武汉地区门诊患者喹诺酮耐药鼠伤寒沙门菌耐药机制分析

目的 研究分析腹泻门诊患者中分离的喹诺酮耐药鼠伤寒沙门菌的耐药机制和遗传关系.方法 对2002-2005年问武汉同济医院腹泻门诊患者中分离的36株喹诺酮耐药鼠伤寒沙门菌进行了耐药谱测定,并通过PCR方法和序列测定对整合子、β内酰胺酶基因、喹诺酮耐药决定区的突变、qnr基因和aac(6')-Ib-cr基因进行了分析,运用脉冲场电泳方法(pulsed-field gel electrophoresis,PFGE)对所收集的菌株进行了分子分型,分析喹诺酮耐药鼠伤寒沙门菌的耐药机制和遗传关系.结果 喹诺酮耐药鼠伤寒沙门菌均为多重耐药株,普遍携带有Ⅰ类整合子,环丙沙星耐药菌株与敏感菌株在PFGE谱型上存在显著性差异,31株环丙沙星耐药菌株在喹诺酮耐药决定区中至少携带GyrA和ParC上的3个点突变,且在这些菌株中均检出了OXA-30基因,这些菌株对头孢吡肟的敏感性出现了不同程度的下降.结论 对环丙沙星耐药的鼠伤寒沙门菌在武汉地区已普遍存在,且这些菌株具有独特的遗传背景,建议在今后的细菌耐药性监测工作中应对这类细菌的耐药谱变化进行重点监测,尤其应加强对氟喹诺酮-三代头孢类抗菌药物均耐药菌株的针对性预警监测.     

临床分离黏质沙雷菌染色体及质粒介导的喹诺酮类耐药机制研究

目的 了解安徽地区临床分离株黏质沙雷菌质粒介导喹诺酮类耐药基因的流行情况以及gyrA、parC基因变异对抗菌药物的耐药性产生的影响.方法 采用琼脂稀释法测定2005至2010年34家医院收集的104株黏质沙雷菌的MIC;采用PCR检测其中31株耐环丙沙星的黏质沙雷菌的qnr、aac(6’) -Ib、qepA基因以及gyrA和parC基因,对阳性扩增产物进行测序分析;对qnr、aac(6’) -Ib-cr阳性菌株做转移接合试验,PCR扩增并测序确定接合子的基因型,测定供体菌、受体菌和接合子对喹诺酮类及其他类型抗菌药物的MIC.结果 共检出31株环丙沙星耐药的黏质沙雷菌,共6株携带qnr基因和(或)aac(6’)-Ib-cr基因,其中2株携带qnrB6亚型,1株携带qnrS2亚型,4株携带aac(6’)-Ib-cr基因(其中1株同时携带qnr基因).9株检测出gyrA基因突变,7株检测出parC基因突变.6株携带qnr基因和(或)aac(6’)-Ib-cr基因的菌株中有5株转移接合成功.接合子与受体菌相比,对喹诺酮类的MIC值均有不同程度的提高.结论 染色体及质粒介导的耐药基因在临床分离的黏质沙雷菌对喹诺酮类药物耐药中起重要的作用,且可以水平传播,故应引起高度重视.     

宋内志贺菌对喹诺酮类抗菌药物的耐药性研究

目的 检测宋内志贺菌对喹诺酮类抗菌药物的耐药情况,探讨染色体介导DNA旋转酶A亚单位(gyrA)和拓扑异构酶ⅣC亚单位(parC)基因突变或(和)质粒介导qnrA基因存在与宋内志贺菌耐喹诺酮类药物的相关性.方法 用K-B纸片扩散法对58株宋内志贺菌进行耐药性检测;PCR法检测宋内志贺菌喹诺酮耐药决定区(QRDR)相关gyrA、parC基因,并根据药敏结果 挑选5株菌扩增片段进行DNA测序;PCR法扩增qnrA基因片段.分析宋内志贺菌gyrA、parC基因突变或(和)qnrA基因存在与喹诺酮类药物耐药性的关系.结果 宋内志贺菌对萘啶酸的耐药率高达94.8%.58株宋内志贺菌中,8株检出qnrA基因,5株测序gyrA、parC基因的菌株中,4株萘啶酸耐药菌株均在gyrA 83位发生有义突变TCG(Ser)→TTG(Leu),但未发生parC基因突变,gyrA基因突变菌株对萘啶酸全耐药.qnrA基因阳性菌株对5种喹诺酮类药物抑菌圈中位数比较都缩小;对NAL、氧氟沙星的耐药率高于qnrA基因阴性菌株(P<0.05),差异有统计学意义.结论 gyrA Ser83分→Leu突变是导致宋内志贺菌临床分离株对喹诺酮类药物耐药的关键突变,宋内志贺菌若携带质粒介导qnrA基因则会导致对喹诺酮类药物的敏感性下降,若两者同存也可引起喹诺酮类药物耐药增强,除萘啶酸耐药外对其他喹诺酮类药物也可呈中介.     

大肠埃希菌临床菌株对氟喹诺酮类的耐药机制

DNA旋转酶编码基因（gyrA和gyrB）和拓扑异构酶编码基因（parC和parE）的染色体突变、多重药物外排泵AcrAB表达水平升高以及存在质粒介导aCC（6’）-Ib-cr和各种qnr基因等耐药机制均可导致氟喹诺酮类药物对大肠埃希菌的MIC升高。已有报道环丙沙星、加替沙星、左氧氟沙星和诺氟沙星对氟喹诺酮类药物耐药大肠埃希菌临床菌株的MIC高,并有很大差异。作者等对153株流行病学信息已知的菌株中选择78株代表不同氟喹诺酮类药物MIC范围的菌株,进行上述各种喹诺酮类药物耐药基因测序,发现：①所有氟喹诺酮类药物耐药菌株（58株）均存在gyrA突变;     

鼠伤寒沙门菌对喹诺酮类耐药机制的研究

目的检测鼠伤寒沙门菌（STM）对喹诺酮类药物的耐药性及耐药机制的分析。方法收集2004年7月1日-10月31日武汉地区4所大型医院的门诊腹泻病人的粪便进行分离培养出鼠伤寒沙门菌33株。琼脂稀释法测其对喹诺酮类药物的MIC，并抽提此菌的基因组DNA，用PCR方法检测喹诺酮类抗菌药作用于鼠伤寒沙门菌的靶位点：Ⅱ型拓扑异构酶，即DNA促旋酶和拓扑异构酶Ⅳ上的基因片段（gyrA，gyrB，parC，parE）突变情况。结果33株鼠伤寒沙门菌中有24株对环丙沙星产生了很强的耐药性（MIC值4～16mg／L），耐药率达72．7％。24株耐药株中gyrA和parC的突变比较常见，其中gyrA位点都存在突变点，且双重突变占92％，并协同parC的点突变造成高水平的耐药；gyrB和parE的突变很少见，本研究中有少数高水平耐药株的parE和gyrB位点发现可疑的碱基插入。结论研究结果表明武汉地区社区内鼠伤寒沙门菌感染对喹诺酮类药物的耐药性严重，其主要机制是喹诺酮类耐药决定区（QRDR）的基因突变，特别是多个位点同时突变导致高水平耐药。     

喹诺酮类耐药福氏志贺菌的相关耐药基因研究

目的了解2010—2014年上海市各区县医院139株福氏志贺菌的耐药性,探讨福氏志贺菌对喹诺酮类药物的耐药机制。方法用纸片扩散法测定菌株对14种抗菌药物的敏感性,用环丙沙星E试验条测定其最低抑菌浓度;采用PCR法检测DNA旋转酶A亚单位(gyrA)、拓扑异构酶ⅣC亚单位(parC)基因的喹诺酮类药物耐药决定区(QRDR),同时对质粒介导喹诺酮类耐药(PMQR)基因qnrA、qnrB、qnrS和氨基糖苷乙酰转移酶变异基因aac(6')-Ib-cr进行筛选。扩增产物进行DNA测序。结果福氏志贺菌对氨苄西林、链霉素、四环素、萘啶酸的耐药率都达到了90%以上,对环丙沙星的耐药率达到了40.3%,同时有30.2%菌株对头孢吡肟产生了耐药。gyrA和parC基因的突变率分别为98.6%和97.8%。gyrA基因存在Ser83、Asp87和His211 3个位点的突变,parC基因只检测到Ser80 1个位点的突变;共检测到9株qnrS和6株aac(6')-Ib-cr喹诺酮耐药质粒。结论上海地区福氏志贺菌耐药情况严重。QRDR相关基因突变率高,Asp87的突变对喹诺酮类抗菌药物的耐药起着主导作用,而耐药质粒起着重要的辅助作用。     

粪肠球菌Ⅱ型拓扑异构酶基因突变与耐氟喹诺酮类药物关系的研究

目的 检测粪肠球菌对氟喹诺酮类药物的敏感性,探讨Ⅱ型拓扑异构酶基因突变与耐氟喹诺酮类药物的关系.方法 用二倍琼脂稀释法检测6种氟喹诺酮类药物对60株粪肠球菌临床分离株的体外抗菌活性,随机筛选出11株对环丙沙星不同程度耐药菌,PCR扩增gyrA,gyrB,parC,parE基因的喹诺酮耐药决定区(QRDR),产物测序后分析.结果 6种药物的相对抗粪肠球菌活性(MIC50,MIC90)从强到弱为:妥舒沙星＞加替沙星,司帕沙星＞左氧氟沙星＞氧氟沙星,环丙沙星;以妥舒沙星抗菌活性最强,氧氟沙星和环丙沙星抗菌活性最差;序列比较发现,有9株耐药株Ⅱ型拓扑异构酶基因发生突变,突变发生在gyrA基因(6株)和parC基因(9株),其中编码gyrA的Ser83→Ile,Arg和编码parC的Ser80→Ile,Arg的密码子表现出高频突变,gyrB和parE编码的氨基酸序列没有改变;未发现gyrA突变单独存在,同时具gyrA和parC突变的MIC值是仅具parC突变菌株MIC值的4倍以上.结论 氟喹诺酮类抗菌药新品种妥舒沙星、加替沙星和司帕沙星的抗粪肠球菌活性较老一代药物更强;粪肠球菌对老一代氟喹诺酮类药物存在不同程度的耐药;gyrA基因83,87位突变及parC基因80,84位突变都可引起粪肠球菌对氟喹诺酮类药物产生耐药,但以parC基因80住突变为主;低耐药株往往是parC基因单位点突变,高耐药株同时合并有gyrA基因双位点突变.     

深圳社区感染沙门菌耐药基因调查与同源性分析

目的 调查深圳社区感染沙门菌耐药特点和分子机制,并进行同源性分析.方法 收集深圳市人民医院2002——2007年临床分离沙门菌共93株,PCR和DNA测序分析沙门菌gyrA、gyrB、parC和parE基因QRDR的突变,PCR检测质粒介导喹诺酮耐药基因qnr和aac(6')-Ib-cr,β内酰胺酶基因blaTEM、blaSHV、blaOXA和blaCTX-M基因,以及Ⅰ类整合子,PFGE对沙门菌进行分子分型.结果 伤寒沙门菌和甲型副伤寒沙门菌对氨苄西林、氯霉素、复方磺胺甲噁唑、头孢曲松和环丙沙尾敏感率为96%～100%;52%(13/25)伤寒沙门菌和95%(61/64)甲型副伤寒沙门菌对萘啶酸耐药.24%(6/25)萘啶酸耐药伤寒沙门菌和94%(60/64)萘啶酸耐药甲型副伤寒沙门菌对环丙沙星敏感性降低(MIC 0.125～μg/ml).75株萘啶酸耐药环丙沙星敏感沙门菌仅GyrA的QRDR均存在第83位或87位单个氨基酸替代,其中Ser83Phe突变占91%(68/75).2株环丙沙星耐药沙门菌在QRDR中均携带GyrA上2个点突变和parC上1个点突变.93株沙门菌均未发现质粒介导的qnr和aac(6')-Ib-cr 基因.1株头孢曲松耐药甲型副伤寒沙门菌检测到blaCTX-M-14基因,且该基因上游存在插入序列ISEcpl.3株多重耐药沙门菌均存在一个1 900 bp的Ⅰ类整合子,其基因盒均为dhfrⅫ-orfF-aadA2,同时携带blaTEM-1或blaOXA-30基因.25株伤寒沙门菌共有22种不同的PFGE带型,64株甲型副伤寒沙门菌的PFGE带型平均相似性为91%.90例患者均系社区感染,6例甲型副伤寒患者发病前30天内曾前往外地旅行.结论 深圳社区感染伤寒和甲型副伤寒沙门菌对萘啶酸耐药率较高,沙门菌GyrA的QRDR点突变是萘啶酸耐药的重要机制,甲型副伤寒沙门菌菌株间遗传同源性极高,来自同一克隆.

Abstract：

Objective To investigate the antimicrobial resistance mechanisms and genetic homogeny of Salmonella from community acquired infections in Shenzhen,China.Methods Ninety-three of Salmonella were isolated from 2002 to 2007 at Shenzhen People's Hospital,China.PCR and DNA sequencing were used to investigate the mutation in QRDR of the gyrA,gyrB,parC and parE.Plasmid mediated quinolone resistance genes including qnr and aac(6')-Ib-cr,β-lactamase genes including blaTEM,blaSHV,blaOXA, blaCTX-M, and class 1 integron were detected. All isolates were typed by PFGE. Results S. enterica typhi and S. enterica paratyphi A were susceptible to ampicillin, chloramphenicol, trimethoprim/sulfamethoxazole, ceftriaxone and ciprofloxacin, with the susceptible rate of 96%-100%. Fifty-two percent (13/25) of S. enterica typhi and 95% (61/64) of S. enterica paratyphi A were resistant to nalidixic acid. Twenty-four percent (6/25) of nalidixic acid-resistant S. enterica typhi and 94% (60/64) of nalidixic acid-resistant S. enterica paratyphi A showed decreased susceptibility to ciprofloxacin (MIC of 0. 125-1 μg/ml).All nalidixic acid-resistant (susceptible to ciprofloxacin ) Salmonella (NARS) isolates had a single substitution in the QRDR of GyrA, and 91% (68/75) of these isolates carried the substitution Ser83Phe in GyrA. Two mutations in the QRDR of GyrA were detected in both of two ciprnfloxacin-resistant Salmonella,with the additional one mutation in the QRDR of parC. Plasmid mediated quinolone resistance genes including qnr and aac(6')-lb-cr were not detected in any isolate. The blaCTX-M-14 gene was detected in a ceftriaxoneresistant isolate of S. enterica paratyphi A, with ISEcpl located on the upstream of it. Three muhidrugresistant strains of Salmonella all carried one 1 900 bp classⅠ integron gene cassette dhfrⅫ-orfF-aadA2,with the additional one β-lactamase gene of blaTEM-1, or blaOXA-30. Twenty-two distinct PFGE patterns were observed among twenty-five S. enterica typhi. The PFGE patterns of sixty-four S. enterica paratyphi A showed limited genetic diversity (average similarity of 91% ). Ninety investigated inpatients were infected in the community. Six patients infected by S. enterica paratyphi A had a travel history before infection. Conclusions Nalidixic acid-resistant S. enterica typhi and S. enterica paratyphi A are highly prevalent in Shenzhen,China. The mutation in the QRDR of GyrA is the prevalent mechanism responsible for the resistance to nalidixic acid in Slmonella. The great genetic similarity among S. enterica paratyphi A isolates indicates endemic disease from the presence of a single clone over 6-year period.     

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ⅣC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gyrA 83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parC 80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。     

gyrA和parC介导的铜绿假单胞菌对环丙沙星耐药机制研究

目的了解十堰地区耐氟喹诺酮类铜绿假单胞菌(PA)的药敏情况及gyrA和parC基因突变情况。方法用Vitek32对110株PA进行鉴定和药敏检测,对临床常用抗生素的药敏情况进行分析,琼脂稀释法测定60株耐环丙沙星(CIP)的PA对CIP的最低抑菌浓度(MIC),限制性长度多态性分析法(PCR-RFLP)检测耐CIP的PAgyrA和parC基因突变情况。结果耐药菌株对哌拉西林/他唑巴坦的敏感率最高(68.3%)。在60株耐CIP的PA中有42株(70.0%)耐药菌株发生gyrA基因的83位点突变,密码子发生ACC→ATC改变,编码83位氨基酸的碱基发生突变Thr→Ile(ACC→ATC),有38株(63.3%)耐药菌株发生parC基因87位点突变Ser→Leu(TCG→TTG),同时发生两种突变的共36株(60.0%)。结论耐氟喹诺酮类PA对临床常用抗生素的敏感性降低,并呈多重耐药,药物作用靶位gyrA和parC的基因突变为其耐氟喹诺酮类药物的主要机制。     

Plasmid-mediated quinolone resistance mechanisms in ESBL positive Escherichia coli and Klebsiella pneumoniae strains at a Tertiary-Care Hospital in Turkey

Plasmid-mediated quinolone resistance mechanisms in extended spectrum beta-lactamase positive and quinolone resistant Escherichia coli and Klebsiella pneumoniae strains isolated from Ege University Hospital were investigated. The presence of qnrA, qnrB, qnrS, aac(6')-Ib and qepA genes were detected by PCR and the products were sequenced. Clonal relationship of isolates was determined by REP-PCR and mutations in gyrA and parC genes were investigated in representative strains. aac(6')-Ib-cr, qnrB and qnrS genes were detected in both E. coli and K. pneumoniae strains, but qnrA detected only in K. pneumoniae strains. qepA determinant is detected in an E. coli strain first time in Turkey. Mutations were observed in both gyrA and parC genes of all representative nalidixic acid and ciprofloxacin resistant E. coli isolates but no mutation was found in parC genes of E. coli and K. pneumoniae strains that were resistant to only nalidixic acid.     

Molecular mechanisms of decreased susceptibility to fluoroquinolones in avian Salmonella serovars and their mutants selected during the determination of mutant prevention concentrations

OBJECTIVES: Salmonella enterica isolates of six serovars and mutants obtained during determination of mutant prevention concentrations (MPCs) were investigated for mechanisms of decreased susceptibility to fluoroquinolones. METHODS: The quinolone resistance determining regions (QRDRs) of gyrA, gyrB, parC and parE genes were sequenced. MIC values were determined in the presence/absence of the efflux pump inhibitors carbonyl cyanide m-chlorophenyl-hydrazone (CCCP) or Phe-Arg-beta-naphthylamide (PA beta N). PCR assays for the quinolone resistance genes qnrA, qnrB, qnrS or aac(6')-Ib-cr were applied. The MPC values of ciprofloxacin (MPC(CIP)) were determined for all isolates and selected mutants were investigated for their quinolone resistance mechanisms. RESULTS: In contrast to 11 nalidixic acid-susceptible isolates, 24 nalidixic acid-resistant isolates exhibited single mutations in gyrA (Asp-87 --> Tyr, Gly, Asn or Ser-83 --> Phe, Tyr) or parC (Thr-57 --> Ser). While CCCP had no influence on the MICs, PA beta N decreased the MIC(CIP) values by 1-3 dilution steps and MIC(NAL) values by up to 6 dilution steps. Of the resistance genes investigated, only qnrS was present, in a single Salmonella Infantis isolate. The MPC(CIP) values were 4-64-fold higher than the MICs and ranged between 1-16 and 0.12-1 mg/L, respectively, for isolates resistant or susceptible to nalidixic acid. Only mutants obtained from formerly nalidixic acid-susceptible isolates developed single mutations in gyrA or gyrB. CONCLUSIONS: In field isolates and mutants, target site mutations and efflux seem to be important mechanisms for decreased fluoroquinolone susceptibility. Mutants derived during MPC determination from field isolates already harbouring single-step mutations in gyrA did not exhibit further mutations in any target genes.     

革兰阴性杆菌临床分离株质粒介导喹诺酮类耐药研究

目的了解质粒介导耐药机制在革兰阴性杆菌临床株对喹诺酮类抗菌药耐药性形成中的作用。方法以PCR方法筛选541株连续分离的所有环丙沙星耐药或中介革兰阴性杆菌中的耐药基因qnrA;以接合试验了解喹诺酮耐药的可转移性;对qnrA阳性株测定氨基糖苷乙酰化酶aac(6′)-Ib-cr基因,分析了gyrA和parC基因的喹诺酮耐药决定区的变异。结果541株革兰阴性杆菌中,7株肠杆菌科细菌qnrA检测阳性,其中4株为阴沟肠杆菌,在不发酵糖菌中未检出qnrA基因。在7株qnrA阳性菌中,4株喹诺酮耐药性可通过质粒转移,接合子对环丙沙星的MIC较受体菌上升12～125倍。4个接合子中环丙沙星MIC较高的2个结合子携带aac(6′)-Ib-cr,7株qnrA阳性临床分离菌中5株耐药决定区gyrA、parC有变异。结论qnrA在肠杆菌属临床分离株中的检出率较高,aac(6′)-Ib-cr基因及靶位改变与qnrA同时存在可能使细菌对喹诺酮类的耐药性进一步上升。     

Epidemiological characteristics and molecular basis of fluoroquinolone-resistant Neisseria gonorrhoeae strains isolated in Korea and nearby countries

OBJECTIVES: This study was performed to examine the cause of the increase in quinolone-resistant Neisseria gonorrhoeae (QRNG) observed in Korea. METHODS: The antimicrobial susceptibilities of 190 isolates of gonococci from Korea in 2000 were examined by NCCLS methods, and subsets of these isolates underwent mutation analysis of the quinolone resistance-determining regions (QRDRs) of gyrA and parC. Molecular epidemiological characterization of 25 Korean isolates and 54 isolates from overseas was performed by pulsed-field gel electrophoresis (PFGE) and the results compared. RESULTS: Most (172, 90.5%) of the 190 gonococci tested displayed reduced susceptibility to ciprofloxacin. All strains with high-level ciprofloxacin resistance (ciprofloxacin MIC >/= 4 mg/L) contained a double amino acid alteration at the 91 and 95 positions in the QRDR of GyrA and a single alteration in ParC. PFGE types of high-level QRNG in Korea were mostly different from those of other nearby countries. CONCLUSIONS: These results suggest that the observed increase in ciprofloxacin-resistant isolates is due to the mutation and spread of Korean multiclonal isolates rather than importation from overseas.     

Comparative Studies of Mutations in Animal Isolates and Experimental In Vitro- and In Vivo-Selected Mutants of Salmonella spp. Suggest a Counterselection of Highly Fluoroquinolone-Resistant Strains in the Field

The occurrence of mutations in the genes coding for gyrase (gyrA and gyrB) and topoisomerase IV (parE and parC) of Salmonella typhimurium experimental mutants selected in vitro and in vivo and of 138 nalidixic acid-resistant Salmonella field isolates was investigated. The sequencing of the quinolone resistance-determining region of these genes in highly fluoroquinolone-resistant mutants (MICs of 4 to 16 microg/ml) revealed the presence of gyrA mutations at codons corresponding to Gly-81 or Ser-83, some of which were associated with a mutation at Asp-87. No mutations were found in the gyrB, parC, and parE genes. An assay combining allele-specific PCR and restriction fragment length polymorphism was developed to rapidly screen mutations at codons 81, 83, and 87 of gyrA. The MICs of ciprofloxacin for the field isolates reached only 2 microg/ml, versus 16 microg/ml for some in vitro-selected mutants. The field isolates, like the mutants selected in vivo, had only a single gyrA mutation at codon 83 or 87. Single gyrA mutations were also found in highly resistant in vitro-selected mutants (MIC of ciprofloxacin, 8 microg/ml), which indicates that mechanisms other than the unique modification of the intracellular targets could participate in fluoroquinolone resistance in Salmonella spp. A comparison of experimental mutants selected in vitro, field strains, and mutants selected in vivo suggests that highly fluoroquinolone-resistant strains are counterselected in field conditions in the absence of selective pressure.     

Molecular basis of high-level ciprofloxacin resistance in Neisseria gonorrhoeae strains isolated in Denmark from 1995 to 1998

In Denmark surveillance of the in vitro susceptibility to ciprofloxacin of Neisseria gonorrhoeae was established in 1990. The proportion of N. gonorrhoeae strains with decreased susceptibility or resistance to ciprofloxacin (MIC >/= 0.06 microg/ml) was low (0.3 to 2.3%) up to 1995. Between 1995 and 1998 the rate of less-susceptible and resistant strains rose from 6.9 to 13.2%. Among ciprofloxacin-resistant strains (MIC >/= 1 microg/ml), 81% were highly resistant (MIC >/= 4 microg/ml). Thirty-five N. gonorrhoeae strains (40 isolates) for which ciprofloxacin MICs were 4 to 32 microg/ml were investigated for the frequency and patterns of mutations within the gyrA and parC genes. The quinolone resistance-determining regions of the gyrA and parC genes were amplified by PCR, and the amplicons were directly sequenced. Alterations at Ser-91 and Asp-95 in GyrA and a single or double alteration in ParC were identified in 32 strains (91%). Ser-91-to-Phe and Asp-95-to-Gly alterations in GyrA were detected in 28 strains (80%). The most common ParC alteration, Asp-86 to Asn, was found in 19 strains (54%). The strains were analyzed for genetic relationship by pulsed-field gel electrophoresis (PFGE). The analysis showed that nine strains with the same mutation pattern in the gyrA and parC genes, originating from different geographical areas over 3 years, had the same PFGE patterns after SpeI as well as NheI digestion (only one strain with one band difference in the NheI pattern), suggesting that a resistant clone had spread worldwide. The results from this study strongly suggest that double gyrA mutations plus a parC mutation(s) play an important role in the development of high-level fluoroquinolone resistance in N. gonorrhoeae.     

An intrinsic pattern of reduced susceptibility to fluoroquinolones in pediatric isolates of Streptococcus pyogenes

A total of 116 clinical isolates collected in 2003 from a tertiary pediatric hospital and a primary pediatric department in Chicago, IL, were screened for reduced susceptibility to selected fluoroquinolones by disc diffusion. Correlation between reduced susceptibility and point mutations in the quinolone resistance-determining region of parC and gyrA genes was evaluated, and point mutations were compared with other reports of isolates derived from adult or mixed patient populations. Nine percent of isolates had reduced susceptibility to 1 or more of these fluoroquinolones by Etest: ciprofloxacin, levofloxacin, and moxifloxacin. A single point mutation (Ser-79) in parC seemed responsible for the reduced susceptibility. Resistant Streptococcus pyogenes isolates were compared using M/emm type, repetitive sequence-based PCR (rep-PCR), and pulsed-field gel electrophoresis (PFGE). Rep-PCR provided no more separation of strains than M/emm typing, and PFGE results with SgrAI were more discriminatory than with SmaI. The majority of these isolates were M/emm type 6. PFGE analysis using SgrAI demonstrated 2 different resistant strains among the M/emm type 6 isolates. The findings suggest that a population of S. pyogenes with an intrinsic reduced susceptibility to fluoroquinolones exists in pediatric clinical isolates. Monitoring of amino acid changes in both parC and gyrA will assist in the prediction of emergence of high-level fluoroquinolone resistance.