF基因的表达。结果5株敏感菌及6株仅耐红霉素的菌株mtrR和IR区基因均未发生突变,21株多重耐药菌均发生了突变(8株低～中等水平耐药株发生mtrR编码基因突变,13株高度多重耐药株IR区发生基因突变或同时存在mtrR单位点突变)。高度多重耐药株mtrF基因表达量(1.019±0.161)显著高于敏感组(0.595±0.064)(P<0.01)和低～中等水平多重耐药组(0.671±0.073)(P<0.01),而后两者间mtrF基因表达量无明显差异。结论在介导产生多重耐药过程中,淋球菌IR区基因突变及mtrF基因的高表达可能发挥着十分重要的作用。     

淋球菌对大观霉素耐药基因的初步检测

【摘要】 目的 检测导致大观霉素耐药的淋球菌16S rRNA基因中的突变位点。 方法 对6株大观霉素耐药淋球菌［最小抑菌浓度（MIC） ≥ 128 mg/L］、20株大观霉素敏感菌株（MIC 32 mg/L和16 mg/L各10株）的16S rRNA基因进行DNA扩增和序列测定,分析16S rRNA基因突变情况。 结果 6株大观霉素耐药淋球菌的16S rRNA基因均发生了突变,其中2株（MIC > 256 mg/L）为C1192T突变,1株（MIC 256 mg/L）为C1344T和T1345A突变,1株（MIC 256 mg/L）为T990G和T991C突变,1株（MIC 128 mg/L）为T990G、G1343C和C1344T突变,1株（MIC 128 mg/L）为T991C突变。20株大观霉素敏感菌株均未发生突变。 结论 淋球菌16S rRNA基因不同位点突变可能与大观霉素不同程度的耐药相关,C1192T突变可能导致高度耐药,其他单一位点或多位点突变与不同程度的耐药相关。 【关键词】 奈瑟球菌,淋病; 壮观霉素; 点突变; RNA,核糖体,16S; 抗药性,细菌     

Analysis of quinolone resistance mechanisms in Neisseria gonorrhoeae isolates in vitro

BACKGROUND AND OBJECTIVES: Gonococcal fluoroquinolone resistance is now a significant problem in Japan. We generated gonococcal mutants resistant to norfloxacin in vitro from norfloxacin sensitive isolates and analysed the contribution of three known mechanisms of quinolone resistance in Neisseria gonorrhoeae. MATERIALS AND METHODS: Three clinical isolates of N gonorrhoeae susceptible to norfloxacin were exposed to increasing concentrations of norfloxacin. To identify mutations in the gyrA and parC genes of the gonococcal mutants, the quinolone resistance determining regions of the gyrA and parC genes were polymerase chain reaction (PCR) amplified and the PCR products were directly sequenced. Norfloxacin accumulation in the gonococcal cells was also measured. RESULTS: The MICs of norfloxacin for three variants containing a single GyrA mutation were 16-fold higher than that for their parent isolates. A variant showing reduced norfloxacin accumulation in the cells, without mutations in the GyrA or ParC proteins, was also less sensitive to norfloxacin, with a 16-fold increase in the MIC, compared with the parent strain. The MIC of norfloxacin for a variant which contained a single GyrA mutation with reduced norfloxacin accumulation in the cells was 128-fold higher than for the parent strain. A variant containing mutations in both GyrA and ParC proteins with reduced accumulation of norfloxacin in the cells showed a 256-fold increase in the norfloxacin MIC compared with the parent strain. There was no variant containing a ParC mutation without the simultaneous presence of a GyrA mutation. CONCLUSIONS: The results from this study suggest that not only a mutation in the gyrA gene but also reduced drug accumulation in cells contributes to the development of fluoroquinolone a mutation in the gyrA gene contributes to a high level of fluoroquinolone resistance in gonococci with decreases in accumulation in cells having an additional but lesser effect.


     

High-frequency of quinolone-resistant Neisseria gonorrhoeae in Austria with a common pattern of triple mutations in GyrA and ParC genes

OBJECTIVE: Quinolones have a broad spectrum of antimicrobial activity and are widely used for the treatment of uncomplicated Neisseria gonorrhoeae infections. A dramatic increase in the number of reported N. gonorrhoeae infections as well as quinolone-resistant isolates in Vienna prompted us to investigate the pattern of mutations in these isolates. GOALS: The goal of this study was to investigate the pattern of mutations in GyrA and ParC genes in quinolone-resistant N. gonorrhoeae clinical isolates in Vienna from 1999 to 2002. STUDY: The antibiotic susceptibility of N. gonorrhoeae clinical isolates and point mutations of the GyrA and ParC genes of 104 clinical isolates were analyzed. RESULTS: Quinolone-resistant N. gonorrhoeae isolates increased from 3.9% (3 of 77) in 1999 to 59.4% (120 of 202) in 2002. As expected, none of the 46 N. gonorrhoeae quinolone-sensitive strains showed mutations at these positions of GyrA and ParC genes with the exception of 1 isolate, which had a single mutation at GyrA 91. Unlike what has been previously reported for other geographic areas, 96.6% (56 of 58) of the quinolone-resistant isolates harbored common triple mutations at Gyr 91, 95, and ParC 86. The majority of these isolates (76.8%) belong to the PPNG phenotype. CONCLUSIONS: Our data indicate that the pattern of mutations in GyrA and ParC subunits of N. gonorrhoeae in Austria differs from that reported from other geographic areas. The differences may either be the result of the difference in bacterial subtypes or various antibiotic regimens used in these regions.     

淋球菌mtr系统的IR区基因突变与其多重耐药性的关系

目的 探索多传递耐药(mtr)系统反向重复序列(IR)区基因突变与淋球菌染色体介导多重耐药性的关系。方法 琼脂稀释法做淋球菌药敏试验,选择耐不同药物的菌株,聚合酶链反应扩增包含IR区的目的基因,然后对扩增产物测序。结果 2株敏感株及5株仅耐青霉素菌株无IR区基因突变,17株多重耐药菌株中,1株同时耐青霉素和阿奇霉素的淋球菌有IR区碱基T/A,T/A插入,其余耐药株IR区均有碱基A/T缺失。结论 淋球菌染色体mtrR启动子区域的IR区基因突变会引起淋球菌多重耐药株的产生,增加淋球菌对抗生素的抗性。     

Molecular characterization of ciprofloxacin resistance of gonococcal strains in Spain

BACKGROUND: Over the past several years, the emergence of gonococcal isolates with intermediate or full resistance to fluoroquinolones has become a significant concern in several countries, including Spain. GOAL: The goal was to determine the occurrence of ciprofloxacin resistance among Neisseria gonorrhoeae strains in Spain during 2000 to 2001 and determine the frequency and patterns of mutations at gyrA, gyrB, and parC genes in these isolates. STUDY DESIGN: Eleven ciprofloxacin-resistant strains (with MICs ranging from 1 to 64 micrograms/mL) and two intermediate isolates (with MICs of 0.12 and 0.5 microgram/mL) were found. Mutations were identified by polymerase chain reaction and direct sequencing of the amplified products. RESULTS AND CONCLUSIONS: Alterations at Ser-91 and Asp-95 in GyrA were detected in all strains except one, an isolate for which the MIC was 0.12 microgram/mL. Alterations in ParC were more variable, and there was no clear correlation between the number of parC mutations and the level of resistance. No alterations at gyrB gene associated with ciprofloxacin resistance were found. The resistance was distributed among different types of strains, suggesting that the increase in the incidence of ciprofloxacin-resistant strains in Spain was not exclusively due to the appearance of a single-strain outbreak.     

Mutation in DNA gyrase of norfloxacin-resistant clinical isolates of Neisseria gonorrhoeae.

BACKGROUND AND OBJECTIVES: Recently a rapid decrease in the susceptibility of Neisseria gonorrhoeae isolates to fluoroquinolones has occurred and gonococcal fluoroquinolone resistance is now a significant problem in the treatment of gonorrhoea in Japan. Thus, in order to investigate the quinolone resistance mechanisms in clinical isolates of N gonorrhoeae we studied an alteration in the DNA gyrase subunit A (GyrA) which is well-known as a common mechanism of bacterial quinolone resistance. MATERIALS AND METHODS: Four clinical isolates of N gonorrhoeae resistant to norfloxacin and 5 strains susceptible to norfloxacin, including 2 clinical isolates and 3 WHO reference strains, were tested in this study. To identify mutations in the GyrA genes of gonococcal strains, polymerase chain reaction and direct DNA sequencing were performed. RESULTS: A single base change (serine codon TCC changed to phenylalanine codon TTC), which resulted in an amino acid change in GyrA at position 91, was identified in all 4 norfloxacin-resistant strains for which the MICs of norfloxacin ranged from 1.0 to 8.0 micrograms/ml, while no mutation within GyrA was detected in 5 norfloxacin-susceptible strains for which the MICs of norfloxacin ranged from 0.004 to 0.063 microgram/ml. CONCLUSIONS: The results from this study suggest that the serine-91 to phenylalanine substitution in GyrA is probably an essential mutation in fluoroquinolone resistance in clinical isolates of N gonorrhoeae.     

淋球菌对阿奇霉素耐药性与mtrR/mtrC基因变异的关联分析

目的探讨mtr系统若干基因变异与淋球菌对阿奇霉素耐药性的相关性。方法对临床分离的淋球菌阿奇霉素耐药性代表株mtrR基因序列及mtrR启动子区域回文结构序列的突变进行分析,使用荧光定量PCR技术对结构基因mtrC的表达水平进行测定。结果所有中等以上耐药菌株均发生mtrR基因H105Y的变异,伴随mtrR启动子区域回文结构的缺失突变;敏感株有一株出现G45D的突变,mtrR启动子区域回文结构未检测出突变;中等以上耐药菌株与敏感菌株相比mtrC基因表达有显著差异(P<0.05)。结论mtrR基因的H105Y变异和回文序列碱基缺失与mtrC表达有负向相关关系,与淋球菌阿奇霉素耐药性显著相关。     

Molecular epidemiology of Neisseria gonorrhoeae exhibiting decreased susceptibility and resistance to ciprofloxacin in Hawaii, 1991-1999

BACKGROUND: Clinically significant resistance to Centers for Disease Control and Prevention (CDC)-recommended doses of fluoroquinolones (ciprofloxacin and ofloxacin) has been reported for Neisseria gonorrhoeae. In Hawaii, fluoroquinolone-resistant gonococcal isolates were first identified in 1991. GOAL: To assess the diversity, based on phenotypic and genotypic characterization, of gonococcal isolates exhibiting decreased susceptibility (CipI; MICs = 0.125-0.5 microg/ml) or clinically significant resistance (CipR; MICs > or = 1 microg/ml) to ciprofloxacin in Hawaii from 1991 through 1999. STUDY DESIGN: Antimicrobial susceptibilities, auxotype/serovar (A/S) class, GyrA/ParC alteration patterns, and plasmid profiles were determined for gonococci isolated in Honolulu from 1991 through 1999 that exhibited intermediate or clinically significant resistance to ciprofloxacin. Strain phenotypes were defined by A/S class, GyrA/ParC alteration pattern, and penicillin-tetracycline resistance phenotype supplemented with plasmid profiles for beta-lactamase-producing isolates. RESULTS: Altogether, 68 isolates exhibiting intermediate or clinically significant resistance to ciprofloxacin belonged to 23 and 19 strain phenotypes, respectively. Among the CipI and CipR isolates, 4 and 13 GyrA/ParC alterations patterns were identified, respectively. The 91,95/Asp-86 alteration pattern occurred most frequently among CipR isolates. Forty-four strain phenotypes were represented by only one isolate. In addition, seven pairs and two clusters of isolates were identified. CONCLUSIONS: From 1991 through 1997, few gonococcal strains exhibiting intermediate or clinically significant resistance to CDC-recommended doses of fluoroquinolones were identified from Hawaii. Isolates belonged to a large number of phenotypic and genotypic types, suggesting that most cases were imported, with only a few instances in which isolate pairs indicated that secondary transmission of infections had occurred in Hawaii. Beginning in 1998, the number of CipR isolates increased markedly, and more isolates belonged to fewer phenotypic and genotypic types, suggesting either more frequent importation of fewer strain types or the possibility that the endemic spread of a few strains is beginning to occur.     

不同耐药淋球菌菌株IR基因突变与mtrC基因转录水平的差异性比较

目的：研究不同耐药淋球菌菌株多传递耐药（mtr）系统反向重复序列（IR）区基因突变与mtrC基因转录水平的差异性,进一步探索mtr系统介导耐药的机制。方法：采用琼脂稀释法测定抗生素对菌株的最小抑菌浓度（MIC）,PCR扩增含IR区的目的基因并对扩增产物测序,同时采用反转录（RT）-PCR检测mtrC基因mRNA表达水平的变化。结果：5株敏感株及5株仅耐青霉素菌株无IR区基因突变,16株多重耐药菌株中IR区均有碱基A／T缺失。敏感株mtrC转录水平显著低于耐药株（P〈0．05）,而耐药菌株组中IR区碱基突变组mtrC转录水平显著高于无突变组（JP〈0．05）。结论：淋球菌染色体mtrR启动子区域的IR区基因突变会引起mtrC基因转录增加,进而提高奈瑟淋球菌的耐药性。