临床痢疾志贺菌整合子检测及耐药基因盒序列分析

目的 检测印度东部1988、1995和2002年部分临床分离痢疾志贺菌中细菌耐药关系密切的1、2、3类整合酶基因及整合子携带的耐药基因盒的分布,分析整合子系统对志贺菌耐药的影响.方法 纸片扩散法检测实验菌株对药物的敏感性;应用PCR方法对16株临床耐药菌株进行1、2、3类整合酶基因(intI)筛选,对阳性样本可变区基因盒序列进行鉴定分析.结果 所有16株菌均耐4种及4种以上药物,包括β-内酰胺类、氨基糖苷类、四环素类、磺胺类、氯霉素类和喹诺酮类.13株菌检出1类整合酶基因,全部菌株含2类整合酶基因,即发现同时存在两种整合子结构菌株,未检测到3类整合酶基因.1类整合酶插入基因盒以blaara30-aadAl基因家族为主,分别对β-内酰胺类抗生素、链霉素、壮观霉素耐药;2类整合酶插人基因盒以dfrAl-satl组合为主,分别对甲氧苄氨嘧啶、链丝菌素耐药,同时在4株菌中发现dfrAl-satl-aadAl基因盒组合.结论 2类整合子普遍存在于临床志贺菌中.整合子与志贺菌的多重耐药具有密切相关性.     

宋内志贺菌对氟喹诺酮类药物敏感性降低的相关耐药基因研究

目的 研究宋内志贺菌对氟喹诺酮类抗菌药物敏感性降低的相关耐药基因的变化情况.方法 采用琼脂稀释法对131株宋内志贺菌进行药物敏感性检测,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),并对PCR结果进行测序分析,同时用PCR法对质粒介导喹诺酮类耐药(PMQR)基因(qnr)和氨基糖苷乙酰转移酶变异基因aac(6’)-Ib-cr]进行筛选.结果 131株宋内志贺菌对萘啶酸的耐药率达100％,而对诺氟沙星、环丙沙星、左氧氟沙星全部敏感,对四环素、复方磺胺甲(噁)唑和氨苄西林的耐药率分别为93.9％、92.8％和93.2％.94％的萘啶酸耐药宋内志贺菌株对氟喹诺酮类药物敏感性出现下降.萘啶酸耐药菌株gyrA均发生单点83位丝氨酸→亮氨酸(Ser→ Leu)突变,但parC未发生突变;未检出qnr和aac(6')-Ib-cr基因.结论 萘啶酸耐药宋内志贺菌对氟喹诺酮类药物敏感性降低,其产生的主要机制为gyrA发生83位Ser→Leu替换.     

福氏志贺菌中质粒介导喹诺酮类耐药qnr基因的检测

目的 了解福氏志贺菌中qnr基因的分布.方法 采用PCR法检测26株福氏志贺菌的qnr基因并对阳性扩增产物进行测序分析,采用琼脂稀释法测定qnr基因阳性菌株对多种抗菌药物的敏感性,采用随机引物PCR法(ERIC-PCR)进行qnr基因阳性菌株同源性检测.结果 26株福氏志贺菌中,qnrAl、qnrS1和qnrS2基因的阳性率分别为30.8%、11.5%和3.8%;qnr基因阳性菌株对氯霉素、奈啶酸耐药.对诺氟沙星、哌拉西林耐药或中介;部分菌株对头孢唑啉、阿米卡星、第二、三代头孢菌素耐药.ERIC-PCR电泳图谱型显示.2株qnrAl基因阳性菌株属于同一谱型.结论 福氏志贺菌中存在qnr基因阳性菌株,部分菌株间存在克隆传播现象,临床应加强检测和监测.     

志贺菌属耐药性上升对临床治疗带来的挑战

本刊这期刊登了2篇有关志贺菌属对喹诺酮类及头孢菌素类抗菌药物的耐药性及耐药机制的研究论文[1-2],说明志贺菌属对抗菌药物的耐药性已受到密切关注.与其他细菌一样,志贺菌属对抗菌药物耐药性的出现及上升并不是新的议题,但该菌对抗菌药物的耐药性逐渐从氨苄西林、四环素类及磺胺类扩展至喹诺酮类及头孢菌素类,却给临床细菌性痢疾(菌痢)的治疗带来了不少影响.近年来国际上有关志贺菌属的耐药性报道较多的是印度等国家,即便与细菌耐药水平位居国际前列的印度相比,我国志贺菌属对抗菌药物的耐药率总体上并不低,这是令人担忧的.     

志贺菌喹诺酮类耐药株gyrA和parC基因突变研究

目的检测志贺菌对喹诺酮类药的耐药情况,指导临床合理用药;探讨志贺菌喹诺酮类耐药株gyrA和parC基因的突变,分析GyrA和ParC氨基酸改变与喹诺酮类耐药的关系。方法 2010~2012年从宿州市3家综合性医院收集志贺菌76株,进行分离培养和血清型鉴定;采用K-B纸片法进行药物敏感试验;采用PCR方法检测志贺菌喹诺酮耐药决定区（QRDR）相关gyrA和parC基因,并挑选部分PCR产物进行DNA测序分析。结果收集的76株志贺菌中福氏志贺菌74株（占97.4%）,宋内志贺菌2株（占2.6%）;药敏结果显示志贺菌耐药情况严重,其中对阿莫西林耐药率最高,达100%;对头孢噻肟耐药率最低,为6.5%。对gyrA基因的序列分析发现3个导致氨基酸改变的基因点突变：Ser83→Leu,Asp87→Gly及His211→Tyr;对parC基因序列分析发现2个导致氨基酸改变的基因点突变：parC Ser80→Ile和Asp197→Asn。结论宿州市志贺菌感染仍以福氏志贺菌为优势菌群,且耐药严重,临床治疗志贺菌感染可优先选择头孢噻肟。志贺菌属对喹诺酮类药物产生耐药性与gyrA和parC基因突变有关,GyrA His211→Tyr和ParC Asp197→Asn氨基酸变异与喹诺酮类耐药的关系需进一步研究。     

福氏志贺菌中超广谱β-内酰胺酶的基因型分析

目的检测福氏志贺菌中超广谱G内酰胺酶（ESBLs）的基因型别。方法琼脂稀释法测定5株福氏志贺菌对多种抗菌药物的敏感性，并进行接合试验；改良三维试验检测产ESBLs菌株，同时对这些菌株进行脉冲场电泳（PFGE）检测；采用TEM、SHV、CTX-M-1组、CTX-M-2组、CTX-M-9组β-内酰胺酶通用引物以及TEM、CTX-M-9组全编码基因引物进行PCR检测，并对全编码基因PCR产物进行DNA序列分析。结果三维试验结果显示，5株福氏志贺菌均为产ESBLs菌株，对青霉素类、第一代、第二代头孢菌素以及四环素、复方磺胺甲嗯唑显著耐药，对第三代头孢菌素中头孢曲松、头孢噻肟耐药或中度敏感，对亚胺培南、头孢美唑、氟喹诺酮类、头孢噻肟克拉维酸、头孢他啶、头孢他啶一克拉维酸显示敏感。对于β-内酰胺类的耐药性可以通过接合方式发生水平转移；ESBLs基因型别为CTX-M-14，5株菌株的PFGE谱型可分为A、B两种谱型。结论5株福氏志贺菌产生CTX-M-14型超广谱β-内酰胺酶，导致对多种β-内酰胺类抗生素耐药，并存在克隆传播，需加强监控。     

北京地区1994-2005年腹泻病原菌的分布及其耐药趋势

目的 监测我院1994-2005年与腹泻有关的肠道致病菌的分布及耐药趋势,为本地区流行病学研究、疫苗制备及临床合理用药提供依据.方法 通过常规大便培养,筛出致病菌后经生化及血清学进一步鉴定到种、群或血清型,并以纸片扩散法测定抗菌药物的敏感性.结果 12年分离到肠道病原菌8426株,患者以男性为主,儿童和青年发病为多,每年7～9月为腹泻发病高峰.病原以志贺菌属居首位(占75.11%),其次是弧菌(占12.70%),再依次为沙门菌属、气单胞菌、致病及侵袭大肠埃希菌分别占6.28%、4.43%及1.25%.6329株志贺菌中,福氏志贺菌占75.62%,其次是宋内菌群(23.98%),痢疾志贺菌及鲍氏志贺菌分别占0.22%及0.01%.12年的变化趋势是,每年肠道病原菌总数在逐渐减少,特别是志贺菌、沙门菌减少明显,而弧菌及气单胞菌相对逐渐增多.各菌属及不同的血清型对抗菌药物的敏感率有差异,福氏志贺菌和气单胞菌属多重耐药较多,而宋内志贺菌和弧菌属对多数抗菌药物敏感.结论 北京地区感染性腹泻的病原种类多,随时间的变迁病原变化明显,耐药性不同,应重视监测.     

枸橼酸杆菌中质粒介导喹诺酮耐药基因的检测

目的 了解枸橼酸杆菌中质粒介导喹诺酮耐药(PMQR)基因的分布,以期发现新型PMQR基因.测定临床分离的PMQR基因阳性枸橼酸杆菌对临床常用抗菌药物的敏感性.方法 收集安徽医科大学第一附属医院检验科2009年临床分离的枸橼酸杆菌,PCR扩增qnr、aac(6′)-Ib-cr和qepA基因,产物纯化测序,测序结果在GenBank上比对并行转移接合实验.对收集的PMQR基因阳性枸橼酸杆菌及其接合子,采用琼脂对倍稀释法进行临床常用抗菌药物的药物敏感试验.结果 收集的枸橼酸杆菌共31株,8株菌株扩增出qnr,qnr基因阳性率为25.8％;其中6株扩增出qnrB.4株qnr阳性菌株的qnr基因转移接合成功.在qnr阳性的枸橼酸杆菌中,测序发现1种PMQR基因新亚型,命名为qnrB24.所有qnr阳性临床菌株对喹诺酮类药物的耐药率为87.5％,对头孢噻肟、阿米卡星、头孢他啶、头孢吡肟和庆大霉素的耐药率分别为75.0％、7.5％、62.5％、37.5％和87.5％,所有qnr阳性菌株对亚胺培南耐药表型为敏感.喹诺酮类药物对qnr阳性的接合子最低抑菌浓度升高10～23倍,敏感性下降.结论 安徽地区枸橼酸杆菌中qnr基因型的检出率较高,以qnrB基因型为主,qnr阳性枸橼酸杆菌对常用抗菌药物耐药性较高.     

天津地区志贺菌属产超广谱β内酰胺酶及其基因型的检测

目的 了解天津地区志贺菌属产超广谱β内酰胺酶(ESBL)的流行基因型、耐药特征和流行病学特征,探讨产ESBL志贺菌属特性与耐药质粒的关系.方法 136株志贺菌来源于2009年5月至2010年9月在天津市儿童医院、天津医科大学第二医院及天津市第一中心医院门诊就诊的以脓血便为主的腹泻患者粪便标本,纸片扩散法筛选可疑产ESBL志贺菌属;对产ESBL志贺菌进行接合试验,供体菌和接合子进行耐药性测定,以确定耐药性质粒传递;采用TEM、SHV、CTX-M-1组、CTX-M-2组、CTX-M'9组β内酰胺酶通用引物对产ESBL菌株的基因型进行PCR检测.使用肠杆菌科基因间的重复序列PCR(ERIC-PCR)分析产ESBL菌株的分子同源性.采用x2检验.结果 136株志贺菌属中,产ESBL志贺菌20株,检出率为14.7％,未检测出产AmpC酶菌株.其中16株志贺菌基因型为CTX-M-14型,4株为CTX-M-15型;产CTX-M型ESBL菌株对多种抗菌药物耐药,但对亚胺培南的敏感率为100％.18株产ESBL志贺菌接合试验获得成功,其接合子对β内酰胺类抗菌药物耐药.结论 天津地区志贺菌属产ESBL基因型以CTX-M型为主,产ESBL是志贺菌属对β内酰胺类耐药的主要原因,产ESBL菌株的传播机制以质粒介导为主.     

广西宋内志贺菌药敏特性及ESBLs基因检测分析

目的了解广西地区宋内志贺菌的药敏特性及携带超广谱β-内酰胺酶(ESBLs)基因与型别特征,为临床合理用药和控制耐药性的传播提供科学依据。方法采用微量肉汤稀释法对宋内志贺菌分离株进行药物敏感试验;采用PCR方法和毛细管电泳方法检测菌株携带相关ESBLs基因;采用近邻相接法构建系统进化树,分析菌株CTX-M型基因型别;对菌株携带ESBLs基因与特定抗菌药物耐药性的关系进行统计学分析。结果90株宋内志贺菌对环丙沙星敏感率为86.67%,无耐药现象;头孢曲松耐药率为96.67%,阿奇霉素耐药率为96.67%,多重耐药率为98.89%。90株宋内志贺菌中50株检出blaCTX-M-9 group基因,检出率为55.56%,经系统发育分析均为CTX-M-14亚型;1株检出blaTEM基因,检出率为1.11%;未检出blaCTX-M-1 group,blaCTX-M-2 group,blaCTX-M-8 group,blaCTX-M-25 group和blaOXA,blaSHV,blaNDM,blaVIM等β-内酰胺酶基因。blaCTX-M-14基因阳性菌株与blaCTX-M-14基因阴性菌株对头孢噻肟的耐药率差异无统计学意义(χ²=0.000,P>0.05);携带blaTEM基因的菌株对头孢他啶不耐药。结论广西地区的宋内志贺菌对主要治疗药物头孢曲松高度耐药,且多重耐药现象严重;菌株普遍携带ESBLs基因,主要为CTX-M-14亚型;携带单一型别的ESBLs基因与菌株对特定β-内酰胺类抗菌药物耐药无明显关联。     

Prevalence of aac(6')-Ib-cr plasmid-mediated and chromosome-encoded fluoroquinolone resistance in Enterobacteriaceae in Italy

The spread of aac(6')-Ib-cr plasmid-mediated quinolone resistance determinants was evaluated in 197 enterobacterial isolates recovered in an Italian teaching hospital. The aac(6')-Ib-cr gene was found exclusively in Escherichia coli strains. The gene was located on a plasmid which presented additional ESBL genes. Most of the clinical strains were clonally related and displayed three point mutations at the topoisomerase level which conferred high resistance to fluoroquinolones.     

Prevalence of plasmid-mediated quinolone resistance in Escherichia coli isolates in Wenzhou, Southern China, 2002-2008

A total of 514 consecutive clinical Escherichia coli isolates, irrespective of resistance background, were collected in the period 2002-2008 in Wenzhou, southern China, to investigate the prevalence of plasmid-mediated quinolone resistance (PMQR). The dominant PMQR gene was aac(6')-Ib-cr, followed by qnr, whereas qepA was absent. A total of 253 (49.2%) of these isolates were aac(6')-Ib-positive. Subsequently, 134 of these isolates were sequenced and 42 (31.3%) found to harbor aac(6')-Ib-cr, 18 to harbor new aac(6')-Ib mutants, and 74 to harbor wild-type aac(6')-Ib. The genes qnrA, qnrB, and qnrS were found in 2 (0.4%), 6 (1.2%), and 14 (2.7%) of 514 isolates, respectively, with 2 isolates co-harboring qnrB and qnrS genes. Sequencing allowed us to identify qnrA1, qnrB4, qnrB6, and qnrS1 in 20 qnr-positive isolates, with qnrS1 being the most prevalent allele. The genes qnrC and qnrD were not found in any isolates. Interestingly, 35% of qnr-positive isolates and 16.7% of aac(6')-Ib-cr-positive isolates were susceptible to ciprofloxacin. PMQR genes are therefore present in both quinolone-resistant and -susceptible isolates and can also be transferred by conjugation experiments, thus suggesting a likely future increase in quinolone resistance.     

Molecular characteristics of extended-spectrum β-lactamases in clinical isolates of Enterobacteriaceae from the Philippines

β-Lactamases, including extended-spectrum β-lactamases (ESBLs) and AmpC β-lactamases, are major resistance mechanisms of Enterobacteriaceae. Emergence of plasmid-mediated quinolone resistance (PMQR) determinants in ESBL-producing isolates poses a global threat. The molecular characterisitcs of ESBL and PMQR determinants in the Philippines are not well characterized. In this study, we investigated ESBLs and AmpC β-lactamases in clinical isolates of Enterobacteriaceae from the Philippines, and analyzed the association between ESBL and PMQR genes. A total of 91 amoxicilin non-susceptible Enterobacteriaceae were collected at the Research Institute for Tropical Medicine of the Philippines from 2006 to 2008. AmpC- or ESBL-producing isolates were screened by detecting a zone diameter for cefoxitin ≤ 14 mm or cefpodoxime ≤ 20 mm, respectively. Possible ESBL-producing strains were assessed by the ESBL confirmation test of the Clinical and Laboratory Standards Institute. PCR and sequencing were performed to detect the ESBL and PMQR genes. The number of ESBL-producers and AmpC-producers confirmed phenotypically was 17 (18.7%) and 61 (67.0%), respectively. Of 17 phenotypic ESBL-producers, 14 isolates had ESBL genes, including 6 of Escherichia coli, 3 of Enterobacter cloacae, 2 of Enterobacter aerogenes, 2 of Klebsiella pneumoniae, and 1 of Klebsiella ozaenae. Among these isolates, there were 13, 4, and 12 with bla(CTX-M), bla(SHV), and bla(TEM), respectively. Of the bla(CTX-M)-positive isolates, bla(CTX-M-15) shows the highest prevalence, followed by bla(CTX-M-3) and bla(CTX-M-14). Of 14 ESBL-producers identified by PCR, 4, 6, and 7 isolates were positive for qnrB, qnrS, and aac(6')-Ib-cr, respectively. The frequency of aac(6')-Ib-cr positivity was significantly higher among CTX-M-15-producing isolates. Thus, we identified bla(CTX-M), aac(6')-Ib-cr, and qnr in ESBL-producing Enterobacteriaceae from the Philippines, and revealed a significant association between bla(CTX-M-15) and aac(6')-Ib-cr. Local epidemiological data are important for implementing appropriate antimicrobial therapy and effective infection control measures. Continuous monitoring of antimicrobial resistance genes in the Philippines will be required.     

Mechanisms of plasmid-mediated resistance to quinolones

Quinolone resistance is caused mainly by chromosomal mutations in gram negative bacteria. In 1998, plasmid-mediated resistance to quinolones in clinical isolates was first reported in a Klebsiella pneumoniae strain. Locus qnr (quinolone resistance) was responsible of the quinolone resistance in this plasmid. qnr codes a protein whose function is protect both DNA-girase and topoisomerase IV from these antimicrobials. Moreover, qnr is located in an integron-like structure upstream of qacEDelta y sul1. A review of the information obtained in the last years about this mechanism of resistance was performed.     

The worldwide emergence of plasmid-mediated quinolone resistance

Fluoroquinolone resistance is emerging in gram-negative pathogens worldwide. The traditional understanding that quinolone resistance is acquired only through mutation and transmitted only vertically does not entirely account for the relative ease with which resistance develops in exquisitely susceptible organisms, or for the very strong association between resistance to quinolones and to other agents. The recent discovery of plasmid-mediated horizontally transferable genes encoding quinolone resistance might shed light on these phenomena. The Qnr proteins, capable of protecting DNA gyrase from quinolones, have homologues in water-dwelling bacteria, and seem to have been in circulation for some time, having achieved global distribution in a variety of plasmid environments and bacterial genera. AAC(6')-Ib-cr, a variant aminoglycoside acetyltransferase capable of modifying ciprofloxacin and reducing its activity, seems to have emerged more recently, but might be even more prevalent than the Qnr proteins. Both mechanisms provide low-level quinolone resistance that facilitates the emergence of higher-level resistance in the presence of quinolones at therapeutic levels. Much remains to be understood about these genes, but their insidious promotion of substantial resistance, their horizontal spread, and their co-selection with other resistance elements indicate that a more cautious approach to quinolone use and a reconsideration of clinical breakpoints are needed.     

Mechanism of plasmid-mediated quinolone resistance

Quinolones are potent antibacterial agents that specifically target bacterial DNA gyrase and topoisomerase IV. Widespread use of these agents has contributed to the rise of bacterial quinolone resistance. Previous studies have shown that quinolone resistance arises by mutations in chromosomal genes. Recently, a multiresistance plasmid was discovered that encodes transferable resistance to quinolones. We have cloned the plasmid-quinolone resistance gene, termed qnr, and found it in an integron-like environment upstream from qacE Delta 1 and sulI. The gene product Qnr was a 218-aa protein belonging to the pentapeptide repeat family and shared sequence homology with the immunity protein McbG, which is thought to protect DNA gyrase from the action of microcin B17. Qnr had pentapeptide repeat domains of 11 and 28 tandem copies, separated by a single glycine with a consensus sequence of A/C D/N L/F X X. Because the primary target of quinolones is DNA gyrase in Gram-negative strains, we tested the ability of Qnr to reverse the inhibition of gyrase activity by quinolones. Purified Qnr-His(6) protected Escherichia coli DNA gyrase from inhibition by ciprofloxacin. Gyrase protection was proportional to the concentration of Qnr-His(6) and inversely proportional to the concentration of ciprofloxacin. The protective activity of Qnr-His(6) was lost by boiling the protein and involved neither quinolone inactivation nor independent gyrase activity. Protection of topoisomerase IV, a secondary target of quinolone action in E. coli, was not evident. How Qnr protects DNA gyrase and the prevalence of this resistance mechanism in clinical isolates remains to be determined.     

Epidemiology of plasmid-mediated quinolone resistance in salmonella enterica serovar typhimurium isolates from food-producing animals in Japan

A total of 225 isolates of Salmonella enterica serovar Typhimurium from food-producing animals collected between 2003 and 2007 were examined for the prevalence of plasmid-mediated quinolone resistance (PMQR) determinants, namely qnrA, qnrB, qnrC, qnrD, qnrS, qepA and aac(6')Ib-cr, in Japan. Two isolates (0.8%) of S. Typhimurium DT104 from different dairy cows on a single farm in 2006 and 2007 were found to have qnrS1 on a plasmid of approximately 9.6-kbp. None of the S. Typhimurium isolates had qnrA, qnrB, qnrC, qnrD, qepA and acc(6')-Ib-cr. Currently in Japan, the prevalence of the PMQR genes among S. Typhimurium isolates from food animals may remain low or restricted. The PFGE profile of two S. Typhimurium DT104 isolates without qnrS1 on the farm in 2005 had an identical PFGE profile to those of two S. Typhimurium DT104 isolates with qnrS1. The PFGE analysis suggested that the already existing S. Typhimurium DT104 on the farm fortuitously acquired the qnrS1 plasmid.     

qnrA prevalence in extended-spectrum beta-lactamase-positive Enterobacteriaceae isolates from Turkey

Quinolone resistance mostly originates from chromosomal mutations. In recent years, however, plasmid-mediated quinolone resistance has been reported in several parts of the world. Plasmid-borne qnrA, qnrB, or qnrS genes are responsible for this kind of resistance. Little is known about the diversity, type, and species range of the qnr genes in Turkey. We screened qnrA, qnrB, and qnrS genes in quinolone-resistant blood culture isolates collected from six different medical centers in Turkey which produced extended-spectrum beta-lactamases (ESBLs). A total of 78 ESBL-positive isolates were enrolled in this study. Of these, 37 (47.4%) were nalidixic-acid resistant or intermediate. qnrA was found on large plasmids isolated from five (6.4%) of the Nal(I/R) isolates. In three of these, the same plasmid also carried bla(CTX-M). Four of the qnrA-positive isolates were Klebsiella pneumoniae from Dokuz Eylul University Hospital, Izmir, and the fifth isolate was Escherichia coli from Istanbul University Hospital. Two of the isolates from Izmir were found by enterobacterial repetitive interegenic consensus sequence-PCR to be clonally related. This is the first report on the qnrA prevalence among ESBL-positive blood culture isolates collected from different regions in Turkey. According to our results, plasmid-mediated resistance is a potential problem for the spread of quinolone resistance, and this mechanism could be emerging strongly among the ESBL-positive Enterobacteriaceae in Turkey.