解脲脲原体的耐药性分析及其耐氟喹诺酮的分子机制研究

目的检测128株临床分离解脲脲原体(UU)对9种药物的敏感性,并检测喹诺酮类耐药UU对5种喹诺酮药物的MIC以及gyrA和parC基因突变情况。方法应用支原体分离鉴定、计数药敏试剂盒检测UU对9种药物的敏感性,用微量肉汤稀释法检测耐药UU菌株对5种喹诺酮类药物的MIC,用PCR和DNA测序及序列比较检测基因突变。结果UU对多西环素最敏感,其次为米诺环素、交沙霉素、司帕沙星及克林霉素,对大环内酯类药物阿奇霉素和罗红霉素高度耐药。而司帕沙星、加替沙星和左氧氟沙星的MIC范围在0.25～8μg/mL,抑制UU活性强于氧氟沙星和环丙沙星。10株喹诺酮类耐药UU中有3株只有parC基因第80位TCA→TTA的突变,氨基酸由丝氨酸→亮氨酸,1株GyrA基因第95位密码子GAC→GAA的突变,氨基酸由天冬氨酸→谷氨酸。6株两者同时存在。结论在UU治疗中可首选多西环素,其次为米诺环素和交沙霉素。gyrA基因95位密码子和parC基因80位的突变密码子与UU耐喹诺酮类药物密切相关。     

人型支原体对8种药物的敏感性及喹诺酮类药物耐药基因gyrA突变的研究

目的：检测人型支原体(Mh)对8种药物的敏感性，并检测喹诺酮类药物耐药株gyrA基因突变情况。方法：应用微量肉汤稀释法检测Mh对8种药物的敏感性，用PCR和DNA测序及序列比较检测基因突变。结果：大环内酯类药物中只有交沙霉素抗Mh活性较强，MIC50和MIC90分别为0．25μg／ml和0．5μg／ml，四环素类药物中多西环素的抗Mh活性优于四环素，MIC50仅为0．06μg／ml，MIC90仅为1μg／ml；喹诺酮类药物环丙沙星和氧氟沙星的抗Mh活性较强。基因序列分析表明gyrA基因碱基113位C到T的突变为有义突变，导致所编码的83位丝氨酸被亮氨酸替代，其余均为同义突变。结论：8种药物中以交沙霉素、多西环素、环丙沙星和氧氟沙星的抗Mh的活性较强；gyrA基因113位碱基C到T的突变与Mh耐喹诺酮类药物密切相关。     

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。     

解脲脲原体与人型支原体对9种抗生素耐药性分析

目的总结东莞地区解脲脲原体(UU)与人型支原体(MH)对9种抗生素的耐药性。方法男性尿道拭子、女性宫颈拭子进行UU和MH检测和药物敏感性试验。结果263例患者共检测到病原体303株,UU247株,MH56株,UU最敏感的是强力霉素和美满霉素,最耐药的是司帕沙星;MH最敏感的也是强力霉素和美满霉素,最耐药的是罗红霉素。UU和MH对9种抗菌素的总体耐药性有统计学意义(χ2=342.63,P=0.000),二者对强力霉素和美满霉素的耐药性无统计学意义(P>0.05),但对其他7种大环内酯类和喹诺酮类的耐药性有统计学意义(P<0.01),MH的耐药性高于UU。结论支原体对多种抗菌素有不同程度的耐药性,MH对强力霉素和美满霉素的耐药性和UU相似,但对其他7种大环内酯类和喹诺酮类药物的耐药性高于UU,治疗MH要尽可能避免使用大环内酯类。     

解脲脲原体临床分离株中喹诺酮类耐药株的筛选及其耐药基因的研究

目的：从临床分离的解脲脲原体中筛选出喹诺酮类药物耐药株,了解耐药株发生的比率。并研究耐药株中喹诺酮抗性决定区域(QRDR)出现基因变异的情况。方法：采用药敏试剂盒及96孔微量培养基稀释法收集解脲脲原体的喹诺酮耐药株。PCR扩增阳性标本测序后比较基因变异情况。结果：从104株解脲脲原体临床分离株中筛选出10株耐药株。测序结果显示有7株QRDR的gyrA基因片段中309位均出现了A→G点变异,导致相应编码的氨基酸由组氨酸变为精氨酸。结论：解脲脲原体对喹诺酮类药物的耐药与QRDR中gyrA基因的点变异可能相关。     

泌尿生殖道解脲支原体和人型支原体感染及对抗生素敏感性调查分析

目的:了解本地区泌尿生殖道解脲支原体(UU)和人型支原体(MH)感染情况以及对抗生素的耐药性,指导临床合理用药。方法:采用珠海浪峰生物技术公司生产的支原体培养、鉴定、计数药敏试剂盒对1000例标本进行培养、鉴定和药敏试验。结果:支原体总阳性感染率为40.5%(405/1000),其中UU305株,阳性率为30.5%;MH18株,阳性率为1.8%;UU和MH混合感染82株,阳性率为8.2%。对抗生素耐药情况,喹诺酮类药物耐药性最高(环丙沙星79.0%、司帕沙星60.7%、可乐必妥52.8%),大环内酯类药物耐药率在3%-31%(交沙霉素3.2%、阿齐霉素14.3%、克拉霉素19.7%、罗红霉素27.1%、环酯红霉素17.2%、红霉素30.6%),四环素类药物耐药率最低在1%-5%(强力霉素为1.7%、美满霉素3.4%)。结论:对泌尿生殖道UU和MH感染患者,四环素和大环内酯类几种抗生素耐药性较低,仍可作本地区一线抗UU和MH药物。     

沙眼衣原体临床分离株药物敏感性测定及耐药基因检测

目的检测本地区泌尿生殖道沙眼衣原体(CT)对四环素类、大环内酯类、喹诺酮类抗生素的体外药物敏感性,并从基因水平上探讨其耐药机制。方法将42株经细胞培养法证实有CT感染的临床株进行McCoy细胞培养,传代至感染率达90%以上,收集标本进行药敏试验。分别检测四环素耐药质粒tetM基因;大环内酯类耐药相关的23S核蛋白体RNA基因、核糖体蛋白基因L4,通过产物测序检测基因突变;限制性片段分析(RFLP)检测gyrA喹诺酮耐药决定区(QRDR)常见的基因点突变。结果药敏MIC(单位均为μg/mL)分别为红霉素0.5～2.0,克拉霉素0.008～0.032,阿奇霉素0.125～0.50,四环素0.157～0.625,多西环素0.063～0.125,米诺环素0.032～0.128,左氧氟沙星0.50～1.00,莫西沙星0.060～0.120,司帕沙星0.064～0.128,其中发现2株红霉素耐药株(MIC值2μg/mL),其23srRNA基因有C2452A,T2611C的突变(大肠杆菌序列编号),红霉素耐药株及敏感株L4基因均发现脯氨酸113(CCG)→亮氨酸(CTG)、脯氨酸156(CCC)→丙氨酸(GCC)两个位点的突变(GenBankNC000117.1),25株临床分离株中检测到tetM基因,未发现gyrA-QRDR基因中Ser83→Ile点突变。结论发现2株红霉素耐药株,未发现其它八种抗菌药的耐药株。克拉霉素显示了较高的体外抗CT活性。C2452A,T2611C的突变导致红霉素低水平耐药,L4基因的点突变可能与红霉素耐药无关。     

淋球菌青霉素结合蛋白PPNG基因与耐青霉素类药物的关系

目的　探讨淋球菌青霉素结合蛋白 2基因 (PenA)突变及产青霉素酶淋球菌 (PPNG)与耐青霉素类药物的关系。方法　采用巢式聚合酶链技术对 97株淋球菌临床分离株进行PPNG基因检测 ,并对 4例PPNG基因检测阴性、而抗生素药敏试验表明对青霉素类药物耐药的淋球菌菌株的PenA基因进行测序。结果　 97株淋球菌菌株中有 67株为PPNG基因检测阳性 ;而 4株PPNG阴性的耐药菌株的PenA基因序列均存在点突变。结论　PPNG所导致的耐药是淋球菌耐青霉素类药物的主要来源 ;PenA基因突变是产生耐药菌株的主要原因。     

泌尿生殖道支原体培养及药敏结果分析

目的了解本地区泌尿道患者支原体的培养及药敏情况。方法用男性尿道拭子、女性宫颈拭子取分泌物进行UU和MH检测及药敏试验。结果 254例样本中,UU阳性者215例,MH阳性者49例,UU与MH同时阳性者24例。UU最敏感的抗生素是交沙霉素,其次强力霉素与美满霉素;MH最敏感的是强力霉素与美满霉素。UU耐药最高的是螺旋霉素与环丙沙星,其次氧氟沙星;耐药最低的是交沙霉素;MH耐药性最高的是罗红霉素,其次是司帕沙星;耐药最低的是强力霉素。结论临床用药应该考虑地区耐药菌株的流行情况,最好按照药敏试验结果合理用药。     

昆明地区泌尿生殖道人型支原体对抗生素的敏感性测定

目的为了解昆明地区泌尿生殖道人型支原体(MH)对抗生素的敏感性。方法应用支原体培养、鉴定、药敏试剂盒,对来自2001~2003年泌尿生殖道标本的52株MH进行了9种常用抗生素的敏感性测定。结果MH对米诺环素最敏感,其次为交沙霉素、多西环素、司帕沙星及克林霉素,而对阿奇霉素、罗红霉素、左旋氧氟沙星及氧氟沙星耐药。结论昆明地区泌尿生殖道MH感染者首选米诺环素。     

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.     

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.     

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.


     

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。     

次抑菌浓度的药物诱导解脲支原体人型支原体耐药与交叉耐药的研究

目的 为了解ＵＵ和ＭＨ的耐药状况，指导临床合理用药。方法 将临床标本分离的６株ＵＵ与６株ＭＨ分别进行了诱导与交叉耐药试验。结果 ＴＣ诱导耐药的ＵＵ和ＭＨ亦对ＤＯＸＹ耐药，但对ＭＣ，喹诺酮类药物和大环内酯类药物无明显交叉耐约性。ＯＦＬＸ诱导耐药的ＵＵ和ＭＨ和ＣＦＬ，Ｘ，ＬＭＬＸ耐药人型支原体耐药与交叉耐药的地四环素类药物和大环内酯类药物没有显著的交叉耐药性。结论 耐ＴＣ的ＵＵ与ＭＨ株对ＤＯＸＹ呈现交     

Phenotypic and genotypic characterization of antibiotic-resistant Propionibacterium acnes isolated from acne patients attending dermatology clinics in Europe, the U.S.A., Japan and Australia

BACKGROUND: Propionibacterium acnes is the target of antimicrobial treatments for acne vulgaris. Acquired resistance to erythromycin, clindamycin and tetracyclines has been reported in strains from diverse geographical loci, but the molecular basis of resistance, via mutations in genes encoding 23S and 16S rRNA, respectively, has so far only been elucidated for isolates from the U.K. OBJECTIVES: To determine whether similar or different resistance mechanisms occur in resistant P. acnes isolates from outside the U.K. METHODS: The phenotypes and genotypes of 73 antibiotic-resistant strains of P. acnes obtained from the skin of acne patients in the U.K., U.S.A., France, Germany, Australia and Japan were compared. Antibiotic susceptibilities were determined by minimum inhibitory concentration (MIC) measurements, and polymerase chain reaction and DNA sequencing were used to identify mutations in genes encoding rRNA. RESULTS: Most erythromycin-resistant isolates (MIC(90) > or = 512 microg mL(-1)) were cross-resistant to clindamycin but at a much lower level (MIC(90) > or = 64 microg mL(-1)). As in the U.K., resistance to erythromycin was associated with point mutations in 23S rRNA in 49 of 58 strains. An A-->G transition at Escherichia coli equivalent base 2058 was present in 24 strains. This gave a unique cross-resistance phenotype against a panel of macrolide, lincosamide and type B streptogramin antibiotics. Two further point mutations (at E. coli equivalent bases 2057 and 2059) were identified (in three and 22 isolates, respectively) and these were also associated with specific cross-resistance patterns originally identified in isolates from the U.K. However, nine of 10 erythromycin resistant-strains from Germany did not exhibit any of the three base mutations identified and, in six cases, cross-resistance patterns were atypical. Consistent with previous U.K. data, 34 of 38 tetracycline-resistant strains carried a base mutation at E. coli 16S rRNA equivalent base 1058. Tetracycline-resistant isolates displayed varying degrees of cross-resistance to doxycycline and minocycline, but isolates from the U.S.A. had higher MICs for minocycline (4--16 microg mL(-1)) than isolates from other countries and, in particular, Australia. All the P. acnes isolates resistant to one or more of the commonly used antiacne antibiotics were sensitive to penicillin, fusidic acid, chloramphenicol and the fluoroquinolone, nadifloxacin. All but one isolate (from the U.K.) were sensitive to trimethoprim. CONCLUSIONS: This study shows that 23S and 16S mutations identified in the U.K. conferring antibiotic resistance in P. acnes are distributed widely. However, resistant strains were isolated in which mutations could not be identified, suggesting that as yet uncharacterized resistance mechanisms have evolved. This is the first report of high-level resistance to minocycline and is of concern as these strains are predicted to be clinically resistant and are unlikely to remain confined to the U.S.A. Epidemiological studies are urgently required to monitor how resistant strains are selected, how they spread and to ascertain whether the prevalence of resistance correlates with antibiotic usage patterns in the different countries.     

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.     

沙眼衣原体对大环内酯类药物敏感性试验及23S rRNA基因突变研究

目的 检测天津地区近年来大环内酯类抗生素对泌尿生殖道沙眼衣原体临床分离株的抗菌活性,筛查耐药株,从基因分子水平上探讨耐药机制。方法 将经McCoy细胞培养法检测出的42株沙眼衣原体临床株,传代培养至感染率达90%以上,收集标本进行3种常用大环内酯类抗生素的药敏试验,用RT-PCR、PCR方法分别扩增与大环内酯类耐药相关的23S核蛋白体RNA基因、核糖体蛋白L4基因,产物直接测序检测基因突变。结果 最小抑菌浓度（MIC）结果为红霉素0.5 ～ 2 mg/L,克拉霉素0.008 ～ 0.032 mg/L,阿奇霉素0.125 ～ 0.5 mg/L。发现2株红霉素耐药株,MIC值为2 mg/L,2株耐药株的23S rRNA基因有C2452A、T2611C的突变（大肠杆菌序列编号）,红霉素耐药株及敏感株L4基因均发现脯氨酸113→亮氨酸、脯氨酸156→丙氨酸两个位点的突变。结论 沙眼衣原体对红霉素已产生一定的耐药性。C2452A、T2611C的突变导致红霉素低水平耐药,L4基因的点突变可能与红霉素耐药无关。