淋球菌mtr外排系统研究进展

淋球菌多重耐药(mtr)外排系统控制着淋球菌对脂溶性因子(HAs)的耐受性。淋球菌的mtr基因系统包括mtr调控基因mtrR和mtrCDE基因复合物。mtrR基因编码的是一个转录抑制蛋白MtrR,调节mtrCDE基因的转录。mtrCDE基因复合物则分别编码淋球菌膜蛋白MtrC,MtrD,MtrE,组成的一个能量依赖型外排泵,能把HAs有效地排出细胞外。mtr基因系统中任何基因的突变、丢失、缺失或其编码蛋白结构的改变,都会影响淋球菌对HAs的耐受性。在转录水平上mtr系统对淋球菌的多重耐药性的调节分为两种机制,一种是mtrR依赖调节,另一种是非mtrR依赖调节,另外还存在有mtrA基因对其进行正向调控。     

淋球菌膜蛋白表达与环丙沙星耐药性的相关性研究

目的：探讨淋球菌细胞膜蛋白表达与环丙沙星耐药性的相关关系。方法：采用SDS－聚丙烯酰胺凝胶电泳（SDS－PAGE）和直接荧光法分别检测淋球菌外膜孔蛋白和内膜泵蛋白的表达以及淋球菌对环丙沙星的摄入蓄积量。结果：耐药菌对环丙沙星的蓄积量明显低于敏感菌；敏感菌外膜上均有33kd孔蛋白的表达，而耐药菌外膜上则缺失33kd蛋白；但大部分耐药菌内膜上却存在泵蛋白的表达。结论：淋球菌外膜孔蛋白表达的缺陷及内膜泵蛋白的表达使淋球菌对环丙沙星的蓄积量明显降低，从而导致淋球菌耐药。     

淋球菌氟喹诺酮耐药性的分子机制探讨

目的：探讨GyrA、ParC基因变异与淋球菌氟喹诺酮耐药性的相关关系。方法：首先对78株淋球菌临床分离株环丙沙星最低抑菌浓度(MIC)进行检测和G、TA、ParC基因进行PCR扩增，然后分别将2株敏感菌、2株中介菌和8株耐药菌的GyrA、ParC基因进行DNA序列测定。结果：所有78株淋球菌均扩增出GyrA、ParC基因；2株敏感菌和1株中介菌的GyrA、ParC基因未发现突变，另外1株中介菌中和8株耐药菌中GyrA、ParC基因发现有一个或多个位点突变。结论：GwA、ParC基因变异可能是导致淋球菌氟喹诺酮耐药的重要分子机制。     

广州地区106株淋球菌中脂蛋白分型与环丙沙星耐药相关性研究

目的:研究广州地区106株淋球菌中脂蛋白分型与环丙沙星耐药的相关性.方法:用糖琼脂稀释法测定淋球菌对环丙沙星的MIC值,PCR分别扩增淋球菌的gyrA、parC和脂蛋白基因并测序分析.结果:广州地区106株淋球菌对环丙沙星的耐药率为95 3%,共有19个脂蛋白亚型,最常见的亚型是17c,有49株(46 2%).对环丙沙星耐药的101株菌中,所有菌株在gyrA基因对应氨基酸的第91和95位点上均发生了突变,而parC基因随着MIC值的升高发生位点突变的概率呈上升趋势.在中低水平耐药组中,最常见的脂蛋白亚型是17c和14d,均有12株(28 6%).在高水平耐药组中,最常见的脂蛋白亚型是17c,有35株(59 3%),其次是14d,有11株(18 6%).结论:淋球菌脂蛋白亚型的分布存在地域性,与环丙沙星耐药程度高低无明显相关性.     

外排泵抑制剂对耐药痤疮丙酸杆菌的体外作用

目的 通过外排泵抑制剂羰基氰氯苯腙(CCCP)和苯丙氨酸-精氨酸-β-萘酰胺(PAβN)联合大环内酯-林可酰胺类抗菌药物进行药敏试验，了解外排泵抑制剂对痤疮丙酸杆菌耐药作用的影响。方法 对前期收集的39株痤疮丙酸杆菌，采用琼脂稀释法进行大环内酯-林可酰胺类抗菌药物体外药敏试验，根据药敏试验结果分组进行外排泵抑制剂联合抗菌药物的外排泵抑制试验，以MIC值降低≥4倍为外排泵表型阳性。结果 39株痤疮丙酸杆菌：多重耐药菌株27株、单一耐药菌株2株、敏感菌株10株。多重耐药组：CCCP联合阿奇霉素筛选出5株外排泵表型阳性菌株；CCCP联合克拉霉素筛选出2株外排泵阳性菌株；CCCP联合红霉素、克林霉素、林可霉素时，菌株外排泵表型均为阴性。PAβN联合克拉霉素筛选出2株外排泵表型阳性菌株；PAβN联合红霉素、阿奇霉素、克林霉素、林可霉素时，菌株外排泵表型均为阴性。单一耐药组：菌株外排泵表型均为阴性。结论 外排泵抑制剂CCCP和PAβN能一定程度上降低多重耐药痤疮丙酸杆对大环内酯-林可酰胺类抗生素的耐药性，痤疮丙酸杆菌耐药与外排泵相关。     

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。     

广州淋球菌基因特征及其多抗原序列分型与环丙沙星耐药相关性研究

目的 探讨2014年广州市97株淋球菌环丙沙星耐药株的基因特征及其多抗原序列分型（NG-MAST）与淋球菌耐环丙沙星的相关性。 方法 用琼脂稀释法测定淋球菌对环丙沙星的最低抑菌浓度（MIC）,PCR分别扩增淋球菌的gyrA、parC基因和NG-MAST分型基因porB、tbpB基因并测序,获取耐药菌株的ST型别。 结果 97株淋球菌中95株（97.9%）对环丙沙星耐药。95株环丙沙星耐药菌株均在gyrA基因对应丝氨酸的第91和95位点上发生了突变,其中93株菌出现了parC基因突变。41株高水平耐药株（MIC ≥ 16 mg/L）中35株（85.4%）出现了parC基因87位点突变,54株低水平耐药株中32株（59.3%）出现此突变,差异有统计学意义（χ2 = 7.64,P < 0.05）。96株淋球菌分离株配对后,50株为网站已编号型别,共35个不同的ST型,其中10个ST型含有2 ~ 4个不同的分离株,ST型别中最常见ST5309。对淋球菌菌株系统进化树分析,淋球菌流行株可分为两群,第1群84株中MIC ≥ 16 mg/L的菌株39株占46.4%,第2群12株中只有1株MIC值为16 mg/L,差异有统计学意义（χ2 = 6.27,P = 0.012）。 结论 淋球菌对环丙沙星的高水平耐药主要与parC基因87位点突变相关。NG-MAST分型与环丙沙星耐药程度高低可能存在相关性。     

抑制性消减杂交结合基因芯片研究淋球菌耐头孢曲松的分子机制

目的 探讨体外人工诱导的耐头孢曲松淋球菌的分子耐药机制。方法 在成功诱导淋球菌标准菌株ATCC49226和临床菌株ZSSY00205对头孢曲松耐药的基础上，分别对标准菌株和临床菌株进行诱导后耐药株对诱导前敏感株的抑制性消减杂交，构建差异基因文库。从文库中随机挑取192个差异基因作为探针点样于基因芯片，用分别标记Cy3、Cy5的敏感株、耐药株基因组DNA的RsaI酶切片段同时与芯片杂交，根据芯片扫描图选取差异荧光探针对应的基因进行测序和Blast分析。结果 分别构建淋球菌标准菌株ATCC49226和临床菌株ZSSY00205的诱导后耐药株DNA特异性的消减文库，并分别获得高分辨的基因芯片扫描图，发现两组菌株间有共同的耐药相关基因mtrR、mtrC、gyrB、rpsJ、PJD1。 结论 淋球菌对头孢曲松耐药与mtrR、mtrC、gyrB、rpsJ等基因有关，很可能通过外排泵活性增强的途径介导耐药。伴随淋球菌对头孢曲松产生耐药的同时，可能引起对青霉素、四环素、红霉素、喹诺酮等多种抗菌药物产生耐药，即多重耐药现象。     

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。     

淋球菌流行株抗生素敏感性监测

目的：监测广州地区2007年度分离的淋球菌对青霉素、四环素、大观霉素、头孢曲松和环丙沙星的敏感性，分析耐药菌株的流行特点。方法：采用琼脂稀释法测定菌株对5种抗生素的最小抑菌浓度（MIC），判断敏感性按WHO西太区淋球菌耐药性监测统一标准。用纸片酸度法检测产β-内酰胺酶淋球菌（PPNG）菌株。结果：123株淋球菌中检出115株对青霉素耐药（93，5％），产β-内酰胺酶淋球菌（PPNG）39株（占31．7％）；四环素耐药率为87％，其中质粒介导高度耐四环素淋球菌（TRNG）为72株，占58、5％；环丙沙星耐药率为91，1％；未发现对大观霉素和头孢曲松耐药菌株。青霉素、四环素和环丙沙星的MIC50及MIC90均已超过耐药标准，尤以青霉素为甚，其MIC508及MIC90均超过耐药标准的8倍和大于32倍。结论：淋球菌对大观霉素和头孢曲松的敏感性较高，可作为治疗的首选药物，对青霉素、四环素和环丙沙星耐药率较高，提示对淋病的治疗作用差。     

Antimicrobial susceptibility and biochemical patterns of Neisseria gonorrhoeae strains in Vejle area, Denmark

Background  Strains of Neisseria gonorrhoeae resistant to penicillin and ciprofloxacin have been isolated worldwide. Increasing number of N. gonorrhoeae that lack the enzyme proline aminopeptidase (proA-negative N. gonorrhoeae ) have been detected in many countries all over the world.
Objectives  This study aims to assess the resistance profiles of N. gonorrhoeae isolates sent to the Department of Clinical Microbiology in Vejle, Denmark, between 2003 and 2007, and to analyse their biochemical patterns.
Methods  Sixty-two strains of N. gonorrhoeae were retrospectively analysed for their susceptibility to penicillin, ciprofloxacin and ceftriaxone. The identification of isolated strains was confirmed using both biochemical and immunological tests.
Results  Twenty-one (33.9%) N. gonorrhoeae isolates were resistant to penicillin and 30 (48.4%) were resistant to ciprofloxacin. All strains were susceptible to ceftriaxone. Fifty-six (90.3%) N. gonorrhoeae strains showed API NH biochemical code 10 010 (produced acid from glucose and proline aminopeptidase). Six strains showed code 10 000 that lack the enzyme proline aminopeptidase (proA-negative N. gonorrhoeae ).
Conclusions  Ceftriaxone should be used as the first-line treatment of gonorrhoea in Vejle community area, Denmark, both for infections with proA-producing and proA-negative N. gonorrhoeae isolates, which circulate in the region.     

Antimicrobial susceptibility of Neisseria gonorrhoeae in Cuba (1995-1999): implications for treatment of gonorrhea

BACKGROUND: Antibiotic-resistant strains of Neisseria gonorrhoeae, especially those resistant to penicillin and tetracycline, have spread with remarkable rapidity in many Caribbean countries. GOAL: The goal of the study was to survey the antibiotic susceptibilities of N gonorrhoeae strains isolated from 1995 to 1999 in Cuba and to discuss the impact of antimicrobial resistance on the management of gonorrhea in the country. STUDY DESIGN: Susceptibility of the strains to penicillin, tetracycline, cefuroxime, ceftriaxone, ciprofloxacin, spectinomycin, and azithromycin were determined by an agar dilution method. RESULTS: Penicillin and tetracycline resistance was noted in 60.8% and 54.2%, respectively, of the N gonorrhoeae strains tested. A total of 63.35 (76/120) of the N gonorrhoeae strains exhibited plasmid-mediated resistance to penicillin, tetracycline, or both. Strains with chromosomally mediated resistance to these antibiotics accounted for 10% (12/120) of the strains. The strains were susceptible to ceftriaxone, cefuroxime, spectinomycin, and ciprofloxacin. One strain's ciprofloxacin MIC was 0.125 mircog/ml. Of the 52 strains tested, 23.1% displayed intermediate resistance to azithromycin. CONCLUSIONS: N gonorrhoeae strains exhibited a high frequency of resistance and multiresistance to penicillin and tetracycline. Therefore, these antibiotics should no longer be used to treat gonococcal infections in Cuba and should be substituted with effective drugs such as third-generation cephalosporins, spectinomycin, and fluoroquinolones. The detection of intermediate resistance to azithromycin and ciprofloxacin underlines the importance of periodic surveillance for susceptibility of N gonorrhoeae strains to antimicrobials agents used as primary therapy for gonorrhea.     

Analysis of the antibiotic sensitivity of Neisseria gonorrhoeae in Guangzhou, Peoples Republic of China

BACKGROUND: Sexually transmitted diseases began to re-emerge in China in the mid 1980s. During the last one and a half decades, Neisseria gonorrhoeae infection has become one of the three most common sexually transmitted diseases in China. At present, resistant strains of N. gonorrhoeae are increasing each year. This study was undertaken to better understand the sensitivity of five antibiotics to N. gonorrhoeae isolates in Guangzhou, China. GOAL: To determine the frequency and diversity of antibiotic resistance, particularly to penicillin and tetracycline, on gonococcal strains in Guangzhou. STUDY DESIGN: Strains of N. gonorrhoeae isolates from 203 patients with uncomplicated urethral gonococcal infections from Guangzhou, China were reviewed from September 1997 to August 1998. All strains were characterized with five different antimicrobials for sensitivity, including penicillin, tetracycline, spectinomycin, ciprofloxacin, and ceftriaxone. RESULTS: Penicillin resistance was present in 121 of 203 isolated strains (59.6%). The plasmid-mediated strains and chromosome-mediated strains among the penicillin-resistant strains that were resistance to penicillin were 5.8% and 94.2%, respectively. Plasmid-mediated strains resistant to penicillin and tetracycline were each 3.4%. Most isolated strains were resistant to ciprofloxacin, accounting for 60.6%. All strains were sensitive to spectinomycin and ceftriaxone. CONCLUSION: N. gonorrhoeae isolates exhibited a high rate of resistance to penicillin and ciprofloxacin. Spectinomycin highly effective for penicillin-producing N. gonorrhoeae, tetracycline-resistant N. gonorrhoeae and the highly resistant strains of ciprofloxacin.     

淋病奈瑟菌mtrC膜蛋白基因的克隆和表达

目的 构建淋病奈瑟菌膜蛋白mtrC表达质粒,探讨淋病奈瑟菌耐药的检测和耐药机制.方法 从淋病奈瑟菌标准株中扩增淋病奈瑟菌膜蛋白mtrC基因片段.酶切后插入pET-28a(+),构建重组表达质粒pET-mtrC.通过质粒双酶切和DNA测序证实该重组质粒构建正确.重组质粒转化入大肠杆菌DE3中.经IPTG诱导表达蛋白.结果 经酶切鉴定和测序分析,质粒构建正确.核苷酸序列与GeneBank(U14993)公布的序列相比较,同源性达到99.5%.通过IPTG诱导,SDS—PAGE可检测到约48500大小的融合蛋白,与预测分子量相同.结论 淋病奈瑟菌膜蛋白mtrC原核表达质粒构建和表达成功,为研究其mtrC外排系统的耐药机制打下基础.     

1999-2006年南京地区淋球菌对环丙沙星耐药现状的分析

目的 了解南京地区1999-2006年淋球菌对环丙沙星的耐药现状.方法 对1999-2006年在南京地区性病门诊患者中分离到的1208株淋球菌,采用琼脂稀释法测定环丙沙星的最小抑菌浓度(MIC).结果 对环丙沙星耐药(MIC 1mg/L)的淋球菌率由1999年的83.93%(94/112)上升至2006年的98.99%(196/198),平均总耐药率为96.61%(1167/1208).其中环丙沙星高度耐药(MIC≥4mg/L)的淋球菌比率达71.61%(865/1208).结论 南京地区流行的淋球菌对环丙沙星的耐药情况极其严重.     

Surveillance of antibiotic resistance of Neisseria gonorrhoeae isolates in China, 1993-1998

BACKGROUND: The prevalence of sexually transmitted diseases (STDs) has been increasing in China since the 1980s. Because gonorrhea is the most frequently reported STD there, information on the antimicrobial susceptibility of Neisseria gonorrhoeae will aid in its control. GOAL: To investigate the antimicrobial susceptibility of N gonorrhoeae isolates in China and to provide data for formulation of treatment guidelines and control policies. METHODS: The agar dilution technique was used to determine antimicrobial susceptibility, and acidimetric method was used to test for penicillinase-producing N gonorrhoeae. RESULTS: A total of 3186 gonococcal isolates were tested during the 6-year study period. The rate of resistance to penicillin was 66.70%; 8.14% of isolates were penicillinase-producing N gonorrhoeae. The percentage of tetracycline-resistant isolates was 92.03%, and that of highly tetracycline-resistant isolates was 4.65%. The rate of resistance to ciprofloxacin was also relatively high (34.25%). The rates of resistance to spectinomycin and ceftriaxone were 0.44% and 0.57%. CONCLUSIONS: The gonococcal isolates in China are relatively highly resistant to penicillin, tetracycline, and ciprofloxacin, but most of them are still susceptible to spectinomycin and ceftriaxone. Standardized treatment of gonorrhea is needed to prevent further spread of resistant gonococcal strains.     

1988-2002年上海分离的淋球菌对抗菌药的敏感性监测

目的 了解上海地区1988-2002年淋球菌对多种抗菌药耐药的发生率、流行情况和耐药特征.方法 采用琼脂稀释法测定抗菌药耐药性,纸片酸度定量法测定青霉素产β-内酰胺酶菌株(PPNG).结果 青霉素敏感性从1988年的11.28%降至2002年的0,MIC50和MIC90分别增加了8倍和4倍,2002年的耐药率达到94.29%,PPNG株达到了50.95%;高度耐四环素株(TRNG)从1995年的0上升到2002年的20.95%;头孢曲松敏感株已由1995年的100%下降至2002年的23.80%;大观霉素的敏感性维持在高点(>97%);环丙沙星敏感性有较大幅度下降,其耐药率在2002年达到了99.05%.分析多重耐药株,同为耐青霉素、环丙沙星和四环素3种药物的菌株从2001年的20.87%上升到2002年的23.30%.同为耐青霉素和环丙沙星2种药物的菌株近2年都已接近70%.结论 近15年来,淋球菌对多种药物产生了耐药,耐药率逐年提高.建议上海地区将大观霉素和头孢曲松作为治疗淋病的首选药物,并且尽早研制出对淋球菌敏感的抗菌药.