阴沟肠杆菌质粒介导耐药机制的研究
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摘要
近年来发现阴沟肠杆菌能引起包括呼吸道感染、泌尿生殖道感染、皮肤软组织感染以及血流感染在内的各种感染,已成为医院感染重要的病原菌。比较上海地区细菌耐药性监测资料,显示本院的肠杆菌属细菌对包括β-内酰胺类、氟喹诺酮类和氨基糖苷类等临床常用的3类抗菌药物的耐药性问题更为突出。据文献报道,肠杆菌科细菌对上述临床常用的3种抗菌药物的耐药机制研究显示：1.产生ESBLs及AmpC酶是该菌对β-内酰胺类抗生素耐药的重要机制,而编码产ESBLs及AmpC酶的基因大多由质粒介导；2.对氨基糖苷类耐药主要因产aac(3)-Ⅰ、Ⅱ、Ⅲ,aac(6’)-Ⅰ、Ⅱ等基因介导的氨基糖苷类钝化酶以及产由rmtB、armA等基因介导的氨基糖苷类甲基化酶所致,以上基因亦通常位于质粒上；3.近年发现的质粒qnr基因可介导喹诺酮类耐药。据文献报道①大多数失活酶和钝化酶的编码基因均由质粒介导。质粒介导产生的酶大多为结构型、固有的,不需要诱导剂即可大量产生,这些酶对p-内酰胺类、氨基糖苷类等相应抗菌药物行水解或修饰钝化作用,造成细菌对抗菌药物高水平耐药；②同一耐药质粒上常同时携带β一内酰胺类、氨基糖苷类、喹诺酮类以及磺胺药、季胺类消毒剂等多种耐药基因,故可引起细菌对多种抗菌药物同时耐药；③质粒介导的耐药基因可随着质粒在肠杆菌科细菌的种和属之间广泛传播,故质粒介导的耐药性在临床上具有十分重要的意义。本课题的前期研究发现临床分离阴沟肠杆菌的PFGE谱显示无明显的流行克隆但其耐药谱竟如此相同,提示这些菌株中可能携带有相同或相似的耐药质粒。因此有必要对阴沟肠杆菌质粒介导的耐药机制进行深入的研究。
本课题对本院2005年临床分离的101株阴沟肠杆菌进行了研究,研究了这些菌株对临床常用抗菌药物的耐药性、同源性,检测了以上菌株对临床上常用的3类抗菌药物质粒介导的耐药基因,并对临床菌株进行了质粒的转移接合试验,检测了质粒所携带的耐药基因和基因的结构。
第一部分阴沟肠杆菌的药物敏感性试验及同源性分析
采用琼脂对倍稀释法测定了头孢噻肟、头孢他啶、环丙沙星、阿米卡星等13种抗菌药物对101株临床分离的阴沟肠杆菌的最低抑菌浓度。结果显示本组101株阴沟肠杆菌对头孢噻肟和头孢他啶的耐药率分别为53.4%和51.4%,但对头孢吡肟较为敏感,细菌耐药率为10.9%。本组资料同时显示该菌对酶抑制剂复方制剂头孢哌酮／舒巴坦和哌拉西林／三唑巴坦十分敏感,细菌耐药率均为16.9%。碳青霉稀类抗生素的抗菌活性最强,细菌敏感率为100%。该菌对环丙沙星和左氧氟沙星的耐药率分别为35.6%和31.7%；对阿米卡星的耐药率较低为19.8%,但对庆大霉素耐药率为41.6%。本组细菌耐药谱显示101株阴沟肠杆菌中对第三代头孢菌素耐药同时对喹诺酮类或氨基糖苷类耐药的菌株为30株(29.7%,30/101),对第三代头孢菌素、喹诺酮类和氨基糖苷均耐药的菌株为22株(21.8%,22/101)。
101株阴沟肠杆菌的PFGE分型结果显示本组细菌同源菌株很少,仅有3组共7株细菌分别属同一克隆株,余94株细菌PFGE图谱均不相同,提示在本院范围内无阴沟肠杆菌同一克隆株的流行,但以上不同克隆株经常表现出相同或相似的耐药谱型,提示这些不同的菌株内可能携带相同或相似的耐药质粒。
第二部分阴沟肠杆菌质粒介导耐药基因的分子生物学研究
本部分研究采用酚／氯仿法对101株临床分离阴沟肠杆菌进行质粒DNA抽提后,经PCR方法扩增检测质粒介导的ESBLs和AmpC酶基因,氨基糖苷类钝化酶和氨基糖苷类甲基化酶基因,以及质粒介导喹诺酮类耐药的三种主要基因。
1.质粒介导的ESBLs和AmpC酶基因检测采用酚／氯仿法制备阴沟肠杆菌质粒DNA模板,运用TEM型、SHV型、CTX-M型引物以及质粒介导AmpC酶的6对引物MOX、CIT、DHA、ACC、EBC、FOX等对101株阴沟肠杆菌进行PCR扩增并测序结果显示72株(71.3%,72/101)含有质粒介导β-内酰胺酶,其中37株(36.6%,37/101)菌株含有TEM-1型β-内酰胺酶基因,54株(53.5%,54/101)携带SHV-12型ESBLs,31株(30.7%,31/101)含CTX-M型ESBLs基因。18株(17.8%,18/101)含DHA-1型AmpC酶。上述72株菌株中仅含TEM-1型基因或仅含DHA-1型AmpC酶基因各1株,余70株(69.3%,70/101)均为产ESBLs菌株,其中46株(45.5%,46/101)含两种或两种以上β-内酰胺酶基因。
2.质粒介导的喹诺酮耐药基因检测本研究以细菌质粒DNA为模板,运用qnrA、qnrB、qnrS、qnrC、qnrD、aac(6')-Ib及qepA基因引物,对101株阴沟肠杆菌采用PCR方法进行了扩增,并对qnr基因进行了测序分型；同时检测了染色体介导的DNA旋转酶及拓扑异构酶IV的gyrA和parC基因突变和相应氨基酸突变。结果显示101株阴沟肠杆菌中共有39株细菌为qnr基因阳性(38.6%,39/101),其中19株(18.8%,19/101)细菌为qnrA基因阳性；18株(17.8%,18/101)细菌为qnrB基因阳性；3株(3.0%,3/101)细菌为qnrS基因阳性。其中1株细菌同时携带qnrB及qnrS两种基因。101株阴沟肠杆菌中未检测出qnrC及qnrD基因。所有qnr基因阳性菌株的扩增产物均经测序并与GenBank上基因序列进行比对,结果显示19株qnrA基因阳性菌株均为qnrAl亚型,18株qnrB基因阳性菌株中有16株为qnrB4亚型,1株为qnrB6亚型,1株为qnrB10亚型；3株qnrS基因阳性菌株中1株为qnrSl亚型,1株为qnrS2亚型,另有1株菌株同时含有qnrB4和qnrSl两种qnr基因。101株阴沟肠杆菌中有44株细菌采用PCR方法扩增到aac(6’)-Ib基因的扩增产物。该44株细菌的aac(6’)-Ib基因的扩增产物经FokI酶切后其中7株细菌因缺乏GGATG位点,无法切开,此7株(6.9%,7/101)同时经序列测定确证为aac(6’)-Ib基因突变株-aac(6’)-Ib-cr菌株,其中4株同时携带qnrB4基因,余37株FokI酶切均可切开,为aac(6’)-Ib野生型菌株。101株阴沟肠杆菌中未扩增到qepA基因。
对39株qnr基因阳性的菌株采用PCR方法扩增了gyrA和parC基因的喹诺酮类耐药决定区域(QRDR),并对其扩增产物进行了测序。与Genbank进行了比对,结果显示有23株qnr基因检测阳性的菌株GyrA和ParC QRDR区域氨基酸无突变,该类菌株均为环丙沙星敏感株(按CLSI2006年标准)。一株细菌的GyrA QRDR区域83位Ser突变为Tyr,该菌株为环丙沙星中敏株。另外在15株qnr基因检测阳性发现QRDR区域发生1-3个位点突变,包括GyrA QRDR区域83位Ser(丝氨酸)突变为Ile(异亮氨酸)或Phe(苯丙氨酸)或Leu(亮氨酸)及87位Asp(天冬氨酸)突变为Ala(丙氨酸),ParC QRDR区域80位Ser(丝氨酸)突变为I1e(异亮氨酸),该类菌株均为环丙沙星耐药株。
101株阴沟肠杆菌中有36株环丙沙星耐药株,其中含qnr基因阳性的菌株为15株,阳性检出率为41.7%(15/36)；在63株环丙沙星敏感菌株中qnr基因阳性菌株23株,阳性率为36.5%(23/63)。两组细菌qnr基因阳性率经卡方检验,差异无统计学意义(P＝0.23),提示在喹诺酮耐药或不耐药的两组菌株中qnr基因检出率相仿。本研究39株qnr阳性菌株中有23株细菌对环丙沙星敏感,这些菌株的GyrA和ParC QRDR区域无氨基酸突变,其环丙沙星的MIC值为0.06mag/L-1mag/L。与野生株相比(通常野生株对环丙沙星的MIC≤0.015mg／L),MIC值明显降低。提示这些细菌对喹诺酮类敏感性的下降可能仅由qnr所致,推测qnr也可能是细菌在对喹诺酮类耐药性形成过程中最早出现的耐药机制。
101中株阴沟肠杆菌中70株细菌含有ESBLs酶基因,其中35株(50%,35/70)qnr基因阳性,而31株非产ESBLs菌株中仅4株(12.9%,4/31)细菌为qnr基因阳性,两组细菌的qnr基因检测阳性率经卡方检验差异具有统计学意义(P=0.005)。在18株质粒介导AmpC酶阳性菌株中14株(77.8%,14/18)细菌出现qnr基因阳性,而83株非产AmpC酶菌株中有25株(30.1%,25/83)细菌qnr基因阳性,两组细菌的qnr基因检测阳性率经卡方检验同样具有统计学意义(P=0.018),上述结果提示产ESBLs或者质粒介导AmpC酶的菌株较非产酶株更易同时携带qnr基因。
3.质粒介导氨基糖苷类钝化酶和氨基糖苷类甲基化酶检测。本研究应用aac(3)-I、aac(3)-II、aac(3)-III、aac(6')-Ib、aac(6')-II、ant(2")-I、ant(3”)-Ⅰ、aph(3’)-Ⅵ等8种氨基糖苷类钝化酶基因和armA及rmtB2种氨基糖苷类甲基化酶基因引物对101株阴沟肠杆菌进行了PCR扩增,结果显示48株(47.5%,48/101)被检测出含有氨基糖苷类钝化酶基因；13株(12.9%,13/101)细菌含有armA或rmtB甲基化酶基因,其中10株(9.9%,10/101)同时检测出含氨基糖苷类钝化酶和氨基糖苷类甲基化酶基因。
48株氨基糖苷类钝化酶基因阳性的菌株中有44株(43.5%,44/101)菌株被检测出含有aac(6’)-Ib基因,其中有7株为aac(6’)-Ib-Cr(aac(6’)-Ib基因突变株)；22株(21.8%,22/101)含ant(2”,)-Ⅰ基因；6株(5.9%,6/101)含aac(3)-Ⅱ基因；1株(1.0%,1/101)含aac(3)-Ⅰ基因；2株(2.0%,2/101)含ant(3”)-Ⅰ基因。而aac(3)-Ⅲ、aac(6’)-Ⅰ和aph(3’)-Ⅵ等3种氨基糖苷类钝化酶基因在本组101株阴沟肠杆菌中均未检出。
在13株(12.9%,13/101)被检测为含有armA或rmtB氨基糖苷类甲基化酶基因的菌株中含armA基因为12株(11.9%,12／101),含rmtB基因为2株(2.0%,2/101)。其中1株同时含有armA和rmtB基因。13株含氨基糖苷类甲基化酶菌株均表现为对阿米卡星和庆大霉素高度耐药,上述两种抗生素的MIC值均>256mg/L。在含氨基糖苷类钝化酶基因的菌株中呈现对氨基糖苷类不同的耐药水平。如含单一aac(6’)-Ib基因的菌株中表现为庆大霉素MIC值从4mg/L到>256mg/L不等。
在101株阴沟肠杆菌中共有70株(70.3%)含有ESBLs酶的细菌中,有47株(67.1%,47/70)细菌呈现氨基糖苷类钝化酶基因阳性,而31株非产ESBLs菌株中仅1株(3.3%,1/31)细菌为氨基糖苷类钝化酶基因阳性,两组细菌的氨基糖苷类钝化酶基因检出阳性率经卡方检验差异具有统计学意义(P=0.0001)。70株ESBLs阳性菌株中有13株(18.6%,13/70)细菌含氨基糖苷类甲基化酶基因,另外31株非产ESBLs菌株均未检测到含氨基糖苷类甲基化酶基因菌株,两组细菌的氨基糖苷类甲基化酶基因检出阳性率经卡方检验差异具有统计学意义(P=0.005)。上述结果显示产ESBLs菌株中产氨基糖苷类钝化酶和氨基糖苷类甲基化酶检出率明显高于非产ESBLs酶菌株,提示在产ESBLs酶菌株中易同时携带有氨基糖苷类钝化酶基因和氨基糖苷类甲基化酶基因。
第三部分阴沟肠杆菌耐药质粒的接合转移
为了解上述101株阴沟肠杆菌中耐药质粒的可转移性,选择①44株同时含有ESBLs(SHV-12型及CTX-M型)及DHA-1型AmpC酶基因的菌株；②12株同时含SHV-12型ESBLs.DHA-1型AmpC.qnr.aae(6')-Ib基因4种基因的临床菌株作为供体菌,对大肠埃希菌J53AZR进行质粒转移接合试验。
44株同时含有ESBLs及AmpC酶基因的菌株中有30株细菌发生了转移接合,耐药质粒的转移接合发生率为70.5%(30/44)。经对接合子的质粒抽提结果显示7株接合子与供体细菌一样均只携带1个质粒。对这7株接合子进一步研究显示①供体菌E-41×J53AZR→接合子41TC发生了CTX-M-1G基因的转移接合；②供体菌E-57×J53 AZR→接合子57TC和供体菌E-70×J53 AZR→70TC发生了CTX-M-9G和aac (6’)-Ib基因的转移接合；③供体菌E-91×J53AZR→接合子91TC和供体菌E-95×J53AZR→95TC发生了qnrB、SHV-12、DHA-1和aac(6’)-Ib基因的转移接合；④供体菌E-206×J53AZR→接合子206TC和供体菌E-209×J53AZR→209TC发生了CTX-M-9G和aac(6’,)-Ib基因的转移接合。转移接合的结果提示①以上CTX-M-1G、CTX-M-3G、CTX-M-9G、SHV-12型ESBLs及DHA-1型AmpC均具有可接合性。②依据7株接合子菌株均含有1个质粒,故提示CTX-M-3G型和aac(6’)-Ib基因、CTX-M-9G和aac(6’)-Ib、SHV-12和aac(6’)-Ib基因可能位于同一质粒上,发生共同转移接合。7株接合子的头孢噻肟和头孢他啶的MIC值在4-32mg/L,与供体菌接近,较受体菌J53AZR(MIC值为≤0.06mag/L)升高66.7-533倍,庆大霉素和阿米卡星的MIC值在4-128mg/L(除1株41TC庆大霉素MIC为0.5mg/L),较受体菌J53AZR(庆大霉素MIC值为0.125mg/L)升高16-1024倍。
12株同时含有qnrB, SHV-12、DHA-1和aac (6’)-Ib基因的临床菌株行转移接合试验后有7株细菌发生了转移接合,耐药质粒的转移接合发生率为58.8%(7/12)。经对接合子的质粒抽提结果显示：7株供体菌基因型类似,均含有SHV-12、DHA-1、qnrB4、aac(6')-Ib、ant (2")-I基因,发生了SHV-12、DHA-1、qnrB4,aac(6')-Ib基因的转移接合,接合子的环丙沙星MIC值比受体菌J53AZR高12-24倍,为0.094 mg/L-0.19mg/L,而受体菌的MIC值仅为0.008mg/L。这一结果同样提示接合子对喹诺酮类敏感性的下降仅由qnrB基因所致,推测qnr可能是细菌对喹诺酮类耐药性形成过程中最早出现的耐药耐药机制。7株接合子的三代头孢菌素的MIC值与供体菌接近,但较头孢噻肟或头孢他啶对受体菌J53AZR(≤0.06 mg／L)的MIC值上升了66.7-1000倍,达到4 mg/L-64mg/L,对氨基糖苷类抗菌药物庆大霉素和阿米卡星均较受体菌J53AZR(0.125 mg/L～0.25mg/L)的MIC上升了16-1024倍,达到2 mg/L-128mg/L。
第四部分阴沟肠杆菌多重耐药质粒的基因结构分析
对上述研究获得的7株含qnrB基因、SHV-12. DHA-1及aac(6’)-Ib基因的转移接合子进行耐药质粒的基因结构及基因周边结构的进一步研究结果显示：经HindⅢ酶切、与载体PUC18连接,再经酶切验证发现一段约14kb的片段连接入载体PUC18中。对其中2株接合子质粒65TC和404TC的14kb片段进行了测序结果显示2个接合子的DNA周边结构相同,且与本研究所前期报道的肺炎克雷伯中含qnrB4基因质粒pSH7结构相仿,包含有sapA-qnrB4-pspF-pspA-pspB-pspC-pspD-orfl-基因序列。qnrB4基因上游为sapA基因,推测其编码一个肽转运系统酶。pspF-pspA-pspB-pspC-pspD为psp操纵子,编码一个噬菌体休克蛋白,其后为orfl读码框,之后为编码AmpC酶的blaDHA-1基因及ampC的调控基因ampR。此外采用PCR检测发现以上7株接合子及其供体菌均含有qac△l-sull耐消毒剂和磺胺基因、整合子遗传标记Integrase I(整合酶基因)、Tn21／Tn501转座子遗传标记(汞离子还原酶基因)基因,提示以上7株接合子除对三代头孢菌素、氨基糖苷类耐药外,还对季胺类消毒剂和磺胺耐药,系多重耐药质粒,故以上7株接合子含有qnrB4基因、SHV-12、DHA-1、aac(6’)-Ib以及qac△l-sull耐消毒剂和磺胺基因、整合子遗传标记Integrase I(整合酶基因)、Tn21／Tn501转座子遗传标记(汞离子还原酶基因)基因,在阴沟肠杆菌中发现含有以上多种耐药基因的质粒尚属首次报道。7株转移接合子中SHV-12、aac (6')-Ib等基因位于质粒的哪个区域还有待进一步的研究。
Recent studies have found that Enterobacter cloacae (E. cloacae) can cause various infections including respiratory tract, urogenital tract, skin soft tissue, and bloodstream infections. It has become an important pathogen of nosocomial infections. Furthermore, contrasting to the surveillance of bacterial resistance of Shanghai to our hospital has shown that Enterobacter has prominent resistance to antibiotics ofβ-lactams, fluoroquinolones and aminoglycosides, which are the three commonly used clinical drugs. Studies on the resistance mechanisms of E. cloacae to the above three antibiotics have indicated that:1. the mainβ-lactam antibiotics-resistant mechanism for E. cloacae is the production of the extended spectrumβ-lactamases (ESBLs) and AmpCβ-lactamases (AmpC enzymes), which is primarily mediated by plasmids; 2. the aminoglycoside resistance of E. cloacae is mainly mediated by aminoglycoside-modifying enzyme genes including aac (3)-I, II, III and aac (6')-I, II as well as aminoglycoside methylase genes such as rmtB and armA, which are also generally located on plasmids.3. the plasmid-mediated quinolone resistance discovered recently is an important mechanism for E. cloacae to resist quinolone antibiotics, which is mediated by genes including qnr. According to the report, the plasmid-mediated resistance has some characteristic:①Generally, modifying enzymes were plasmid mediated. Plasmid-mediated enzymes were constitutive hyperproduction without inducers. These enzymes can hydrolyze or modifyβ-lactams or aminoglycosides and cause high level resistance;②one plasmid can containβ-lactams, fluoroquinolones, aminoglycosides resistance gene, together with quaternary ammonium disinfectants and sulfonamides resistance gene simultaneously, it will cause one strain can resist many kinds of antibiotics;③The gene for plasmid-mediated resistance enzymes can disseminate between Enterobacter spp. Due to above reason, plasmid-mediated resistance is very important on the study of bacterial resistance. The results of pulsed-field gel electrophoresis (PFGE) of E. cloacae clinical isolates showed that there were no clone transmission but the resistance pattern was the same, it implied that these strains can have the same or similar plasmids. Therefore, it is necessary to conduct deep studies on the plasmid-mediated mechanisms of E. cloacae.
Based on the above situations, the present study investigated a total of 101 strains of E. cloacae clinically isolated in 2005 in our hospital. In this study, drug resistance and homology of these strains were studied; plasmid-mediated resistance genes in these strains to the three clinically commonly used antibiotics were detected; plasmid conjugation experiments of these clinical strains were performed, and the resistance genes and structural genes carried by plasmids were tested.
Part 1 Drug sensitivity test and homology analysis of Enterobacter cloacae
The two-fold agar dilution method was used to determine the minimum inhibitory concentration (MIC) of 14 anti-infective drugs on the 101 strains of clinically isolated E. cloacae. Results showed that the resistance rates of the 101 E. cloacae strains in this group to cefotaxime and ceftazidime were up to 53.4%and 51.4%respectively; while the strains were sensitive to cefepime with the resistance rate of only 10.9%; and the resistance rates to cefoperazone/sulbactam and piperacillin/tazobactam were both 16.9%. The strains were highly sensitive to carbapenem antibiotics without resistance. The resistance rates to quinolone antibiotics and ciprofloxacin were 35.6%and 31.7%; to amikacin and gentamicin were 19.8%and 41.6%respectively. Of 101 strais,30 (29.7%,30/101)strains were resistant to the third cephosporins, together with resistance to quinolone or aminoglycosides, 22 (21.8%,22/101) were resistant to the third cephosporins, quinolone and aminoglycosides.
Results of pulsed-field gel electrophoresis (PFGE) characterization for the 101 E. cloacae strains showed that there were a very small number of homogenous strains in this group. Only 3 strains were of the same clone, while 94 strains had different PFGE fingerprint patterns, indicating that no prevalence of the same clone strains of E. cloacae within our hospital. However, the above strains of different clones often showed the same or similar type of drug resistance spectrums, suggesting that these different strains were likely to carry the same or similar drug resistance plasmids.
Part 2 molecular biology study of plasmid-mediated resistance genes in E. cloacae
After the extraction of bacterial plasmid DNA templates by phenol/chloroform method, PCR was used to detect plasmid-mediated ESBLs and AmpC, aminoglycoside-modifying enzymes and the plasmid-mediated 16S rRNA aminoglycoside methylase, three kinds of quinolone resistance genes and detecting the amino acid mutations of gyrA and parC genes.
1. detecting plasmid-mediated ESBLs and AmpC. After the extraction of bacterial plasmid DNA templates by phenol/chloroform method, the PCR method using CTX-M, SHV and TEM-1 primers, the multiplex PCR using MOX, CIT, ACC, EBC, DHA or FOX-type AmpC primers, found 72 (71.3%) of the 101 E. cloacae strains contained plasmid-mediatedβ-lactamases.37 (36.6%, 37/101) had TEM-1 typeβ-lactamase genes,54 strains (53.5%,54/101) contained SHV-12 type ESBLs genes,31 strains (30.7%,31/101) contained CTX-M-type ESBLs genes.18 strains (17.8%,18/101) were found to contain DHA-1 type plasmid-mediated AmpC enzyme. In 72 containingβ-lactamases strains,1 contained only DHA-1 type AmpC enzymes and 1 contained only TEM-1 type 0-lactamase gene, the other 70 strains (69.3%,70/101) were all ESBLs stains. Among these 70 strains,46 strains (45.5%,46/101) contained two or more than two kinds ofβ-lactamase.
2. detecting plasmid-mediated quinolone resistant genes. In this study, with bacterial plasmid DNA as template, using qnrA, qnrB, qnrS, qnrC, qnrD, aac (6')-Ib-cr and qepA primers, plasmid-mediated quinolone resistant genes of the 101 E. cloacae strains were detected by PCR. The qnr genes were sequenced for typing, and the amino acid mutations of gyrA and parC genes encoding DNA gyrase and topoisomerase IV were detected.
Of the 101 E. cloacae strains,39 had positive qnr genes (positive rate 38.6%).19 strains (18.8%) were positive for qnrA gene,18 (17.8%) positive for qnrB, and 3 (3.0%) positive for qnrS; 1 of them carried both qnrB and qnrS genes; qnrC and qnrD genes were not found. Through gene sequencing and alignment on GenBank for all qnr positive strains, all qnr A gene positive strains contained qnrAl subtype genes. Among qnrB gene positive strains,16 contained qnrB4 subtype,1 contained qnrB6 subtype, and 1 contained qnrB10 subtype. Among qnrS gene positive strains,1 contained qnrSl subtype,1 contained qnrS2 subtype, and 1 had both qnrB4 and qnrSl genes.44 of the 101 strains were positive for aac (6')-Ib gene by PCR detection. After the PCR products were digested by FokI enzyme, the products of 7 strains could not be cut due to the lack of GGATG site. The 7 strains were determined by sequencing and found to be positive for aac (6')-Ib-cr gene with the positive rate of 6.9%, of which 4 strains were also carried qnrB4 gene. No qepA gene was found through amplification.
GyrA and ParC genes of the 39 qnr gene positive strains were tested by PCR, and they were sequenced and aligned on Genbank.23 strains that were positive for qnr gene, having no amino acid mutations at the quinolone resistance determining region (QRDR) of the GyrA and ParC genes, were found sensitive to ciprofloxacin (according to 2006 standard). Another strain with Ser 83 mutating to Tyr at the QRDR of GyrA, was a ciprofloxacin sensitive strain. In the other 15 qnr gene positive strains which 1-3 point mutations of each were found at the QRDR, including Ser 83 mutated to Ile, Phe or Leu, Asp 87 mutated to Ala at the GyrA QRDR, and Ser 80 mutated to Ile at the ParC QRDR, were ciprofloxacin resistant strais.
Of the 36 ciprofloxacin-resistant strains,15 were qnr gene positive with the positive rate of 41.7%; of the 63 ciprofloxacin-sensitive strains, 23 were qnr gene positive with the positive rate of 36.5%. The difference of the qnr gene positive rate between the two groups was not statistically significant by the chi-square test (P-0.23), indicating that the detection rate of qnr genes between the quinolone resistant and sensitive groups was similar. In this study,23 of the 39 qnr positive strains were ciprofloxacin-sensitive, but the MIC values were up to 0.06-1mg/l, suggesting that the decreased sensitivity to quinolones for these bacteria may be caused only by qnr, and qnr may be the first appeared resistance mechanism during the formation of quinolone resistance for bacteria.
Among 101 strains,70 contained ESBLs, of the 70 ESBLs strains,35(50%, 35/70) were qnr positive strains. Of the other 31 non-ESBLs strains, only 4(12.9%,4/31) strains were qnr positive strains. The difference of the qnr gene positive rate between the two groups was statistically significant by the chi-square test (P=0.005). Of the 18 AmpC strains,14 (77.8%,14/18) were qnr positive strains, of the other 83 non-AmpC strains, 25 strains (30.1%,25/83) were qnr positive strains. It was also statistically significant by the chi-square test between the two groups (P=0.018). It suggested that qnr was more frequent in ESBLs or AmpC strains.
3.Detecting aminoglycoside-modifying enzymes and the plasmid-mediated 16S rRNA aminoglycoside methylase. In this study, eight aminoglycoside-modifying enzyme genes of aac (3)-Ⅰ, aac (3)-Ⅱ, aac (3)-Ⅲ, aac (6')-Ⅰb, aac (6')-Ⅱ, ant (2")-Ⅰ, ant (3")-Ⅰ, and aph (3')-Ⅵ, as well as two methylase genes of armA and rmtB were amplified by PCR. Among the 101 E. cloacae strains,48 (47.5%,48/101) contained aminoglycoside-modifying enzyme genes,13 (12.9%,13/101) contained armA or rmtB, in which 10 (9.9%,10/101) contained both aminoglycoside-modifying enzymes and aminoglycoside methylase genes.
In the 48 strains containing aminoglycoside-modifying enzyme genes, the most commonly found gene was aac (6')-Ⅰb, which existed in 44 strains (43.6%,44/101). After digestion,7 strains (6.9%) were found to contain aac (6')-Ⅰb-Cr gene, 22 (21.8%) contained ant (2")-Ⅰgene, 6 (5.9%) had aac (3)-Ⅱ, 1 (1.0%) had aac (3)-Ⅰ, and 2 (2.0%) contained ant (3")-Ⅰ. The aac (3)-Ⅲ, aac (6')-Ⅱ, or aph (3')-Ⅵgenes were not found in the 101 strains of E. cloacae in this group.
Through PCR detection,13 strains (12.9%) were found to contain methylase genes armA or rmtB, of which 12 (11.9%) contained armA gene, 2 (2.0%) contained rmtB gene, and 1 contained both armA and rmtB genes. The 13 strains with aminoglycoside methylases all showed a high degree of resistance to amikacin and gentamicin, and the MIC values were all above 256ug/ml. The aminoglycoside resistance mediated by aminoglycoside modifying enzymes showed different resistance levels, for example, strains containing aac (6')-Ib gene had MIC values for gentamicin from 4ug/ml to above 256ug/ml.
Among 101 strains,70 contained ESBLs, of the 70 ESBLs strains, 47(67.1%,47/70) strains were aminoglycoside-modifying enzyme genes positive. Of the other 31 non-ESBLs strains, only 1(3.3%,1/31) strain were aminoglycoside-modifying enzyme genes positive. The difference of the aminoglycoside-modifying enzyme genes positive rate between the two groups was statistically significant by the chi-square test (P=0.0005). Of the 70 ESBLs strains,13 (18.6%,13/70)strains were aminoglycoside methylases positive strains. Of the other 31 non-ESBLs strains, none strain were aminoglycoside methylases enzyme genes positive. It was also statistically significant by the chi-square test between the two groups (P=0.0003). It suggested that aminoglycoside-modifying and aminoglycoside methylases enzyme genes were more frequent in ESBLs strains.
Part 3 Plasmid conjuction of multi-resistant Enterobacter Cloacae resistance genes
To investigate the transferability of resistant plasmid in 101 strains, we selected①44 strains with ESBLs(SHV-12 and CTX-M) and AmpC (DHA-1) enzyme genes;②12 strains with SHV-12, DHA-1, aac (6')-Ib and qnrB4 as as donor strains, E. Coli J53AzR as receptor to do plasmid conjugation tests.
Plasmid conjugation tests were conducted for a total of 44 strains with ESBLs or AmpC enzyme genes and 30 strains got conjugants, conjugated rate was 68.2%(30/44). Seven conjugants were found to have only one plasmid each, so further study had been done to the seven conjugants, the result showed:①donor strain E-41 X J53AZR→conjugant 41TC (CTX-M-1G gene conjugation);②donor strain E-57 X J53 AzR→conjugant 57TC and donor strain E-70×J53 AZR→conjugant 70TC (CTX-M-9G and aac (6')-Ib genes conjugation);③donor strain E-91 X J53AzR→conjugant 91TC and donor strain E-95×J53 AZR→95TC (qnrB, SHV-12, DHA-1 and aac(6')-Ib genes conjugation);④donor strain E-206×J53AZR→conjugant 206TC and donor strain E-209 X J53AZR→conjugant 209TC (CTX-M-9G and aac(6')-Ib genes conjugation).
The result of conjugation suggested①OTX-M-1G, CTX-M-3G, CTX-M-9G, SHV-12 and DHA-1 could be transmitted by plasmid conjugation.②Since 7 conjugated strains containing only one plasmid, it implied that CTX-M-3G together with aac(6')-Ib, CTX-M-9G together with aac(6')-Ib, SHV-12 together with aac(6')-Ib can be cotransmitted by one plasmid. The 7 conjugants MIC of cefotaxime and ceftazidim was 4-32mg/l, like donor strains, but it was 66.7-533 times than receptor J53AzR (MIC≤0.06mg/L) The conjugants MIC of Gentamycin and Amikacin was 4-128mg/l, it was 16-1024 times than receptor J53AZR (MIC 0.125mg/L)
Plasmid conjugation tests were conducted for 12 strains harboring qnrB, SHV-12, DHA-1 and aac(6')-Ib and 7 strains got conjugants, conjugated rate was 58.8%(7/12).The genes of 7 donor strains were similar, all of them had qnrB4, SHV-12, DHA-1, aac(6')-Ib and ant (21-I genes. The conjugants contained qnrB4, SHV-12, DHA-1, aac(6')-Ib genes. Ciprofloxacin MIC of 7 conjugants was 0.094 mg/L～0.19mg/L, which was 12-24 times more than receptor J53AZR (MIC 0.008mg/L). It also suggested that the decreased sensitivity to quinolones for these bacteria may be caused only by qnr, and qnr may be the first appeared resistance mechanism during the formation of quinolone resistance for bacteria. The 7 conjugants MIC of cefotaxime and ceftazidim was 4-64mg/l, like donor strains, but it was 66.7-1000 times than receptor J53AZR (MIC≤0.06mg/L) The conjugants MIC of Gentamycin and Amikacin was 4-64mg/l, it was 16-1024 times than receptor J53AZR (MIC 0.125mg/L-0.25mg/L)
Part 4 Construction analysis of multi-resistant plasmid of Enterobacter Cloacae
The conjugated plasmids containing qnrB, SHV-12, DHA-1 and aac(6')-Ib was futher investigated in the structural features of the plasmids. The plasmids were digested by HindIII and ligated with PUC18. Then the ligated PUC18 was digested again and was found a 14kb fragment was ligated in the PUC18. The 14kb fragment of conjugants 65TC and 404TC was sequenced. Sequencing results showed that the two plasmids had the same structure at the surrounding DNA regions. The structure was similar to the Plasmid pSH7 containing qnrB4 gene in Klebsiella pneumoniae, which was reported previously by our institute, containing sapA-qnrB4-pspF-pspA-pspB-pspC-orfl-pspD-blaBGA-1-ampR gene sequences. The sapA gene was at the upstream of qnrB4, suggesting that it encoded a peptide transport system enzyme. The pspF-pspA-pspB-pspC-pspD was the operon of psp, encoding a phage shock protein, followed by orfl reading frame, and the blaBHA-1 gene encoding AmpC enzyme, as well as the ampC regulatory gene ampR. Detected by PCR, the seven conjugants also contained qacΔ1-sull disinfectant and sulfonamide resistance genes, integron genetic marker Integrase I (integrase gene), and Tn21/Tn501 transposon genetic marker gene (mercury ion reductase gene), indicating that the seven zygotes were resistant to quaternary ammonium disinfectants and sulfonamides, in addition to the third generation of cephalosporins and aminoglycoside antibiotics. Therefore, they were multiple drug resistance plasmids. The seven zygotes harbor qnrB4, SHV-12, DHA-1, aac(6')-Ib, qacΔ1-sull disinfectant and sulfonamide resistance genes, integron genetic marker Integrase I (integrase gene), and Tn21/Tn501 transposon genetic marker gene. This is the first report a plasmid from E. cloacae contain such kinds of resistance genes. SHV-12, aac(6')-Ib locate in which place in plasmid needs futher research.
本课题对本院2005年临床分离的101株阴沟肠杆菌进行了研究,研究了这些菌株对临床常用抗菌药物的耐药性、同源性,检测了以上菌株对临床上常用的3类抗菌药物质粒介导的耐药基因,并对临床菌株进行了质粒的转移接合试验,检测了质粒所携带的耐药基因和基因的结构。
第一部分阴沟肠杆菌的药物敏感性试验及同源性分析
采用琼脂对倍稀释法测定了头孢噻肟、头孢他啶、环丙沙星、阿米卡星等13种抗菌药物对101株临床分离的阴沟肠杆菌的最低抑菌浓度。结果显示本组101株阴沟肠杆菌对头孢噻肟和头孢他啶的耐药率分别为53.4%和51.4%,但对头孢吡肟较为敏感,细菌耐药率为10.9%。本组资料同时显示该菌对酶抑制剂复方制剂头孢哌酮／舒巴坦和哌拉西林／三唑巴坦十分敏感,细菌耐药率均为16.9%。碳青霉稀类抗生素的抗菌活性最强,细菌敏感率为100%。该菌对环丙沙星和左氧氟沙星的耐药率分别为35.6%和31.7%；对阿米卡星的耐药率较低为19.8%,但对庆大霉素耐药率为41.6%。本组细菌耐药谱显示101株阴沟肠杆菌中对第三代头孢菌素耐药同时对喹诺酮类或氨基糖苷类耐药的菌株为30株(29.7%,30/101),对第三代头孢菌素、喹诺酮类和氨基糖苷均耐药的菌株为22株(21.8%,22/101)。
101株阴沟肠杆菌的PFGE分型结果显示本组细菌同源菌株很少,仅有3组共7株细菌分别属同一克隆株,余94株细菌PFGE图谱均不相同,提示在本院范围内无阴沟肠杆菌同一克隆株的流行,但以上不同克隆株经常表现出相同或相似的耐药谱型,提示这些不同的菌株内可能携带相同或相似的耐药质粒。
第二部分阴沟肠杆菌质粒介导耐药基因的分子生物学研究
本部分研究采用酚／氯仿法对101株临床分离阴沟肠杆菌进行质粒DNA抽提后,经PCR方法扩增检测质粒介导的ESBLs和AmpC酶基因,氨基糖苷类钝化酶和氨基糖苷类甲基化酶基因,以及质粒介导喹诺酮类耐药的三种主要基因。
1.质粒介导的ESBLs和AmpC酶基因检测采用酚／氯仿法制备阴沟肠杆菌质粒DNA模板,运用TEM型、SHV型、CTX-M型引物以及质粒介导AmpC酶的6对引物MOX、CIT、DHA、ACC、EBC、FOX等对101株阴沟肠杆菌进行PCR扩增并测序结果显示72株(71.3%,72/101)含有质粒介导β-内酰胺酶,其中37株(36.6%,37/101)菌株含有TEM-1型β-内酰胺酶基因,54株(53.5%,54/101)携带SHV-12型ESBLs,31株(30.7%,31/101)含CTX-M型ESBLs基因。18株(17.8%,18/101)含DHA-1型AmpC酶。上述72株菌株中仅含TEM-1型基因或仅含DHA-1型AmpC酶基因各1株,余70株(69.3%,70/101)均为产ESBLs菌株,其中46株(45.5%,46/101)含两种或两种以上β-内酰胺酶基因。
2.质粒介导的喹诺酮耐药基因检测本研究以细菌质粒DNA为模板,运用qnrA、qnrB、qnrS、qnrC、qnrD、aac(6')-Ib及qepA基因引物,对101株阴沟肠杆菌采用PCR方法进行了扩增,并对qnr基因进行了测序分型；同时检测了染色体介导的DNA旋转酶及拓扑异构酶IV的gyrA和parC基因突变和相应氨基酸突变。结果显示101株阴沟肠杆菌中共有39株细菌为qnr基因阳性(38.6%,39/101),其中19株(18.8%,19/101)细菌为qnrA基因阳性；18株(17.8%,18/101)细菌为qnrB基因阳性；3株(3.0%,3/101)细菌为qnrS基因阳性。其中1株细菌同时携带qnrB及qnrS两种基因。101株阴沟肠杆菌中未检测出qnrC及qnrD基因。所有qnr基因阳性菌株的扩增产物均经测序并与GenBank上基因序列进行比对,结果显示19株qnrA基因阳性菌株均为qnrAl亚型,18株qnrB基因阳性菌株中有16株为qnrB4亚型,1株为qnrB6亚型,1株为qnrB10亚型；3株qnrS基因阳性菌株中1株为qnrSl亚型,1株为qnrS2亚型,另有1株菌株同时含有qnrB4和qnrSl两种qnr基因。101株阴沟肠杆菌中有44株细菌采用PCR方法扩增到aac(6’)-Ib基因的扩增产物。该44株细菌的aac(6’)-Ib基因的扩增产物经FokI酶切后其中7株细菌因缺乏GGATG位点,无法切开,此7株(6.9%,7/101)同时经序列测定确证为aac(6’)-Ib基因突变株-aac(6’)-Ib-cr菌株,其中4株同时携带qnrB4基因,余37株FokI酶切均可切开,为aac(6’)-Ib野生型菌株。101株阴沟肠杆菌中未扩增到qepA基因。
对39株qnr基因阳性的菌株采用PCR方法扩增了gyrA和parC基因的喹诺酮类耐药决定区域(QRDR),并对其扩增产物进行了测序。与Genbank进行了比对,结果显示有23株qnr基因检测阳性的菌株GyrA和ParC QRDR区域氨基酸无突变,该类菌株均为环丙沙星敏感株(按CLSI2006年标准)。一株细菌的GyrA QRDR区域83位Ser突变为Tyr,该菌株为环丙沙星中敏株。另外在15株qnr基因检测阳性发现QRDR区域发生1-3个位点突变,包括GyrA QRDR区域83位Ser(丝氨酸)突变为Ile(异亮氨酸)或Phe(苯丙氨酸)或Leu(亮氨酸)及87位Asp(天冬氨酸)突变为Ala(丙氨酸),ParC QRDR区域80位Ser(丝氨酸)突变为I1e(异亮氨酸),该类菌株均为环丙沙星耐药株。
101株阴沟肠杆菌中有36株环丙沙星耐药株,其中含qnr基因阳性的菌株为15株,阳性检出率为41.7%(15/36)；在63株环丙沙星敏感菌株中qnr基因阳性菌株23株,阳性率为36.5%(23/63)。两组细菌qnr基因阳性率经卡方检验,差异无统计学意义(P＝0.23),提示在喹诺酮耐药或不耐药的两组菌株中qnr基因检出率相仿。本研究39株qnr阳性菌株中有23株细菌对环丙沙星敏感,这些菌株的GyrA和ParC QRDR区域无氨基酸突变,其环丙沙星的MIC值为0.06mag/L-1mag/L。与野生株相比(通常野生株对环丙沙星的MIC≤0.015mg／L),MIC值明显降低。提示这些细菌对喹诺酮类敏感性的下降可能仅由qnr所致,推测qnr也可能是细菌在对喹诺酮类耐药性形成过程中最早出现的耐药机制。
101中株阴沟肠杆菌中70株细菌含有ESBLs酶基因,其中35株(50%,35/70)qnr基因阳性,而31株非产ESBLs菌株中仅4株(12.9%,4/31)细菌为qnr基因阳性,两组细菌的qnr基因检测阳性率经卡方检验差异具有统计学意义(P=0.005)。在18株质粒介导AmpC酶阳性菌株中14株(77.8%,14/18)细菌出现qnr基因阳性,而83株非产AmpC酶菌株中有25株(30.1%,25/83)细菌qnr基因阳性,两组细菌的qnr基因检测阳性率经卡方检验同样具有统计学意义(P=0.018),上述结果提示产ESBLs或者质粒介导AmpC酶的菌株较非产酶株更易同时携带qnr基因。
3.质粒介导氨基糖苷类钝化酶和氨基糖苷类甲基化酶检测。本研究应用aac(3)-I、aac(3)-II、aac(3)-III、aac(6')-Ib、aac(6')-II、ant(2")-I、ant(3”)-Ⅰ、aph(3’)-Ⅵ等8种氨基糖苷类钝化酶基因和armA及rmtB2种氨基糖苷类甲基化酶基因引物对101株阴沟肠杆菌进行了PCR扩增,结果显示48株(47.5%,48/101)被检测出含有氨基糖苷类钝化酶基因；13株(12.9%,13/101)细菌含有armA或rmtB甲基化酶基因,其中10株(9.9%,10/101)同时检测出含氨基糖苷类钝化酶和氨基糖苷类甲基化酶基因。
48株氨基糖苷类钝化酶基因阳性的菌株中有44株(43.5%,44/101)菌株被检测出含有aac(6’)-Ib基因,其中有7株为aac(6’)-Ib-Cr(aac(6’)-Ib基因突变株)；22株(21.8%,22/101)含ant(2”,)-Ⅰ基因；6株(5.9%,6/101)含aac(3)-Ⅱ基因；1株(1.0%,1/101)含aac(3)-Ⅰ基因；2株(2.0%,2/101)含ant(3”)-Ⅰ基因。而aac(3)-Ⅲ、aac(6’)-Ⅰ和aph(3’)-Ⅵ等3种氨基糖苷类钝化酶基因在本组101株阴沟肠杆菌中均未检出。
在13株(12.9%,13/101)被检测为含有armA或rmtB氨基糖苷类甲基化酶基因的菌株中含armA基因为12株(11.9%,12／101),含rmtB基因为2株(2.0%,2/101)。其中1株同时含有armA和rmtB基因。13株含氨基糖苷类甲基化酶菌株均表现为对阿米卡星和庆大霉素高度耐药,上述两种抗生素的MIC值均>256mg/L。在含氨基糖苷类钝化酶基因的菌株中呈现对氨基糖苷类不同的耐药水平。如含单一aac(6’)-Ib基因的菌株中表现为庆大霉素MIC值从4mg/L到>256mg/L不等。
在101株阴沟肠杆菌中共有70株(70.3%)含有ESBLs酶的细菌中,有47株(67.1%,47/70)细菌呈现氨基糖苷类钝化酶基因阳性,而31株非产ESBLs菌株中仅1株(3.3%,1/31)细菌为氨基糖苷类钝化酶基因阳性,两组细菌的氨基糖苷类钝化酶基因检出阳性率经卡方检验差异具有统计学意义(P=0.0001)。70株ESBLs阳性菌株中有13株(18.6%,13/70)细菌含氨基糖苷类甲基化酶基因,另外31株非产ESBLs菌株均未检测到含氨基糖苷类甲基化酶基因菌株,两组细菌的氨基糖苷类甲基化酶基因检出阳性率经卡方检验差异具有统计学意义(P=0.005)。上述结果显示产ESBLs菌株中产氨基糖苷类钝化酶和氨基糖苷类甲基化酶检出率明显高于非产ESBLs酶菌株,提示在产ESBLs酶菌株中易同时携带有氨基糖苷类钝化酶基因和氨基糖苷类甲基化酶基因。
第三部分阴沟肠杆菌耐药质粒的接合转移
为了解上述101株阴沟肠杆菌中耐药质粒的可转移性,选择①44株同时含有ESBLs(SHV-12型及CTX-M型)及DHA-1型AmpC酶基因的菌株；②12株同时含SHV-12型ESBLs.DHA-1型AmpC.qnr.aae(6')-Ib基因4种基因的临床菌株作为供体菌,对大肠埃希菌J53AZR进行质粒转移接合试验。
44株同时含有ESBLs及AmpC酶基因的菌株中有30株细菌发生了转移接合,耐药质粒的转移接合发生率为70.5%(30/44)。经对接合子的质粒抽提结果显示7株接合子与供体细菌一样均只携带1个质粒。对这7株接合子进一步研究显示①供体菌E-41×J53AZR→接合子41TC发生了CTX-M-1G基因的转移接合；②供体菌E-57×J53 AZR→接合子57TC和供体菌E-70×J53 AZR→70TC发生了CTX-M-9G和aac (6’)-Ib基因的转移接合；③供体菌E-91×J53AZR→接合子91TC和供体菌E-95×J53AZR→95TC发生了qnrB、SHV-12、DHA-1和aac(6’)-Ib基因的转移接合；④供体菌E-206×J53AZR→接合子206TC和供体菌E-209×J53AZR→209TC发生了CTX-M-9G和aac(6’,)-Ib基因的转移接合。转移接合的结果提示①以上CTX-M-1G、CTX-M-3G、CTX-M-9G、SHV-12型ESBLs及DHA-1型AmpC均具有可接合性。②依据7株接合子菌株均含有1个质粒,故提示CTX-M-3G型和aac(6’)-Ib基因、CTX-M-9G和aac(6’)-Ib、SHV-12和aac(6’)-Ib基因可能位于同一质粒上,发生共同转移接合。7株接合子的头孢噻肟和头孢他啶的MIC值在4-32mg/L,与供体菌接近,较受体菌J53AZR(MIC值为≤0.06mag/L)升高66.7-533倍,庆大霉素和阿米卡星的MIC值在4-128mg/L(除1株41TC庆大霉素MIC为0.5mg/L),较受体菌J53AZR(庆大霉素MIC值为0.125mg/L)升高16-1024倍。
12株同时含有qnrB, SHV-12、DHA-1和aac (6’)-Ib基因的临床菌株行转移接合试验后有7株细菌发生了转移接合,耐药质粒的转移接合发生率为58.8%(7/12)。经对接合子的质粒抽提结果显示：7株供体菌基因型类似,均含有SHV-12、DHA-1、qnrB4、aac(6')-Ib、ant (2")-I基因,发生了SHV-12、DHA-1、qnrB4,aac(6')-Ib基因的转移接合,接合子的环丙沙星MIC值比受体菌J53AZR高12-24倍,为0.094 mg/L-0.19mg/L,而受体菌的MIC值仅为0.008mg/L。这一结果同样提示接合子对喹诺酮类敏感性的下降仅由qnrB基因所致,推测qnr可能是细菌对喹诺酮类耐药性形成过程中最早出现的耐药耐药机制。7株接合子的三代头孢菌素的MIC值与供体菌接近,但较头孢噻肟或头孢他啶对受体菌J53AZR(≤0.06 mg／L)的MIC值上升了66.7-1000倍,达到4 mg/L-64mg/L,对氨基糖苷类抗菌药物庆大霉素和阿米卡星均较受体菌J53AZR(0.125 mg/L～0.25mg/L)的MIC上升了16-1024倍,达到2 mg/L-128mg/L。
第四部分阴沟肠杆菌多重耐药质粒的基因结构分析
对上述研究获得的7株含qnrB基因、SHV-12. DHA-1及aac(6’)-Ib基因的转移接合子进行耐药质粒的基因结构及基因周边结构的进一步研究结果显示：经HindⅢ酶切、与载体PUC18连接,再经酶切验证发现一段约14kb的片段连接入载体PUC18中。对其中2株接合子质粒65TC和404TC的14kb片段进行了测序结果显示2个接合子的DNA周边结构相同,且与本研究所前期报道的肺炎克雷伯中含qnrB4基因质粒pSH7结构相仿,包含有sapA-qnrB4-pspF-pspA-pspB-pspC-pspD-orfl-基因序列。qnrB4基因上游为sapA基因,推测其编码一个肽转运系统酶。pspF-pspA-pspB-pspC-pspD为psp操纵子,编码一个噬菌体休克蛋白,其后为orfl读码框,之后为编码AmpC酶的blaDHA-1基因及ampC的调控基因ampR。此外采用PCR检测发现以上7株接合子及其供体菌均含有qac△l-sull耐消毒剂和磺胺基因、整合子遗传标记Integrase I(整合酶基因)、Tn21／Tn501转座子遗传标记(汞离子还原酶基因)基因,提示以上7株接合子除对三代头孢菌素、氨基糖苷类耐药外,还对季胺类消毒剂和磺胺耐药,系多重耐药质粒,故以上7株接合子含有qnrB4基因、SHV-12、DHA-1、aac(6’)-Ib以及qac△l-sull耐消毒剂和磺胺基因、整合子遗传标记Integrase I(整合酶基因)、Tn21／Tn501转座子遗传标记(汞离子还原酶基因)基因,在阴沟肠杆菌中发现含有以上多种耐药基因的质粒尚属首次报道。7株转移接合子中SHV-12、aac (6')-Ib等基因位于质粒的哪个区域还有待进一步的研究。
Recent studies have found that Enterobacter cloacae (E. cloacae) can cause various infections including respiratory tract, urogenital tract, skin soft tissue, and bloodstream infections. It has become an important pathogen of nosocomial infections. Furthermore, contrasting to the surveillance of bacterial resistance of Shanghai to our hospital has shown that Enterobacter has prominent resistance to antibiotics ofβ-lactams, fluoroquinolones and aminoglycosides, which are the three commonly used clinical drugs. Studies on the resistance mechanisms of E. cloacae to the above three antibiotics have indicated that:1. the mainβ-lactam antibiotics-resistant mechanism for E. cloacae is the production of the extended spectrumβ-lactamases (ESBLs) and AmpCβ-lactamases (AmpC enzymes), which is primarily mediated by plasmids; 2. the aminoglycoside resistance of E. cloacae is mainly mediated by aminoglycoside-modifying enzyme genes including aac (3)-I, II, III and aac (6')-I, II as well as aminoglycoside methylase genes such as rmtB and armA, which are also generally located on plasmids.3. the plasmid-mediated quinolone resistance discovered recently is an important mechanism for E. cloacae to resist quinolone antibiotics, which is mediated by genes including qnr. According to the report, the plasmid-mediated resistance has some characteristic:①Generally, modifying enzymes were plasmid mediated. Plasmid-mediated enzymes were constitutive hyperproduction without inducers. These enzymes can hydrolyze or modifyβ-lactams or aminoglycosides and cause high level resistance;②one plasmid can containβ-lactams, fluoroquinolones, aminoglycosides resistance gene, together with quaternary ammonium disinfectants and sulfonamides resistance gene simultaneously, it will cause one strain can resist many kinds of antibiotics;③The gene for plasmid-mediated resistance enzymes can disseminate between Enterobacter spp. Due to above reason, plasmid-mediated resistance is very important on the study of bacterial resistance. The results of pulsed-field gel electrophoresis (PFGE) of E. cloacae clinical isolates showed that there were no clone transmission but the resistance pattern was the same, it implied that these strains can have the same or similar plasmids. Therefore, it is necessary to conduct deep studies on the plasmid-mediated mechanisms of E. cloacae.
Based on the above situations, the present study investigated a total of 101 strains of E. cloacae clinically isolated in 2005 in our hospital. In this study, drug resistance and homology of these strains were studied; plasmid-mediated resistance genes in these strains to the three clinically commonly used antibiotics were detected; plasmid conjugation experiments of these clinical strains were performed, and the resistance genes and structural genes carried by plasmids were tested.
Part 1 Drug sensitivity test and homology analysis of Enterobacter cloacae
The two-fold agar dilution method was used to determine the minimum inhibitory concentration (MIC) of 14 anti-infective drugs on the 101 strains of clinically isolated E. cloacae. Results showed that the resistance rates of the 101 E. cloacae strains in this group to cefotaxime and ceftazidime were up to 53.4%and 51.4%respectively; while the strains were sensitive to cefepime with the resistance rate of only 10.9%; and the resistance rates to cefoperazone/sulbactam and piperacillin/tazobactam were both 16.9%. The strains were highly sensitive to carbapenem antibiotics without resistance. The resistance rates to quinolone antibiotics and ciprofloxacin were 35.6%and 31.7%; to amikacin and gentamicin were 19.8%and 41.6%respectively. Of 101 strais,30 (29.7%,30/101)strains were resistant to the third cephosporins, together with resistance to quinolone or aminoglycosides, 22 (21.8%,22/101) were resistant to the third cephosporins, quinolone and aminoglycosides.
Results of pulsed-field gel electrophoresis (PFGE) characterization for the 101 E. cloacae strains showed that there were a very small number of homogenous strains in this group. Only 3 strains were of the same clone, while 94 strains had different PFGE fingerprint patterns, indicating that no prevalence of the same clone strains of E. cloacae within our hospital. However, the above strains of different clones often showed the same or similar type of drug resistance spectrums, suggesting that these different strains were likely to carry the same or similar drug resistance plasmids.
Part 2 molecular biology study of plasmid-mediated resistance genes in E. cloacae
After the extraction of bacterial plasmid DNA templates by phenol/chloroform method, PCR was used to detect plasmid-mediated ESBLs and AmpC, aminoglycoside-modifying enzymes and the plasmid-mediated 16S rRNA aminoglycoside methylase, three kinds of quinolone resistance genes and detecting the amino acid mutations of gyrA and parC genes.
1. detecting plasmid-mediated ESBLs and AmpC. After the extraction of bacterial plasmid DNA templates by phenol/chloroform method, the PCR method using CTX-M, SHV and TEM-1 primers, the multiplex PCR using MOX, CIT, ACC, EBC, DHA or FOX-type AmpC primers, found 72 (71.3%) of the 101 E. cloacae strains contained plasmid-mediatedβ-lactamases.37 (36.6%, 37/101) had TEM-1 typeβ-lactamase genes,54 strains (53.5%,54/101) contained SHV-12 type ESBLs genes,31 strains (30.7%,31/101) contained CTX-M-type ESBLs genes.18 strains (17.8%,18/101) were found to contain DHA-1 type plasmid-mediated AmpC enzyme. In 72 containingβ-lactamases strains,1 contained only DHA-1 type AmpC enzymes and 1 contained only TEM-1 type 0-lactamase gene, the other 70 strains (69.3%,70/101) were all ESBLs stains. Among these 70 strains,46 strains (45.5%,46/101) contained two or more than two kinds ofβ-lactamase.
2. detecting plasmid-mediated quinolone resistant genes. In this study, with bacterial plasmid DNA as template, using qnrA, qnrB, qnrS, qnrC, qnrD, aac (6')-Ib-cr and qepA primers, plasmid-mediated quinolone resistant genes of the 101 E. cloacae strains were detected by PCR. The qnr genes were sequenced for typing, and the amino acid mutations of gyrA and parC genes encoding DNA gyrase and topoisomerase IV were detected.
Of the 101 E. cloacae strains,39 had positive qnr genes (positive rate 38.6%).19 strains (18.8%) were positive for qnrA gene,18 (17.8%) positive for qnrB, and 3 (3.0%) positive for qnrS; 1 of them carried both qnrB and qnrS genes; qnrC and qnrD genes were not found. Through gene sequencing and alignment on GenBank for all qnr positive strains, all qnr A gene positive strains contained qnrAl subtype genes. Among qnrB gene positive strains,16 contained qnrB4 subtype,1 contained qnrB6 subtype, and 1 contained qnrB10 subtype. Among qnrS gene positive strains,1 contained qnrSl subtype,1 contained qnrS2 subtype, and 1 had both qnrB4 and qnrSl genes.44 of the 101 strains were positive for aac (6')-Ib gene by PCR detection. After the PCR products were digested by FokI enzyme, the products of 7 strains could not be cut due to the lack of GGATG site. The 7 strains were determined by sequencing and found to be positive for aac (6')-Ib-cr gene with the positive rate of 6.9%, of which 4 strains were also carried qnrB4 gene. No qepA gene was found through amplification.
GyrA and ParC genes of the 39 qnr gene positive strains were tested by PCR, and they were sequenced and aligned on Genbank.23 strains that were positive for qnr gene, having no amino acid mutations at the quinolone resistance determining region (QRDR) of the GyrA and ParC genes, were found sensitive to ciprofloxacin (according to 2006 standard). Another strain with Ser 83 mutating to Tyr at the QRDR of GyrA, was a ciprofloxacin sensitive strain. In the other 15 qnr gene positive strains which 1-3 point mutations of each were found at the QRDR, including Ser 83 mutated to Ile, Phe or Leu, Asp 87 mutated to Ala at the GyrA QRDR, and Ser 80 mutated to Ile at the ParC QRDR, were ciprofloxacin resistant strais.
Of the 36 ciprofloxacin-resistant strains,15 were qnr gene positive with the positive rate of 41.7%; of the 63 ciprofloxacin-sensitive strains, 23 were qnr gene positive with the positive rate of 36.5%. The difference of the qnr gene positive rate between the two groups was not statistically significant by the chi-square test (P-0.23), indicating that the detection rate of qnr genes between the quinolone resistant and sensitive groups was similar. In this study,23 of the 39 qnr positive strains were ciprofloxacin-sensitive, but the MIC values were up to 0.06-1mg/l, suggesting that the decreased sensitivity to quinolones for these bacteria may be caused only by qnr, and qnr may be the first appeared resistance mechanism during the formation of quinolone resistance for bacteria.
Among 101 strains,70 contained ESBLs, of the 70 ESBLs strains,35(50%, 35/70) were qnr positive strains. Of the other 31 non-ESBLs strains, only 4(12.9%,4/31) strains were qnr positive strains. The difference of the qnr gene positive rate between the two groups was statistically significant by the chi-square test (P=0.005). Of the 18 AmpC strains,14 (77.8%,14/18) were qnr positive strains, of the other 83 non-AmpC strains, 25 strains (30.1%,25/83) were qnr positive strains. It was also statistically significant by the chi-square test between the two groups (P=0.018). It suggested that qnr was more frequent in ESBLs or AmpC strains.
3.Detecting aminoglycoside-modifying enzymes and the plasmid-mediated 16S rRNA aminoglycoside methylase. In this study, eight aminoglycoside-modifying enzyme genes of aac (3)-Ⅰ, aac (3)-Ⅱ, aac (3)-Ⅲ, aac (6')-Ⅰb, aac (6')-Ⅱ, ant (2")-Ⅰ, ant (3")-Ⅰ, and aph (3')-Ⅵ, as well as two methylase genes of armA and rmtB were amplified by PCR. Among the 101 E. cloacae strains,48 (47.5%,48/101) contained aminoglycoside-modifying enzyme genes,13 (12.9%,13/101) contained armA or rmtB, in which 10 (9.9%,10/101) contained both aminoglycoside-modifying enzymes and aminoglycoside methylase genes.
In the 48 strains containing aminoglycoside-modifying enzyme genes, the most commonly found gene was aac (6')-Ⅰb, which existed in 44 strains (43.6%,44/101). After digestion,7 strains (6.9%) were found to contain aac (6')-Ⅰb-Cr gene, 22 (21.8%) contained ant (2")-Ⅰgene, 6 (5.9%) had aac (3)-Ⅱ, 1 (1.0%) had aac (3)-Ⅰ, and 2 (2.0%) contained ant (3")-Ⅰ. The aac (3)-Ⅲ, aac (6')-Ⅱ, or aph (3')-Ⅵgenes were not found in the 101 strains of E. cloacae in this group.
Through PCR detection,13 strains (12.9%) were found to contain methylase genes armA or rmtB, of which 12 (11.9%) contained armA gene, 2 (2.0%) contained rmtB gene, and 1 contained both armA and rmtB genes. The 13 strains with aminoglycoside methylases all showed a high degree of resistance to amikacin and gentamicin, and the MIC values were all above 256ug/ml. The aminoglycoside resistance mediated by aminoglycoside modifying enzymes showed different resistance levels, for example, strains containing aac (6')-Ib gene had MIC values for gentamicin from 4ug/ml to above 256ug/ml.
Among 101 strains,70 contained ESBLs, of the 70 ESBLs strains, 47(67.1%,47/70) strains were aminoglycoside-modifying enzyme genes positive. Of the other 31 non-ESBLs strains, only 1(3.3%,1/31) strain were aminoglycoside-modifying enzyme genes positive. The difference of the aminoglycoside-modifying enzyme genes positive rate between the two groups was statistically significant by the chi-square test (P=0.0005). Of the 70 ESBLs strains,13 (18.6%,13/70)strains were aminoglycoside methylases positive strains. Of the other 31 non-ESBLs strains, none strain were aminoglycoside methylases enzyme genes positive. It was also statistically significant by the chi-square test between the two groups (P=0.0003). It suggested that aminoglycoside-modifying and aminoglycoside methylases enzyme genes were more frequent in ESBLs strains.
Part 3 Plasmid conjuction of multi-resistant Enterobacter Cloacae resistance genes
To investigate the transferability of resistant plasmid in 101 strains, we selected①44 strains with ESBLs(SHV-12 and CTX-M) and AmpC (DHA-1) enzyme genes;②12 strains with SHV-12, DHA-1, aac (6')-Ib and qnrB4 as as donor strains, E. Coli J53AzR as receptor to do plasmid conjugation tests.
Plasmid conjugation tests were conducted for a total of 44 strains with ESBLs or AmpC enzyme genes and 30 strains got conjugants, conjugated rate was 68.2%(30/44). Seven conjugants were found to have only one plasmid each, so further study had been done to the seven conjugants, the result showed:①donor strain E-41 X J53AZR→conjugant 41TC (CTX-M-1G gene conjugation);②donor strain E-57 X J53 AzR→conjugant 57TC and donor strain E-70×J53 AZR→conjugant 70TC (CTX-M-9G and aac (6')-Ib genes conjugation);③donor strain E-91 X J53AzR→conjugant 91TC and donor strain E-95×J53 AZR→95TC (qnrB, SHV-12, DHA-1 and aac(6')-Ib genes conjugation);④donor strain E-206×J53AZR→conjugant 206TC and donor strain E-209 X J53AZR→conjugant 209TC (CTX-M-9G and aac(6')-Ib genes conjugation).
The result of conjugation suggested①OTX-M-1G, CTX-M-3G, CTX-M-9G, SHV-12 and DHA-1 could be transmitted by plasmid conjugation.②Since 7 conjugated strains containing only one plasmid, it implied that CTX-M-3G together with aac(6')-Ib, CTX-M-9G together with aac(6')-Ib, SHV-12 together with aac(6')-Ib can be cotransmitted by one plasmid. The 7 conjugants MIC of cefotaxime and ceftazidim was 4-32mg/l, like donor strains, but it was 66.7-533 times than receptor J53AzR (MIC≤0.06mg/L) The conjugants MIC of Gentamycin and Amikacin was 4-128mg/l, it was 16-1024 times than receptor J53AZR (MIC 0.125mg/L)
Plasmid conjugation tests were conducted for 12 strains harboring qnrB, SHV-12, DHA-1 and aac(6')-Ib and 7 strains got conjugants, conjugated rate was 58.8%(7/12).The genes of 7 donor strains were similar, all of them had qnrB4, SHV-12, DHA-1, aac(6')-Ib and ant (21-I genes. The conjugants contained qnrB4, SHV-12, DHA-1, aac(6')-Ib genes. Ciprofloxacin MIC of 7 conjugants was 0.094 mg/L～0.19mg/L, which was 12-24 times more than receptor J53AZR (MIC 0.008mg/L). It also suggested that the decreased sensitivity to quinolones for these bacteria may be caused only by qnr, and qnr may be the first appeared resistance mechanism during the formation of quinolone resistance for bacteria. The 7 conjugants MIC of cefotaxime and ceftazidim was 4-64mg/l, like donor strains, but it was 66.7-1000 times than receptor J53AZR (MIC≤0.06mg/L) The conjugants MIC of Gentamycin and Amikacin was 4-64mg/l, it was 16-1024 times than receptor J53AZR (MIC 0.125mg/L-0.25mg/L)
Part 4 Construction analysis of multi-resistant plasmid of Enterobacter Cloacae
The conjugated plasmids containing qnrB, SHV-12, DHA-1 and aac(6')-Ib was futher investigated in the structural features of the plasmids. The plasmids were digested by HindIII and ligated with PUC18. Then the ligated PUC18 was digested again and was found a 14kb fragment was ligated in the PUC18. The 14kb fragment of conjugants 65TC and 404TC was sequenced. Sequencing results showed that the two plasmids had the same structure at the surrounding DNA regions. The structure was similar to the Plasmid pSH7 containing qnrB4 gene in Klebsiella pneumoniae, which was reported previously by our institute, containing sapA-qnrB4-pspF-pspA-pspB-pspC-orfl-pspD-blaBGA-1-ampR gene sequences. The sapA gene was at the upstream of qnrB4, suggesting that it encoded a peptide transport system enzyme. The pspF-pspA-pspB-pspC-pspD was the operon of psp, encoding a phage shock protein, followed by orfl reading frame, and the blaBHA-1 gene encoding AmpC enzyme, as well as the ampC regulatory gene ampR. Detected by PCR, the seven conjugants also contained qacΔ1-sull disinfectant and sulfonamide resistance genes, integron genetic marker Integrase I (integrase gene), and Tn21/Tn501 transposon genetic marker gene (mercury ion reductase gene), indicating that the seven zygotes were resistant to quaternary ammonium disinfectants and sulfonamides, in addition to the third generation of cephalosporins and aminoglycoside antibiotics. Therefore, they were multiple drug resistance plasmids. The seven zygotes harbor qnrB4, SHV-12, DHA-1, aac(6')-Ib, qacΔ1-sull disinfectant and sulfonamide resistance genes, integron genetic marker Integrase I (integrase gene), and Tn21/Tn501 transposon genetic marker gene. This is the first report a plasmid from E. cloacae contain such kinds of resistance genes. SHV-12, aac(6')-Ib locate in which place in plasmid needs futher research.
引文
[1]周光,沈定霞,罗燕萍等.临床阴沟肠杆菌感染的分布与耐药特性研究[J].中华医院感染学杂志,2006,16(3)：340-342.
[2]朱德妹,汪复,张婴元等.2005年上海地区细菌耐药性监测[J].中国感染与化疗杂志,2006,6：371-376.
[3]胡付品,叶信予,吴培澄等.2005年上海华山医院细菌耐药性监测[J].中国感染与化疗杂志,2007,7：233-237.
[4]D. M. Livermore. Defining an extended-spectrum β-lactamase[J]. Clin Microbiol Infect,2008,14 (Suppl.1):3-10.
[5]George A. Jacoby. AmpCβ-Lactamases[J]. Clin Microbiol Review,2009, 22(1):161-182.
[6]Shaw KJ, Rather PN, Hare RS, et al. Molecular genetics of aminoglycoside resistance genes and familial relationships of the aminoglycoside-modifying enzymes[J]. Microbiol Rev,1993,57:138-163.
[7]Llano-Sotelo B, AzueenaEFJr, KotraLP, etal. Aminoglyeosides modified by resistance enzymes aisphy diminished binding to the bacterial ribosomal aminoneyl-tRNA site[J]. ChemBiol,2002,9(4):455-463.
[8]Galimand, M, Courvalin, etal. Plasmid mediated high-level resistance to aminoglycosides in Enterobacteriaceae due to 16S rRNA methylation[J]. Antimicrob Agents Chemother,2003,47(6):2565-2571.
[9]Martinez-Martinez L, Pascual A, Jacoby GA. Quinolone resistance from a transferable plasmid[J]. Lancet,1998,351(9105):797-799.
[10]Courvalin P. New plasmid-mediated resistances to antimicrobial agents[J]. Arch Microbiol,2008,189(4):289-91.
[11]Ping Shen, Yan Jiang, Zhihui Zhou. Complete nucleotide sequence of pKP96, a 67850 bp multiresistance plasmid encoding qnrAl, aac(6')-Ib-cr and blaCTX-M-24 from Klebsiella pneumoniae[J]. J Antimicrob Chemother,2008, 62:1252-1256.
[12]HataM, Suzuki M, Matsumoto M, etal. Cloning of a novel gene for quinolone resistance from a transferable plasmid in Shigella flexneri[J]. Antimicrob Agents Chemother,2005,49(2):801-803.
[13]National Committee for ClinicalLaboratory Standards. Performance standards for antimicrobial suscep tibility testing [S].2005, M100-S14.
[14]Barry AL, Jones R.V. Criteria for disk susceptibility tests and quality control guidelines for the cefoperazone-sulbactam combination[J]. J Clin Microbiol,1988,26(1):13-7
[15]Tenover FC, Arbeit RD, Goering RV,et al. Interpreting Chromosomal DNA Restriction Patterns Produced by Pulsed-Field Gel Electrophoresis:Criteria for Bacterial Strain Typing [J]. J Clin Microbiol,1995,33(9):2233-2239.
[16]Deguchi T, Fukuoka A, Yasuda M, et al. Alterations in the GyrA subunit of DNA Gyrase and tile ParG subunit of topoisomerase IV in quinolone-resistant clinical isolates of Klebsiella pneumoniae[J]. Antimicmb Agents Chemother,1997,41:699-701.
[17]Robicsek A, Strahilevitz J, Jacoby GA, et al. Fluoroquinolone modifying enzyme:a novel adaptation of a common aminoglycoside acetyltransferase[J]. Nat Med,2006,12:83-88.
[18]Yamane K, Wachino J, Suzuki S, et al. New plasmid-mediated fluoroquinolone efflux pump, QepA, found in an Escherichia coli clinical isolate[J]. Antimicrob Agents Chemother,2007,51 (9):3354-3360.
[19]Weill FX, Perrier-Gros-Claude JD, Demartin M, et al. Characterization of extended-spectrum-beta-lactamase (CTX-M-15)-producing strains of Salmonella enterica isolated in France and Senegal [J]. FEMS Microbiol Lett, 2004,238(2):353-358.
[20]Costa D, Poeta P, Saenz Y, et al. Detection of Escherichia coli harbouring extended-spectrum beta-lactamases of the CTX-M, TEM and SHV classes in faecal samples of wild animals in Portugal[J]. J Antimicrob Chemother,2006,58(6):1311-2.
[21]Liu JH, Wei SY, Ma JY, et al. Detection and characterisation of CTX-M and CMY-2 beta-lactamases among Escherichia coli isolates from farm animals in Guangdong Province of China[J]. Int J Antimicrob gents,2007,9(5):576-581
[22]F. Javier Perez-Perez, Nancy D. Hanson. Detection of Plasmid-Mediated AmpC-Lactamase Genes in Clinical Isolates by Using Multiplex PCR. J Clin Microbiol,2002,40(6):2153-2162
[23]Robicsek A, Strahilevitz J, Sahm DF, et al. qnr Prevalence in Ceftazidime-Resistant Enterobacteriaceae Isolates from the United States [J]. Antimicrob Agents Chemother,2006,50(8):2872-2874.
[24]Chi HP, Robicsek A, Jacoby GA, et al. Prevalence in the United States of aac(6')-Ib-cr Encoding a Ciprof loxacin-Modifying Enzyme [J]. Antimicrob Agents Chemother,2006,50(11):3953-3955.
[25]杜鑫淼,冯玉麟,俞云松等.亚胺培南耐药铜绿假单胞菌氨基糖苷类修饰酶基因型分布[J].中国呼吸与危重监护杂志,2006,5(2)：136-139.
[26]Yohei Doi, Angela C. R, Ghilardi, et al. High Prevalence of Metallo-Lactamase and 16S rRNA Methylase Coproduction among Imipenem-Resistant Pseudomonas aeruginosa Isolates in Brazil [J]. Antimicrob Agents Chemother,2007,51(9):3388-3390.
[27]Frederick MA, Robert EK, Roger B et al颜子颖,王海林等译[M].精编分子生物学实验指南.第2版,北京：科学出版社,2002.
[28]Jones ME, Peters E, Weersink A, et al. Widespread occurrence of integrons causing multiple resistance in bacteria[J]. Lancet,1997,349:1742-1743.
[29]潘高辉,毛彩萍.科玛嘉尿道定位显色培养基的应用评价[J].中国微生态学杂志,2006,29(2)：218-220
[30]Goldstein C, Lee M. D, Sanchez A, et al. Incidence of class 1 and class 2 integrons in clinical and commensal bacteria from livestock, companion animals, and exotics [J]. Antimicrob Agents Chemother,2001,45 (3):723-725
[31]胡庆丰,吕火祥,糜祖煌.泛耐药肺炎克雷伯菌的耐药基因研究[J].中华医院感染杂志,2009,19(13)：1634-1636
[32]Hu FP, Xu XG, Zhu DM, et al. Coexistence of qnrB4 and qnrS1 in a clinical strain of Klebsiella pneumoniae[J]. Acta Pharmacol Sin 2008,29:320-4.
[33]Giannouli M, Cuccurullo S, Crivaro V et al. Molecular epidemiology of multidrug-resistant Acinetobacter baumannii in a tertiary care hospital in Naples, Italy, shows the emergence of a novel epidemic clone[J].J Clin Microbiol,2010,48(4):1223-30.
[34]Johnson JK, Smith G, Lee MS, et al. The role of patient-to-patient transmission in the acquisition of imipenem-resistant Pseudomonas aeruginosa colonization in the intensive care unit[J].J Infect Dis,2009, 200(6):900-905.
[35]周强,黄宪章,张文.147株阴沟肠杆菌AmpC酶及ESBLs的表型测定[J].广东医学,2007,28(9)：1444-1446.
[36]李岩,许淑珍,苏建荣.阴沟肠杆菌产超广谱p-内酰胺酶、AmpC酶的检测及耐药性分析[J].中华医院感染学杂志,2007,17(12)：1586-1589.
[37]National Committee for ClinicalLaboratory Standards. Performance standards for antimicrobial suscep tibility testing [S].2010, M100-S14.
[38]Wu JJ, Ko WC, Tsai SH, et al. Prevalence of plasmid-mediated quinolone resistance determinants QnrA, QnrB, and QnrS among clinical isolates of Enterobacter cloacae in a Taiwanese hospital[J]. Antimicrob Agents Chemother,2007,51 (4):1223-1227.
[39]Yamane K, J Wachino, S Suzuki, Y Arakawa. Plasmid-mediated qepA gene among Escherichia coli clinical isolates from Japan[J]. Antimicrob Agents Chemother,2008,52(4):1564-1566.
[40]Jing-Jou Yan, Jiunn-Jong Wu, Wen-Chien. Plasmid-mediated 16S rRNA methylases conferring high-levelaminoglycoside resistance in Escherichia coli and Klebsiella pneumoniae isolates from two Taiwanese hospitals[J]. J Antimicrob Chemother,2004(54):1007-1012.
[41]Qiong Wu, Yibo Zhang, Lizhong Han, et al. Plasmid-Mediated 16S rRNA Methylases in Aminoglycoside-Resistant Enterobacteriaceae Isolates in Shanghai, China[J]. Antimicrob Agents Chemother,2009,53(1):271-272.
[42]Hyukmin Lee, Dongeun Yong, Jong Hwa Yum.Dissemination of 16S rRNA methylase-mediated highly amikacin-resistant isolates of Klebsiella pneumoniae and Acinetobacter baumannii in Korea[J]. Diag Microbiol Infect Dis,2006 (56):305-312.
[43]黄支密,单浩,糜祖煌.阴沟肠杆菌16S rRNA甲基化酶基因及氨基糖苷类修饰酶基因研究[J].中华流行病学杂志,2008,29(4)：369-371
[44]casin I, Bordon F, Bertin P, et al. Aminoglycoside 6'-N-acetyltransferase variants of the Ib type with altered substrate profile in clinical isolates of Enterobacter cloacae and Citrobacter freundii[J]. Antimicrob Agents Chemother,1998,42(2):209-215.
[1]朱德妹,汪复,张婴元等.2005年上海地区细菌耐药性监测[J].中国感染与化疗杂志,2006,6：371-376.
[2]Philippon A, Labia R, Jacoby G. Extended-spectrum beta-lactamases [J]. Antimicrob Agents Chemother,1989,33:1131-1136.
[3]Crowley B, Ratcliff G. Extended-spectrum β-lactamases in Enterobacter cloacae:underestimated but clinically significant![J]. J Antimicrob Chemother,2003,51 (5):1316-1317
[4]Bush K, Jacoby GA. Medeiros AA. A functional classification scheme for beta-lactamases and its correlation with molecular structure [J]. Antimicrob Agents Chemther.1995,39(6):1211-1233.
[5]Ambler, R. P. The structure of β-lactamases[M]. Philos. Trans. R. Soc. Lond. B 1980,289:321-331.
[6]]George A. Jacoby. AmpC β-Lactamases[J]. Clin Microbiol Review,2009, 22(1):161-182
[7]Nelson EC, Elisha BG. Molecular basis of AmpC hyperproduction in clinical isolates of Escherichia coli [J]. Antimicrob Agents Chemother,1999,43(4): 957-959.
[8]Siu LK, Lu PI, Chen JY, et al. High-level expression of ampC β-lactamase due to insertion of nucleotides between-10 and-35 promoter sequences in Escherichia coli clinical isolates:cases not responsive to extended-spectrum cephalosporin treatment [J]. Antimicrob Agents Chemother, 2003,47(7):2138-2144.
[9]Corvec S,Caroff N,Espaze E, et al. Mutation in the ampC promoter increasing resistance to beta-lactams in a clinical Escherichia coli strain[J]. Antimicrob Agents Chemother,2002,46(10):3265-3267.
[10]Hanson ND, C. C. Sanders. Regulation of inducible AmpC-lactamase expression among Enterobacteriaceae[J]. Curr Pharm Des,1999,5:881-894.
[11]Kuga A, Okamoto R, Inoue M. ampR gene mutations that greatly increase class C beta-lactamase activity in Enterobacter cloacae[J]. Antimicrob Agents Chemother,2000,44(3):561-567.
[12]Schmidtke AJ, Hanson ND. Model system to evaluate the effect of ampD mutations on AmpCmediated beta-lactam resistance[J]. Antimicrob Agents Chemother,2006,50(6):2030-7.
[13]Korfmann G, Wiedemann B. Genetic control of beta-lactamase production in Enterobacter cloacae[J]. Rev Infect Dis,1988,10(4):793-799.
[14]Schmidt H, Korfmann G, Barth H, et al. The signal transducer encoded by ampG is essential for induction of chromosomal AmpC beta-lactamase in Escherichia coli by beta-lactam antibiotics and "unspecific" inducers[J]. Microbiology,1995,141 (Pt5):1085-1092.
[15]Lj Y, Li Q, Du Y, et al. Prevalence of plasmid-mediated AmpC beta-lactamases in a Chinese university hospital from 2003 to 2005:first report of CMY-2-type AmpC beta-lactamase resistance in China[J]. J Clin Microbiol,2008,46(4): 1317-1321.
[16]Barnaud G, G Arlet, C Verdet, et al. Salmonella enteritidis:AmpC plasmid-mediated inducible beta-lactamase (DHA-1) with an ampR gene from Morganella morganii [J]. Antimicrob Agents Chemother,1998,42:2352-2358.
[17]Reisbig M. D., N. D. Hanson. The ACT-1 plasmid-encoded AmpC beta-lactamase is inducible:detection in a complex beta-lactamase background[J]. J Antimicrob Chemother,2002,49:557-560.
[18]Chen, Y. T., T. L. Lauderdale, T. L. Liao, et al. Sequencing and comparative genomic analysis of pK29, a 269-kilobase conjugative plasmid encoding CMY-8 and CTX-M-3 beta-lactamases in Klebsiella pneumoniae[J]. Antimicrob Agents Chemother,2007,51:3004-3007.
[19]Gniadkowski M. Evolution and epidemiology of extended-spectrum beta-lactamases (ESBLs) and ESBL-producing microorganisms[J]. Clin Microbiol Infect,2001,7:597-608.
[20]Bush K. New beta-lactamases in gram-negative bacteria:diversity and impact on the selection of antimicrobial therapy [J]. Clin Infect Dis,2001, 32:1085-1089.
[21]Ping Shen, Yan Jiang, Zhihui Zhou. Complete nucleotide sequence of pKP96, a 67850 bp multiresistance plasmid encoding qnrAl, aac(6')-Ib-cr and blaCTX-M-24 from Klebsiella pneumoniae[J]. J Antimicrob Chemother,2008, 62:1252-1256.
[22]Jacoby GA. Epidemiology of extended-spectrum beta-lactamases[J]. Clin Infect Dis,1998,27:81-83.
[23]Du Bois SK, Marriott MS, Amyes SG. TEM-and SHV-derived extended-spectrum beta-lactamases:relationship between selection, structure and function[J]. J Antimicrob Chemother,1995,35:7-22.
[24]Livermore DM. beta-Lactamases in laboratory and clinical resistance [J]. Clin Microbiol Rev,1995,8:557-584.
[25]Barthelemy M, Peduzzi J, Labia R. Distinction between the primary structures of TEM-1 and TEM-2 beta-lactamases[J]. Ann Inst Pasteur Microbiol, 1985,136:311-321.
[26]Sirot D, Recule C, Chaibi EB, et al. A complex mutant of TEM-1 beta-lactamase with mutations encountered in both IRT-4 and extended-spectrum TEM-15, produced by an Escherichia coli clinical isolate[J]. Antimicrob Agents Chemother,1997,41:1322-1325.
[27]Yang Y, Rasmussen BA, Shlaes DM. Class A beta-lactamases-enzyme-inhibitor interactions and res i stance [J]. Pharmacol Ther,1999,83:141-151.
[28]Matthew M, Hedges RW, Smith JT. Types of beta-lactamase determined by plasmids in gram-negative bacteria [J]. J Bacteriol,1979,138:657-662.
[29]Bradford PA. Extended-spectrum beta-lactamases in the 21st century: characterization, epidemiology, and detection of this important resistance threat[J]. Clin Microbiol Rev,2001,14:933-951.
[30]余丹阳,刘又宁.超广谱β-内酰胺酶CTX-M-3和HSHV-12在临床分离阴沟肠杆菌中的流行[J].中国抗生素杂志,2003,1：32-38.
[31]Kim J, Kwon Y, Pai H, et al. Survey of Klebsiella pneumoniae strains producing extended-spectrum Beta-lactamases:prevalence of SHV-12 and SHV-2a in Korea [J]. J clin Microbiol,1998,36:1446-1449.
[32]张哲,蒋晓飞,李敏等.SHV-12型超广谱β-内酰胺酶特性的研究[J].检验医学,2006,21(1)：31-34.
[33]Huletsky A, Knox JR, Levesque Rc. Role of ser-238 and Lys-240 in the hydrolysis of third-generation cephalosporins by SHV-type beta-lactamases probed by site-directed mutagenesis and three-dimensional modeling[J]. J Biol Chem,1993,268:3690-3697.
[34]Tzouvelekis LS, Tzelepi E, Tassios PT, et al. CTX-M-type beta-lactamases: an emerging group of extended-spectrum enzymes [J]. Int J Antimicrob Agents, 2000,14:137-142.
[35]Boyd DA, Tyler S, Christianson S, et al. Complete nucleotide sequence of a 92-kilobase plasmid harboring the CTX-M-15 extended-spectrum beta-lactamase involved in an outbreak in long-term care facilities in Toronto, Canada [J]. Antimicrob Agents Chemother,2004,48:3758-3764.
[36]Naas T,. Nordmann P. OXA-type beta-lactamases[J]. Curr Pharm Des,1999, 5:865-79.
[37]Bradford PA. What's New in beta-lactamases [J]? Curr Infect Dis Rep,2001, 3:13-19.
[38]Nordmann P, Mariotte S, Naas T, et al. Biochemical properties of a carbapenem-hydrolyzing beta-lactamase from Enterobacter cloacae and cloning of the gene into Escherichia coli[J]. Antimicrob Agents Chemother,1993, 37:939-46.
[39]Rasmussen BA, Bush KKeeney D, et al. Characterization of IMI-1 Beta-lactamase, a class A carbapenem-hydrolyzing enzyme from Enterobacter cloacae[J]. Antimicrob Agents Chemother,1996,40:2080-2086.
[40]Jeong SH, Lee K, Chong Y, et al. Characerization of a new integron containing VIM-2, a metallo-beta-lactamase gene cassette, in a clinical isolate of Enterobacter cloacae[J]. J Antimicrob Chemother,2003,51:397-400.
[41]Lnzzaro F, Docquier JD, Colinon C, et al. Emergence in Klebsiella pneumoniae and Enterobacter cloacae clinical isolates of the VIM-4 metallo-lactamase Encoded by a conjugative plasmid[J]. Antimicrob Agents Chemother,2004,48:648-650.
[42]Deguchi T, Fukuoka A, Yasuda M, et al. Alterations in the GyrA subunit of DNA Gyrase and the ParC subunit of topoisomerase IV in quinolone-resistant clinical isolates of Klebsiella pneumoniae[J]. Antimicmb Agents Chemother, 1997,41:699-701.
[43]Ruiz J. Mechanisms of resistance to quinolones:targe, alterations, decreased accumulation and DNA gyrase protection[J]. J Antimiemb Chemother,2003,51:1109-1117.
[44]Brisse S, Milatovic D, Fluit AC, et al. Comparative in vitro activities of ciprofloxacin, clinafloxacin, gatifloxacin, levofloxacin, moxifloxacin and trovafloxacin against Klebsiella pneumoniae, Klebsiella oxytoca, Enterobacter cloacae, and Enterobacter aerogenes clinical isolates with alterations in GyrA and ParC proteins [J]. Ant imicrob Agents Chemother,1999, 43:2051-2055.
[45]Jacoby GA. Mechanisms of resistance to quinolones [J]. Clin Infect Dis, 2005,41(Suppl 2):S120-126.
[46]Martinez-Martinez L, Pascual A, Jacoby GA. Quinolone resistance from a transferable plasmid[J]. Lancet,1998,351 (9105):797-799.
[47]Tran JH, Jacoby GA. Mechanism of plasmid-mediated quinolone resistance[J]. Proc Natl Acad Sci USA,2002,99:5638-5642.
[48]HataM, Suzuki M, Matsumoto M, et al. Cloning of a novel gene for quinolone resistance from a transferable plasmid in Shigella flexneri. Antimicrob Agents Chemother,2005,49:801-803.
[49]Jacoby GA, Walsh KE, Mills DM, et al. qnrB, another plasmid-mediated gene for quinolone resistance[J]. Antimicrob Agents Chemother,2006,50: 1178-1182.
[50]Wang M, Guo Q, Xu X, et al. New plasmid-mediated quinolone resistance gene, qnrC, found in a clinical isolate of Proteus mirabilis[J]. Antimicrob Agents Chemother,2009,53:1892-1897.
[51]Robicsek A, Strahilevitz J, Jacoby GA, et al. Fluoroquinolone modifying enzyme:a novel adaptation of a common aminoglycoside acetyltransferase[J]. Nat Med,2006,12:83-88.
[52]Yang H, Chen H, Yang Q, Chen M, Wang H.High prevalence of plasmid-mediated quinolone resistance genes qnr and aac (6')-Ib-cr in clinical isolates of Enterobacteriaceae from nine teaching hospitals in China[J]. Antimicrob Agents Chemother.2008 Dec;52(12):4268-73
[53]Xu Zhao, Xiaogang Xu,Demei Zhu, Xinyu Ye and Minggui Wang. Decreased quinolone susceptibility in high percentage of Enterobacter cloacae clinical isolates caused only by Qnr determinants[J]. Diag Microbiol Infect Dis,2010, 67 (1):110-113.
[54]Yamane K, Wachino J, Suzuki S, et al. New plasmid-mediated fluoroquinolone efflux pump, QepA, found in an Escherichia coli clinical isolate[J]. Antimicrob Agents Chemother,2007,51:3354-3360.
[55]Shaw KJ, Rather PN, Hare RS, et al. Molecular genetics of aminoglycoside resistance genes and familial relationships of the aminoglycoside-modifying enzymes [J]. Microbiol Rev,1993,57:138-63.
[56]Alvarez M, Mendoza MC. Epidemiological survey of genes encoding aminoglycoside phosphotransferases APH (3') I and APH (3') II using DNA probes. J Chemother,1992,4(4):203-210.
[57]Lambert T, Gerbaud G, CourvalinP. Characterization of transposon Tnl528, which confers amikacin resistance by synthesis of aminoglycoside 3'-0-phosphotransferase type VI[J]. Antimicrob Agents Chemother,1994, 38(4):702-706
[58]Teran FJ, Suarez JE, Mendoza MC. Cloning, sequencing, and use as a molecular probe of a gene encoding an aminoglycoside 6'-N-acetyltransferase of broad substrate profile. Antimicmb Agents Chemother,1991,35 (4):714-719.
[59]M Galimand, T Lambert, G Gerbaud, et al. Characterization of the aac(6')-Ib gene encoding an aminoglycoside 6'-N-acetyltransferase in Pseudomonas aeruginosa BM2656[J]. Antimicrob Agents Chemother.1993,37(7): 1456-1462.
[60]Casin I, Bordon F, Bertin P, et al. Aminoglycoside 6'-N-acetyltransferase variants of the Ib type with altered substrate profile in clinical isolates of Enterobacter cloacae and Citrobacter freundii[J]. Antimicrob Agents Chemother,1998,42(2):209-15.
[61]G. A Jacoby, M. J Blaser, P Santanam, et al. Appearance of amikacin and tobramycin resistance due to 4'-aminoglycoside nucleotidyltransferase ANT (4')-I I in gram-negative pathogens [J]. Antimicrob Agents Chemother,1990, 34(12):2381-2386.
[62]Wright Edward, Serpersu Engin H. Enzyme-substrate interactions with an antibiotic resistance enzyme:Aminoglycoside nucleotidyltransferase (2")-Ia characterized by kinetic and thermodynamic methods[J]. Biochemistry, 2005,44:11581-11591.
[63]Doi Y, Arakawa Y.16S ribosomal RNA methylation:emerging resistance mechanism against aminoglycosides[J]. Clin Infect Dis,2007,45(1):88-94.
[64]Galimand, M, Courvalin, et al. Plasmid mediated high-level resistance to aminoglycosides in Enterobacteriaceae due to 16S rRNA methylation[J]. Antimicrob Agents Chemother,2003,47(6):2565-2571.
[65]Jing-Jou Yan, Jiunn-Jong Wu, Wen-Chien Ko Plasmid-mediated 16S rRNA methylases conferring high-levelaminoglycoside resistance in Escherichia coli and Klebsiella pneumoniae isolates from two Taiwanese hospitals[J]. J Antimicrob Chemother,2004(54):1007-1012
[66]Llano-Sotelo B, AzueenaEFJr, KotraLP, etal. Aminoglyeosides modified by resistance enzymes aisphy diminished binding to the bacterial ribosomal aminoneyl-tRNA site[J]. Chem Biol,2002,9(4):455-463.
[67]Lin Chen, Zhang-Liu Chen, Jian-Hua Liu. Emergence of RmtB methylase-producing Escherichia coli and Enterobacter cloacae isolates from pigs in China[J]. J Antimicrobial Chemother,2007,59:880-885.
[68]Kotra LP, Haddad J, Mobashery S. Aminoglycosides:perspectives on mechanisms of action and resistance and strategies to counter resistance. Antimicmb Agents Chemother,2000,44(12):3249-3256.
[69]Qiong Wu, Yibo Zhang, Lizhong Han, et al. Plasmid-Mediated 16S rRNA Methylases in Aminoglycoside-Resistant Enterobacteriaceae Isolates in Shanghai, China[J]. Antimicrob Agents Chemother.2009,53(1):271-272.
[70]Goldsten C, Lee M D, Sanchez A, et al. Incidence of class 1 and class 2 integrons in clinical and commensal bacteria from livestock, companion animals, and exotics [J]. Antimicrob Agents Chemother,2001,45 (3) 723-725
[2]朱德妹,汪复,张婴元等.2005年上海地区细菌耐药性监测[J].中国感染与化疗杂志,2006,6：371-376.
[3]胡付品,叶信予,吴培澄等.2005年上海华山医院细菌耐药性监测[J].中国感染与化疗杂志,2007,7：233-237.
[4]D. M. Livermore. Defining an extended-spectrum β-lactamase[J]. Clin Microbiol Infect,2008,14 (Suppl.1):3-10.
[5]George A. Jacoby. AmpCβ-Lactamases[J]. Clin Microbiol Review,2009, 22(1):161-182.
[6]Shaw KJ, Rather PN, Hare RS, et al. Molecular genetics of aminoglycoside resistance genes and familial relationships of the aminoglycoside-modifying enzymes[J]. Microbiol Rev,1993,57:138-163.
[7]Llano-Sotelo B, AzueenaEFJr, KotraLP, etal. Aminoglyeosides modified by resistance enzymes aisphy diminished binding to the bacterial ribosomal aminoneyl-tRNA site[J]. ChemBiol,2002,9(4):455-463.
[8]Galimand, M, Courvalin, etal. Plasmid mediated high-level resistance to aminoglycosides in Enterobacteriaceae due to 16S rRNA methylation[J]. Antimicrob Agents Chemother,2003,47(6):2565-2571.
[9]Martinez-Martinez L, Pascual A, Jacoby GA. Quinolone resistance from a transferable plasmid[J]. Lancet,1998,351(9105):797-799.
[10]Courvalin P. New plasmid-mediated resistances to antimicrobial agents[J]. Arch Microbiol,2008,189(4):289-91.
[11]Ping Shen, Yan Jiang, Zhihui Zhou. Complete nucleotide sequence of pKP96, a 67850 bp multiresistance plasmid encoding qnrAl, aac(6')-Ib-cr and blaCTX-M-24 from Klebsiella pneumoniae[J]. J Antimicrob Chemother,2008, 62:1252-1256.
[12]HataM, Suzuki M, Matsumoto M, etal. Cloning of a novel gene for quinolone resistance from a transferable plasmid in Shigella flexneri[J]. Antimicrob Agents Chemother,2005,49(2):801-803.
[13]National Committee for ClinicalLaboratory Standards. Performance standards for antimicrobial suscep tibility testing [S].2005, M100-S14.
[14]Barry AL, Jones R.V. Criteria for disk susceptibility tests and quality control guidelines for the cefoperazone-sulbactam combination[J]. J Clin Microbiol,1988,26(1):13-7
[15]Tenover FC, Arbeit RD, Goering RV,et al. Interpreting Chromosomal DNA Restriction Patterns Produced by Pulsed-Field Gel Electrophoresis:Criteria for Bacterial Strain Typing [J]. J Clin Microbiol,1995,33(9):2233-2239.
[16]Deguchi T, Fukuoka A, Yasuda M, et al. Alterations in the GyrA subunit of DNA Gyrase and tile ParG subunit of topoisomerase IV in quinolone-resistant clinical isolates of Klebsiella pneumoniae[J]. Antimicmb Agents Chemother,1997,41:699-701.
[17]Robicsek A, Strahilevitz J, Jacoby GA, et al. Fluoroquinolone modifying enzyme:a novel adaptation of a common aminoglycoside acetyltransferase[J]. Nat Med,2006,12:83-88.
[18]Yamane K, Wachino J, Suzuki S, et al. New plasmid-mediated fluoroquinolone efflux pump, QepA, found in an Escherichia coli clinical isolate[J]. Antimicrob Agents Chemother,2007,51 (9):3354-3360.
[19]Weill FX, Perrier-Gros-Claude JD, Demartin M, et al. Characterization of extended-spectrum-beta-lactamase (CTX-M-15)-producing strains of Salmonella enterica isolated in France and Senegal [J]. FEMS Microbiol Lett, 2004,238(2):353-358.
[20]Costa D, Poeta P, Saenz Y, et al. Detection of Escherichia coli harbouring extended-spectrum beta-lactamases of the CTX-M, TEM and SHV classes in faecal samples of wild animals in Portugal[J]. J Antimicrob Chemother,2006,58(6):1311-2.
[21]Liu JH, Wei SY, Ma JY, et al. Detection and characterisation of CTX-M and CMY-2 beta-lactamases among Escherichia coli isolates from farm animals in Guangdong Province of China[J]. Int J Antimicrob gents,2007,9(5):576-581
[22]F. Javier Perez-Perez, Nancy D. Hanson. Detection of Plasmid-Mediated AmpC-Lactamase Genes in Clinical Isolates by Using Multiplex PCR. J Clin Microbiol,2002,40(6):2153-2162
[23]Robicsek A, Strahilevitz J, Sahm DF, et al. qnr Prevalence in Ceftazidime-Resistant Enterobacteriaceae Isolates from the United States [J]. Antimicrob Agents Chemother,2006,50(8):2872-2874.
[24]Chi HP, Robicsek A, Jacoby GA, et al. Prevalence in the United States of aac(6')-Ib-cr Encoding a Ciprof loxacin-Modifying Enzyme [J]. Antimicrob Agents Chemother,2006,50(11):3953-3955.
[25]杜鑫淼,冯玉麟,俞云松等.亚胺培南耐药铜绿假单胞菌氨基糖苷类修饰酶基因型分布[J].中国呼吸与危重监护杂志,2006,5(2)：136-139.
[26]Yohei Doi, Angela C. R, Ghilardi, et al. High Prevalence of Metallo-Lactamase and 16S rRNA Methylase Coproduction among Imipenem-Resistant Pseudomonas aeruginosa Isolates in Brazil [J]. Antimicrob Agents Chemother,2007,51(9):3388-3390.
[27]Frederick MA, Robert EK, Roger B et al颜子颖,王海林等译[M].精编分子生物学实验指南.第2版,北京：科学出版社,2002.
[28]Jones ME, Peters E, Weersink A, et al. Widespread occurrence of integrons causing multiple resistance in bacteria[J]. Lancet,1997,349:1742-1743.
[29]潘高辉,毛彩萍.科玛嘉尿道定位显色培养基的应用评价[J].中国微生态学杂志,2006,29(2)：218-220
[30]Goldstein C, Lee M. D, Sanchez A, et al. Incidence of class 1 and class 2 integrons in clinical and commensal bacteria from livestock, companion animals, and exotics [J]. Antimicrob Agents Chemother,2001,45 (3):723-725
[31]胡庆丰,吕火祥,糜祖煌.泛耐药肺炎克雷伯菌的耐药基因研究[J].中华医院感染杂志,2009,19(13)：1634-1636
[32]Hu FP, Xu XG, Zhu DM, et al. Coexistence of qnrB4 and qnrS1 in a clinical strain of Klebsiella pneumoniae[J]. Acta Pharmacol Sin 2008,29:320-4.
[33]Giannouli M, Cuccurullo S, Crivaro V et al. Molecular epidemiology of multidrug-resistant Acinetobacter baumannii in a tertiary care hospital in Naples, Italy, shows the emergence of a novel epidemic clone[J].J Clin Microbiol,2010,48(4):1223-30.
[34]Johnson JK, Smith G, Lee MS, et al. The role of patient-to-patient transmission in the acquisition of imipenem-resistant Pseudomonas aeruginosa colonization in the intensive care unit[J].J Infect Dis,2009, 200(6):900-905.
[35]周强,黄宪章,张文.147株阴沟肠杆菌AmpC酶及ESBLs的表型测定[J].广东医学,2007,28(9)：1444-1446.
[36]李岩,许淑珍,苏建荣.阴沟肠杆菌产超广谱p-内酰胺酶、AmpC酶的检测及耐药性分析[J].中华医院感染学杂志,2007,17(12)：1586-1589.
[37]National Committee for ClinicalLaboratory Standards. Performance standards for antimicrobial suscep tibility testing [S].2010, M100-S14.
[38]Wu JJ, Ko WC, Tsai SH, et al. Prevalence of plasmid-mediated quinolone resistance determinants QnrA, QnrB, and QnrS among clinical isolates of Enterobacter cloacae in a Taiwanese hospital[J]. Antimicrob Agents Chemother,2007,51 (4):1223-1227.
[39]Yamane K, J Wachino, S Suzuki, Y Arakawa. Plasmid-mediated qepA gene among Escherichia coli clinical isolates from Japan[J]. Antimicrob Agents Chemother,2008,52(4):1564-1566.
[40]Jing-Jou Yan, Jiunn-Jong Wu, Wen-Chien. Plasmid-mediated 16S rRNA methylases conferring high-levelaminoglycoside resistance in Escherichia coli and Klebsiella pneumoniae isolates from two Taiwanese hospitals[J]. J Antimicrob Chemother,2004(54):1007-1012.
[41]Qiong Wu, Yibo Zhang, Lizhong Han, et al. Plasmid-Mediated 16S rRNA Methylases in Aminoglycoside-Resistant Enterobacteriaceae Isolates in Shanghai, China[J]. Antimicrob Agents Chemother,2009,53(1):271-272.
[42]Hyukmin Lee, Dongeun Yong, Jong Hwa Yum.Dissemination of 16S rRNA methylase-mediated highly amikacin-resistant isolates of Klebsiella pneumoniae and Acinetobacter baumannii in Korea[J]. Diag Microbiol Infect Dis,2006 (56):305-312.
[43]黄支密,单浩,糜祖煌.阴沟肠杆菌16S rRNA甲基化酶基因及氨基糖苷类修饰酶基因研究[J].中华流行病学杂志,2008,29(4)：369-371
[44]casin I, Bordon F, Bertin P, et al. Aminoglycoside 6'-N-acetyltransferase variants of the Ib type with altered substrate profile in clinical isolates of Enterobacter cloacae and Citrobacter freundii[J]. Antimicrob Agents Chemother,1998,42(2):209-215.
[1]朱德妹,汪复,张婴元等.2005年上海地区细菌耐药性监测[J].中国感染与化疗杂志,2006,6：371-376.
[2]Philippon A, Labia R, Jacoby G. Extended-spectrum beta-lactamases [J]. Antimicrob Agents Chemother,1989,33:1131-1136.
[3]Crowley B, Ratcliff G. Extended-spectrum β-lactamases in Enterobacter cloacae:underestimated but clinically significant![J]. J Antimicrob Chemother,2003,51 (5):1316-1317
[4]Bush K, Jacoby GA. Medeiros AA. A functional classification scheme for beta-lactamases and its correlation with molecular structure [J]. Antimicrob Agents Chemther.1995,39(6):1211-1233.
[5]Ambler, R. P. The structure of β-lactamases[M]. Philos. Trans. R. Soc. Lond. B 1980,289:321-331.
[6]]George A. Jacoby. AmpC β-Lactamases[J]. Clin Microbiol Review,2009, 22(1):161-182
[7]Nelson EC, Elisha BG. Molecular basis of AmpC hyperproduction in clinical isolates of Escherichia coli [J]. Antimicrob Agents Chemother,1999,43(4): 957-959.
[8]Siu LK, Lu PI, Chen JY, et al. High-level expression of ampC β-lactamase due to insertion of nucleotides between-10 and-35 promoter sequences in Escherichia coli clinical isolates:cases not responsive to extended-spectrum cephalosporin treatment [J]. Antimicrob Agents Chemother, 2003,47(7):2138-2144.
[9]Corvec S,Caroff N,Espaze E, et al. Mutation in the ampC promoter increasing resistance to beta-lactams in a clinical Escherichia coli strain[J]. Antimicrob Agents Chemother,2002,46(10):3265-3267.
[10]Hanson ND, C. C. Sanders. Regulation of inducible AmpC-lactamase expression among Enterobacteriaceae[J]. Curr Pharm Des,1999,5:881-894.
[11]Kuga A, Okamoto R, Inoue M. ampR gene mutations that greatly increase class C beta-lactamase activity in Enterobacter cloacae[J]. Antimicrob Agents Chemother,2000,44(3):561-567.
[12]Schmidtke AJ, Hanson ND. Model system to evaluate the effect of ampD mutations on AmpCmediated beta-lactam resistance[J]. Antimicrob Agents Chemother,2006,50(6):2030-7.
[13]Korfmann G, Wiedemann B. Genetic control of beta-lactamase production in Enterobacter cloacae[J]. Rev Infect Dis,1988,10(4):793-799.
[14]Schmidt H, Korfmann G, Barth H, et al. The signal transducer encoded by ampG is essential for induction of chromosomal AmpC beta-lactamase in Escherichia coli by beta-lactam antibiotics and "unspecific" inducers[J]. Microbiology,1995,141 (Pt5):1085-1092.
[15]Lj Y, Li Q, Du Y, et al. Prevalence of plasmid-mediated AmpC beta-lactamases in a Chinese university hospital from 2003 to 2005:first report of CMY-2-type AmpC beta-lactamase resistance in China[J]. J Clin Microbiol,2008,46(4): 1317-1321.
[16]Barnaud G, G Arlet, C Verdet, et al. Salmonella enteritidis:AmpC plasmid-mediated inducible beta-lactamase (DHA-1) with an ampR gene from Morganella morganii [J]. Antimicrob Agents Chemother,1998,42:2352-2358.
[17]Reisbig M. D., N. D. Hanson. The ACT-1 plasmid-encoded AmpC beta-lactamase is inducible:detection in a complex beta-lactamase background[J]. J Antimicrob Chemother,2002,49:557-560.
[18]Chen, Y. T., T. L. Lauderdale, T. L. Liao, et al. Sequencing and comparative genomic analysis of pK29, a 269-kilobase conjugative plasmid encoding CMY-8 and CTX-M-3 beta-lactamases in Klebsiella pneumoniae[J]. Antimicrob Agents Chemother,2007,51:3004-3007.
[19]Gniadkowski M. Evolution and epidemiology of extended-spectrum beta-lactamases (ESBLs) and ESBL-producing microorganisms[J]. Clin Microbiol Infect,2001,7:597-608.
[20]Bush K. New beta-lactamases in gram-negative bacteria:diversity and impact on the selection of antimicrobial therapy [J]. Clin Infect Dis,2001, 32:1085-1089.
[21]Ping Shen, Yan Jiang, Zhihui Zhou. Complete nucleotide sequence of pKP96, a 67850 bp multiresistance plasmid encoding qnrAl, aac(6')-Ib-cr and blaCTX-M-24 from Klebsiella pneumoniae[J]. J Antimicrob Chemother,2008, 62:1252-1256.
[22]Jacoby GA. Epidemiology of extended-spectrum beta-lactamases[J]. Clin Infect Dis,1998,27:81-83.
[23]Du Bois SK, Marriott MS, Amyes SG. TEM-and SHV-derived extended-spectrum beta-lactamases:relationship between selection, structure and function[J]. J Antimicrob Chemother,1995,35:7-22.
[24]Livermore DM. beta-Lactamases in laboratory and clinical resistance [J]. Clin Microbiol Rev,1995,8:557-584.
[25]Barthelemy M, Peduzzi J, Labia R. Distinction between the primary structures of TEM-1 and TEM-2 beta-lactamases[J]. Ann Inst Pasteur Microbiol, 1985,136:311-321.
[26]Sirot D, Recule C, Chaibi EB, et al. A complex mutant of TEM-1 beta-lactamase with mutations encountered in both IRT-4 and extended-spectrum TEM-15, produced by an Escherichia coli clinical isolate[J]. Antimicrob Agents Chemother,1997,41:1322-1325.
[27]Yang Y, Rasmussen BA, Shlaes DM. Class A beta-lactamases-enzyme-inhibitor interactions and res i stance [J]. Pharmacol Ther,1999,83:141-151.
[28]Matthew M, Hedges RW, Smith JT. Types of beta-lactamase determined by plasmids in gram-negative bacteria [J]. J Bacteriol,1979,138:657-662.
[29]Bradford PA. Extended-spectrum beta-lactamases in the 21st century: characterization, epidemiology, and detection of this important resistance threat[J]. Clin Microbiol Rev,2001,14:933-951.
[30]余丹阳,刘又宁.超广谱β-内酰胺酶CTX-M-3和HSHV-12在临床分离阴沟肠杆菌中的流行[J].中国抗生素杂志,2003,1：32-38.
[31]Kim J, Kwon Y, Pai H, et al. Survey of Klebsiella pneumoniae strains producing extended-spectrum Beta-lactamases:prevalence of SHV-12 and SHV-2a in Korea [J]. J clin Microbiol,1998,36:1446-1449.
[32]张哲,蒋晓飞,李敏等.SHV-12型超广谱β-内酰胺酶特性的研究[J].检验医学,2006,21(1)：31-34.
[33]Huletsky A, Knox JR, Levesque Rc. Role of ser-238 and Lys-240 in the hydrolysis of third-generation cephalosporins by SHV-type beta-lactamases probed by site-directed mutagenesis and three-dimensional modeling[J]. J Biol Chem,1993,268:3690-3697.
[34]Tzouvelekis LS, Tzelepi E, Tassios PT, et al. CTX-M-type beta-lactamases: an emerging group of extended-spectrum enzymes [J]. Int J Antimicrob Agents, 2000,14:137-142.
[35]Boyd DA, Tyler S, Christianson S, et al. Complete nucleotide sequence of a 92-kilobase plasmid harboring the CTX-M-15 extended-spectrum beta-lactamase involved in an outbreak in long-term care facilities in Toronto, Canada [J]. Antimicrob Agents Chemother,2004,48:3758-3764.
[36]Naas T,. Nordmann P. OXA-type beta-lactamases[J]. Curr Pharm Des,1999, 5:865-79.
[37]Bradford PA. What's New in beta-lactamases [J]? Curr Infect Dis Rep,2001, 3:13-19.
[38]Nordmann P, Mariotte S, Naas T, et al. Biochemical properties of a carbapenem-hydrolyzing beta-lactamase from Enterobacter cloacae and cloning of the gene into Escherichia coli[J]. Antimicrob Agents Chemother,1993, 37:939-46.
[39]Rasmussen BA, Bush KKeeney D, et al. Characterization of IMI-1 Beta-lactamase, a class A carbapenem-hydrolyzing enzyme from Enterobacter cloacae[J]. Antimicrob Agents Chemother,1996,40:2080-2086.
[40]Jeong SH, Lee K, Chong Y, et al. Characerization of a new integron containing VIM-2, a metallo-beta-lactamase gene cassette, in a clinical isolate of Enterobacter cloacae[J]. J Antimicrob Chemother,2003,51:397-400.
[41]Lnzzaro F, Docquier JD, Colinon C, et al. Emergence in Klebsiella pneumoniae and Enterobacter cloacae clinical isolates of the VIM-4 metallo-lactamase Encoded by a conjugative plasmid[J]. Antimicrob Agents Chemother,2004,48:648-650.
[42]Deguchi T, Fukuoka A, Yasuda M, et al. Alterations in the GyrA subunit of DNA Gyrase and the ParC subunit of topoisomerase IV in quinolone-resistant clinical isolates of Klebsiella pneumoniae[J]. Antimicmb Agents Chemother, 1997,41:699-701.
[43]Ruiz J. Mechanisms of resistance to quinolones:targe, alterations, decreased accumulation and DNA gyrase protection[J]. J Antimiemb Chemother,2003,51:1109-1117.
[44]Brisse S, Milatovic D, Fluit AC, et al. Comparative in vitro activities of ciprofloxacin, clinafloxacin, gatifloxacin, levofloxacin, moxifloxacin and trovafloxacin against Klebsiella pneumoniae, Klebsiella oxytoca, Enterobacter cloacae, and Enterobacter aerogenes clinical isolates with alterations in GyrA and ParC proteins [J]. Ant imicrob Agents Chemother,1999, 43:2051-2055.
[45]Jacoby GA. Mechanisms of resistance to quinolones [J]. Clin Infect Dis, 2005,41(Suppl 2):S120-126.
[46]Martinez-Martinez L, Pascual A, Jacoby GA. Quinolone resistance from a transferable plasmid[J]. Lancet,1998,351 (9105):797-799.
[47]Tran JH, Jacoby GA. Mechanism of plasmid-mediated quinolone resistance[J]. Proc Natl Acad Sci USA,2002,99:5638-5642.
[48]HataM, Suzuki M, Matsumoto M, et al. Cloning of a novel gene for quinolone resistance from a transferable plasmid in Shigella flexneri. Antimicrob Agents Chemother,2005,49:801-803.
[49]Jacoby GA, Walsh KE, Mills DM, et al. qnrB, another plasmid-mediated gene for quinolone resistance[J]. Antimicrob Agents Chemother,2006,50: 1178-1182.
[50]Wang M, Guo Q, Xu X, et al. New plasmid-mediated quinolone resistance gene, qnrC, found in a clinical isolate of Proteus mirabilis[J]. Antimicrob Agents Chemother,2009,53:1892-1897.
[51]Robicsek A, Strahilevitz J, Jacoby GA, et al. Fluoroquinolone modifying enzyme:a novel adaptation of a common aminoglycoside acetyltransferase[J]. Nat Med,2006,12:83-88.
[52]Yang H, Chen H, Yang Q, Chen M, Wang H.High prevalence of plasmid-mediated quinolone resistance genes qnr and aac (6')-Ib-cr in clinical isolates of Enterobacteriaceae from nine teaching hospitals in China[J]. Antimicrob Agents Chemother.2008 Dec;52(12):4268-73
[53]Xu Zhao, Xiaogang Xu,Demei Zhu, Xinyu Ye and Minggui Wang. Decreased quinolone susceptibility in high percentage of Enterobacter cloacae clinical isolates caused only by Qnr determinants[J]. Diag Microbiol Infect Dis,2010, 67 (1):110-113.
[54]Yamane K, Wachino J, Suzuki S, et al. New plasmid-mediated fluoroquinolone efflux pump, QepA, found in an Escherichia coli clinical isolate[J]. Antimicrob Agents Chemother,2007,51:3354-3360.
[55]Shaw KJ, Rather PN, Hare RS, et al. Molecular genetics of aminoglycoside resistance genes and familial relationships of the aminoglycoside-modifying enzymes [J]. Microbiol Rev,1993,57:138-63.
[56]Alvarez M, Mendoza MC. Epidemiological survey of genes encoding aminoglycoside phosphotransferases APH (3') I and APH (3') II using DNA probes. J Chemother,1992,4(4):203-210.
[57]Lambert T, Gerbaud G, CourvalinP. Characterization of transposon Tnl528, which confers amikacin resistance by synthesis of aminoglycoside 3'-0-phosphotransferase type VI[J]. Antimicrob Agents Chemother,1994, 38(4):702-706
[58]Teran FJ, Suarez JE, Mendoza MC. Cloning, sequencing, and use as a molecular probe of a gene encoding an aminoglycoside 6'-N-acetyltransferase of broad substrate profile. Antimicmb Agents Chemother,1991,35 (4):714-719.
[59]M Galimand, T Lambert, G Gerbaud, et al. Characterization of the aac(6')-Ib gene encoding an aminoglycoside 6'-N-acetyltransferase in Pseudomonas aeruginosa BM2656[J]. Antimicrob Agents Chemother.1993,37(7): 1456-1462.
[60]Casin I, Bordon F, Bertin P, et al. Aminoglycoside 6'-N-acetyltransferase variants of the Ib type with altered substrate profile in clinical isolates of Enterobacter cloacae and Citrobacter freundii[J]. Antimicrob Agents Chemother,1998,42(2):209-15.
[61]G. A Jacoby, M. J Blaser, P Santanam, et al. Appearance of amikacin and tobramycin resistance due to 4'-aminoglycoside nucleotidyltransferase ANT (4')-I I in gram-negative pathogens [J]. Antimicrob Agents Chemother,1990, 34(12):2381-2386.
[62]Wright Edward, Serpersu Engin H. Enzyme-substrate interactions with an antibiotic resistance enzyme:Aminoglycoside nucleotidyltransferase (2")-Ia characterized by kinetic and thermodynamic methods[J]. Biochemistry, 2005,44:11581-11591.
[63]Doi Y, Arakawa Y.16S ribosomal RNA methylation:emerging resistance mechanism against aminoglycosides[J]. Clin Infect Dis,2007,45(1):88-94.
[64]Galimand, M, Courvalin, et al. Plasmid mediated high-level resistance to aminoglycosides in Enterobacteriaceae due to 16S rRNA methylation[J]. Antimicrob Agents Chemother,2003,47(6):2565-2571.
[65]Jing-Jou Yan, Jiunn-Jong Wu, Wen-Chien Ko Plasmid-mediated 16S rRNA methylases conferring high-levelaminoglycoside resistance in Escherichia coli and Klebsiella pneumoniae isolates from two Taiwanese hospitals[J]. J Antimicrob Chemother,2004(54):1007-1012
[66]Llano-Sotelo B, AzueenaEFJr, KotraLP, etal. Aminoglyeosides modified by resistance enzymes aisphy diminished binding to the bacterial ribosomal aminoneyl-tRNA site[J]. Chem Biol,2002,9(4):455-463.
[67]Lin Chen, Zhang-Liu Chen, Jian-Hua Liu. Emergence of RmtB methylase-producing Escherichia coli and Enterobacter cloacae isolates from pigs in China[J]. J Antimicrobial Chemother,2007,59:880-885.
[68]Kotra LP, Haddad J, Mobashery S. Aminoglycosides:perspectives on mechanisms of action and resistance and strategies to counter resistance. Antimicmb Agents Chemother,2000,44(12):3249-3256.
[69]Qiong Wu, Yibo Zhang, Lizhong Han, et al. Plasmid-Mediated 16S rRNA Methylases in Aminoglycoside-Resistant Enterobacteriaceae Isolates in Shanghai, China[J]. Antimicrob Agents Chemother.2009,53(1):271-272.
[70]Goldsten C, Lee M D, Sanchez A, et al. Incidence of class 1 and class 2 integrons in clinical and commensal bacteria from livestock, companion animals, and exotics [J]. Antimicrob Agents Chemother,2001,45 (3) 723-725