鲍曼不动杆菌整合子相关耐药基因研究

目的了解我院鲍曼不动杆菌整合子流行情况，证实整合子与鲍曼不动杆菌多重耐药性的关系，建立一种快速简便的整合酶聚合酶链反应（PCR）检测方法。方法收集我院2005年9月至2006年2月临床分离的鲍曼不动杆菌共52株，用纸片扩散法检测鲍曼不动杆菌耐药性及用整合酶PCR检测其整合子基因。结果在52株鲍曼不动杆菌中，整合酶PCR检测出Ⅰ类整合子33株（63％），未检测出Ⅱ类整合子。统计学分析结果显示，整合子阴性和阳性的鲍曼不动杆菌对各种抗生素的耐药率存在明显差异，前者仅对几种抗生素耐药而后者对多种抗生素耐药。结论我院多重耐药鲍曼不动杆菌中主要为Ⅰ类整合子，整合酶PCR是一种快速、简便、易在临床开展的检测方法，同时也是控制医院感染的有效检测手段。     

鲍曼不动杆菌的耐药性与Ⅰ类整盒子的相关研究

目的了解该院鲍曼不动杆菌的流行趋势以及鲍曼不动杆菌与Ⅰ类整合子的耐药关系。方法用该院临床常用22种抗菌药物来检测临床分离的鲍曼不动杆菌的敏感性。用PCR检测鲍曼不动杆菌Ⅰ类整合子酶基因,再扩增部分Ⅰ类整合子酶基因的可变区,对整合子可变区进行基因测序分析。结果鲍曼不动杆菌的耐药现象十分严重,呈多重耐药性。鲍曼不动杆菌对CPZ/SB耐药率为1.2%,对替加环素、左旋氧氟沙星、亚胺培南、比阿培南、阿米卡星等几种抗菌药物的耐药率为15.4%~69.5%,对其他抗菌药物均在71%以上。102株中有72株菌株含Ⅰ类整合子(阳性率为70.2%),Ⅰ类整合子阳性株的耐药性均高于阴性株,对Ⅰ类整合子可变区采用双酶切分析产生类似的酶切条带,说明该院鲍曼不动杆菌具有同源性。Ⅰ类整合子基因盒序列分析表明该院鲍曼不动杆菌Ⅰ类整合子携带aacA4、catB8和aadA1 3种耐药基因。结论该院2010~2013年,鲍曼不动杆菌对头孢菌素类、广谱青霉素、氨基糖苷类和氟喹诺酮类呈高度耐药及严重的多重耐药现状,说明治疗鲍曼不动杆菌所致感染的抗菌药物选择已十分有限。Ⅰ类整合子与鲍曼不动杆菌多重耐药性密切相关。     

鲍曼不动杆菌整合子流行现状与多重耐药相关性研究

[目的]了解我院鲍曼不动杆菌整合子流行情况,并探讨整合子与鲍曼不动杆菌多重耐药的关系。[方法]收集我院2009年9月至2010年1月临床分离的鲍曼不动杆菌共56株,用纸片扩散法检测鲍曼不动杆菌对19种抗生素的敏感性,用整合酶PCR方法检测I类、Ⅱ类和Ⅲ类整合子基因。[结果]鲍曼不动杆菌耐药现象十分严重。56株菌株中有41株检出I类整合子,阳性率73．2％,其中8株为I类和Ⅱ类杂交整合子,2株为I类和Ⅲ类杂交整合子。I类整合子阳性株对多种药物的耐药率均高于阴性株,且I类整合子阳性株多重耐药率（90．2％）明显高于阴性株（30％）（P〈0．0L）。[结论]I类整合子在我院鲍曼不动杆菌中检出率很高并与其多重耐药性关系密切。     

鲍曼不动杆菌整合子与耐药性的相关性研究

目的调查鲍曼不动杆菌中Ⅰ类、Ⅱ类和Ⅲ类整合子的存在情况,分析整合子与鲍曼不动杆菌耐药性的关系。方法测定93株鲍曼不动杆菌对抗菌药物的敏感性;应用简并引物PCR方法,同时扩增整合子5’保守区的I类、Ⅱ类和Ⅲ类整合酶基因,对阳性PCR产物用限制性内切酶Hinf I作限制片段长度多态性（RFLP）分析进行整合子分类。结果 22株鲍曼不动杆菌检测出Ⅰ类整合子,未检出Ⅱ类和Ⅲ类整合子。Ⅰ类整合子阳性的菌株对抗菌药物的耐药率普遍比整合子阴性的菌株高。结论鲍曼不动杆菌临床分离株的耐药性强,细菌的多重耐药性与Ⅰ类整合子的存在相关。     

分析9年不动杆菌院内感染的耐药性变化及其与I类整合子的相关性

目的了解我院9年间鲍曼不动杆菌和洛菲不动杆菌的耐药性变化,并探讨其耐药性与I类整合子的相关性。方法收集2001年1月至2009年12月我院分离培养的不动杆菌共1552株,采用VITEK-2全自动细菌鉴定仪进行检验及耐药分析。采用PCR检测60株鲍曼不动杆菌和30株洛菲不动杆菌的I类整合子,分析I类整合子与细菌耐药性的相关性。结果1552株不动杆菌中以鲍曼不动杆菌1360株（87．6％）,洛菲不动杆菌154株（9．9％）为主。药敏结果显示不动杆菌的耐药率总体呈逐年上升的趋势。PCR检测结果显示38株（63-3％）鲍曼不动杆菌和17株（56．7％）洛菲不动杆菌为I类整合子阳性,I类整合子阳性菌株的耐药率及多重耐药（mulfidrug resistance,MDR）率显著高于I类整合子阴性菌株,且差异均有统计学意义（P均〈0．05）。结论经验性用药或抗生素滥用导致了9年间不动杆菌的耐药率呈逐年上升的趋势。I类整合子在不动杆菌的耐药率及MDR率升高中起重要作用。     

整合子在鲍曼不动杆菌耐药中的作用研究

目的调查本院鲍曼不动杆菌耐药谱并了解其整合子流行情况;检测鉴定整合子类型并分析其与耐药的相关性。方法收集本院2007年临床分离鲍曼不动杆菌85株,KB法测定其药敏结果。PCR法扩增整合子的整合酶基因,并进一步鉴定整合子种类。分析鲍曼不动杆菌整合子携带与其耐药之间的关系。PCR法扩增整合子开放阅读框,观测其多态性并挑选个别产物测序。结果 85株鲍曼不动杆菌除对亚胺培南、头孢哌酮/舒巴坦耐药率低于10%,其他15种常用抗菌药物的耐药率均超过30%。67.1%(57/85)的鲍曼不动杆菌中检出整合子;进一步鉴定结果显示均为Ⅰ类整合子,未检出Ⅱ类和Ⅲ类整合子。携带整合子鲍曼不动杆菌株对14种抗菌药物耐药率高于不携带整合子鲍曼不动杆菌株耐药率。开放阅读框可变区扩增产物为0.3～2.5 KB不等条带,测序结果证实含有多药耐药基因编码。结论本院鲍曼不动杆菌耐药率高,以多药耐药菌株为主,携带Ⅰ类整合子率较高;携带整合子鲍曼不动杆菌对14种抗菌药的耐药率明显增高,整合子与鲍曼不动杆菌的多重耐药性具有密切相关性。     

临床革兰阴性杆菌整合子携带情况及其与耐药性的相关性分析

目的研究临床分离的4种革兰阴性杆菌耐药性与整合子的关系。方法收集江苏大学附属医院2017年10月—12月临床分离的肺炎克雷伯菌56株、大肠埃希菌49株、铜绿假单胞菌45株及鲍曼不动杆菌48株,Vitek 2 Compact全自动鉴定和药敏分析仪及K-B法进行药敏试验;多重PCR法检测细菌携带Ⅰ类、Ⅱ类和Ⅲ类整合子情况,并比较细菌耐药性与携带整合子之间的关系。结果 4种革兰阴性杆菌中共有37.37%的菌株检出Ⅰ类整合酶基因,其中鲍曼不动杆菌的阳性率为54.17%,大肠埃希菌的阳性率为46.94%,肺炎克雷伯菌的阳性率为39.29%,铜绿假单胞菌的阳性率为6.67%,所有菌株均未检测到Ⅱ类和Ⅲ类整合酶基因。4种革兰阴性杆菌中,铜绿假单胞菌、大肠埃希菌和肺炎克雷伯菌携带Ⅰ类整合子菌株对部分药物的耐药率较未携带整合子菌株差异有统计学意义,而携带Ⅰ类整合子的鲍曼不动杆菌的耐药率较未携带菌株差异均无统计学意义。结论Ⅰ类整合子在镇江地区临床分离革兰阴性杆菌中普遍存在,且与铜绿假单胞菌、大肠埃希菌和肺炎克雷伯菌耐药性存在一定的相关性。     

镇江医院感染革兰阴性杆菌耐药性与整合子关系研究*

目的 研究江苏大学附属医院分离的4种革兰阴性杆菌耐药性与携带整合子的关系。方法 收集江苏大学附属医院2017年10~12月临床分离的肺炎克雷伯菌56株、铜绿假单胞菌45株、大肠埃希菌49株及鲍曼不动杆菌48株,对其进行药敏试验；采用多重PCR法检测细菌携带Ⅰ类、Ⅱ类和Ⅲ类整合子情况,并比较细菌耐药性与携带整合子之间的关系。结果 4种革兰阴性杆菌中共有37.4%的菌株检出Ⅰ类整合酶基因,其中鲍曼不动杆菌的阳性率为54.17%,铜绿假单胞菌的阳性率为6.67%,大肠埃希菌的阳性率为46.94%,肺炎克雷伯杆菌的阳性率为39.29%,所有菌株均未检测到Ⅱ类和Ⅲ类整合酶基因。4种革兰阴性杆菌中,铜绿假单胞菌、大肠埃希菌和肺炎克雷伯菌携带Ⅰ类整合子细菌耐药性均较未携带菌株有显著性差异,而携带Ⅰ类整合子的鲍曼不动杆菌较未携带菌株耐药性无显著差异。结论 Ⅰ类整合子在镇江地区临床分离革兰阴性杆菌中普遍存在,与上述细菌耐药性存在不同程度联系。     

多重耐药不动杆菌16S rRNA甲基化酶和同源性研究

目的 调查2007年7月-2008年5月山西医科大学第二附属医院临床分离的61株多重耐药不动杆菌中介导氨基糖苷类抗生素高水平耐药的16S rRNA甲基化酶基因和Ⅰ类整合子携带耐药基因的分布.方法 利用blaOXA-51基因及16S rRNA-23S rRNA序列进行菌株鉴定,琼脂稀释法测定12种抗菌药物对61株不动杆菌的MIC,PCR筛选6种16S rRNA甲基化酶基因以及整合子基因盒,脉冲场凝胶电泳(PFGE)分析菌株同源性.结果 61株临床分离不动杆菌中55株为鲍曼不动杆菌、3株为3TU不动杆菌、l株13TU不动杆菌、1株醋酸钙不动杆菌、l株溶血不动杆菌.48株不动杆菌对阿米卡星、庆大霉素、妥布霉素均耐药,其中有47株检出armA基因;未检出rmtA、rmtB、rmtC、mad和npmA基因.armA基因阳性的菌株中Ⅰ类整合子阳性27株,分别携带arr-3、accA4、aacCl、catB8、aadA1和dfrA12基因.PFGE条带分析发现47株armA阳性菌株分为5个克隆,其中A、B为主要克隆,分布在我院多个科室中.结论 16S rRNA甲基化酶基因armA在多重耐药不动杆菌中广泛存在,armA基因不位于Ⅰ类整合子中,不动杆菌Ⅰ类整合子携带耐药基因主要介导对氨基糖苷类及氯霉素的耐药性.PFGE结果显示armA基因阳性菌株在我院呈克隆播散,必须采取有效的措施来控制耐药菌的传播.     

Ⅰ类整合子与鲍曼不动杆菌耐药性相关性研究

目的 了解鲍曼不动杆菌整合子分布情况,探讨Ⅰ类整合子与其耐药性的关系.方法 琼脂稀释法检测65株鲍曼不动杆菌耐药率,聚合酶链反应(PCR)检测Ⅰ～Ⅲ类整合子基因;对Ⅰ类整合子阳性菌株进行可变区扩增及序列分析.结果 Ⅰ类整合子检出率为60%(39/65),未检出Ⅱ、Ⅲ类整合子;Ⅰ类整合子阳性菌株对14种抗菌药物的耐药率高于阴性菌株(P＜0.05);92%(36/39)的Ⅰ类整合子阳性菌株可变区扩增阳性,序列分析显示携带3种耐药基因aacA4、aadA1和catB8.结论 Ⅰ类整合子与鲍曼不动杆菌耐药性密切相关,在鲍曼不动杆菌多药耐药性形成机制中起重要作用.     

鲍曼不动杆菌Ⅰ类整合子系统与其耐药性关系的研究

目的了解Ⅰ类整合子系统在鲍曼不动杆菌中的分布状况,探讨其与该菌耐药性之间的关系。方法用琼脂稀释法测定鲍曼不动杆菌对14种抗生素的敏感性。用PCR法检测Ⅰ类整合子系统。结果29.2%(21/72)的菌株检测出Ⅰ类整合子系统。含与不含Ⅰ类整合子系统的菌株在耐药性上存在显著性差异(P<0.05)。不同类型Ⅰ类整合子的耐药表型存在一定的差异。结论Ⅰ类整合子系统与鲍曼不动杆菌多重耐药性以及耐药表型之间有密切的关系。     

耐亚胺培南鲍曼不动杆菌碳青霉烯酶及整合子分布

目的 了解耐亚胺培南鲍曼不动杆菌碳青霉烯酶及整合子分布情况.方法 收集天津医科大学总医院2008年1月至2010年3月期间,103株亚胺培南耐药鲍曼不动杆菌临床标本.用Vitek-2系统鉴定细菌,并进行药敏试验,通过改良Hodge试验、改良三维试验和2-巯基丙酸协同试验初筛碳青霉烯酶,多重PCR同时检测4种OXA型碳青霉烯酶基因、2种金属酶基因及整合酶基因,对整合子可变区进行PCR检测及序列分析.结果 103株鲍曼不动杆菌中,改良Hodge试验检出碳青霉烯酶阳性75株(72.8%),改良三维试验检出产碳青霉烯酶菌株80株(77.7%),未检出产金属酶菌株.PCR检出blaOXA-51-like+bsaOXA-23-like+int11基因84株,blaOxA-51-like+blaOXA-23-like阳性5株,blaOXA-51-like+intll阳性8株,blaOXA-51-like+blaOXA-24-like阳性2株,仅blaOXA-51-like阳性4株,blaOXA-58-like、金属酶基因(IMP-1、VIM-2)及Ⅱ类整合酶基因(intI2)均阴性.89株(96.7%)Ⅰ类整合酶阳性株均扩增出可变区,检出2种耐药基因盒组合形式:aacA4-catB8-aadAl(2 300 bp)81株,aacCl-orfX-orfX-orfX'-aadAla(3 000 bp)8株.结论 鲍曼不动杆菌对碳青霉烯类耐药及多重耐药主要与其携带的OXA-23型碳青霉烯酶和Ⅰ类整合子有关.

Abstract：

Objective To investigate the carbapenemases and integrons in imipenem-resistant Acinetobacter baumannii. Methods One hundred and three Acinetobacter baumannii were collected from Janurary 2008 to March 2010 in Tianjin Medical University General Hospital. The identification of strains and antimicrobial susceptibility test were performed by using Vitek-2 compact automatic system. Isolates of imipenem-resistant A. baumannii were screened for carbapenemases by modified Hodge test, improved threedimensional test and 2-mercaptopropionic acid synergy test. Isolates were then subjected to the multiplex PCR targeting genes encoding for OXA type carbapenemases, metallo-β-lactamases (MBLs) and integrases. The variable regions of integrons were amplificated and sequenced. Results Among the 103 isolates, 75 (72. 8% ) demonstrated positive in the modified Hodge test, 80 (77.7%)were positive in the improved three-dimensional test. No MBLs was found in the 2-mercaptopropionic acid synergy test. Eightyfour isolates were positive for blaOXA-51-like, blaOXA-23-like, and intI1; five were positive for blaOXA-51-like and blaOXA-23-like ;eight were positive for blaOXA-51-like and int11 ;two were positive for blaOXA-51-like and blaOXA-24-like ;four were only found positive for blaOXA-51-like. The blaOXA-58-like, IMP-1, VIM-2 and intI2 genes were all negative. Eighty-nine(96. 7% )of the intI1 positive strains owned the variable region. Two different cassettes arrangements were identified within class 1 integrons:81 isolates harbored aacA4-catB8-aadAI (2 300 bp) and 8 harbored aacCl-orX-orfX-orX'-aadAla (3 000 bp ) . Conclusion The presence of OXA-23 carbapenermase and class Ⅰ integrons are correlated with Acinetobacter baumannii resistant to carbapenems and multi-drug resistance.     

Evolution of multidrug-resistant Acinetobacter baumannii isolates obtained from elderly patients with respiratory tract infections

OBJECTIVES: To study the evolution between 1999 and 2002 and mechanisms of antibiotic resistance in a multidrug-resistant Acinetobacter baumannii clone predominant in isolates from elderly patients with respiratory tract infections. METHODS: Susceptibility to antimicrobials was determined using an agar dilution method. Bacterial clones were identified by PCR-fingerprinting and PFGE with ApaI. Carbapenemases were detected by phenotypic tests; by PCR with primers specific for bla (OXA-40), bla(IMP), bla(VIM-1) and bla(VIM-2); and by hybridization with DNA probes. Class 1 integrons were detected using PCR. RESULTS: In 1999 isolates were grouped into two main genotypes: clone I (33%) and clone II (55%). These were also detected in 2002 with a different distribution: clone I (69%), clone II (22%). Resistance to amikacin, meropenem and imipenem increased significantly in clone I over this time, whereas clone II was not affected. In 2002, the incidence of bla(OXA-40) rose to 91% in clone I isolates with some also harbouring bla(VIM-2) and bla(IMP) genes. Different class 1 integrons were detected ranging in size from 550 to 1200 bp. No relationship was found between carbapenemases and class 1 integrons. CONCLUSIONS: In elderly patients, a single clone became predominant among A. baumannii isolates, coinciding with an increase in antibiotic resistance rates. The majority of isolates harboured the bla(OXA-40) carbapenemase gene and some of them also harboured bla(VIM-2) and bla(IMP) genes. The presence of class 1 integrons also increased over time.     

Molecular characterization of integrons in Acinetobacter baumannii: description of a hybrid class 2 integron

Twenty Acinetobacter baumannii strains resistant to various antibiotics were analyzed for integron content and sequences of the amplification products. Sixteen clinical isolates had a class 1 integron, 2 contained an additional class 1 or class 2 integron, but no class 3 integron was detected. Thirteen strains had integrons with a single cassette: aac(3)-Ia (9 strains), ant(2")-Ia (2 strains), or aac(6')-Ib (2 strains); 1 had aac(6')-Ib and oxa20 cassettes and an unknown gene; and 1 had an integron containing ant(2")-Ia and an oxa3 cassette truncated by IS6100. The remaining strains harbored class 1 integrons with gene cassettes previously found in Enterobacteriaceae. One integron had a hybrid structure composed of intI2 and the 3' conserved segment of class 1 integrons. These data indicate that integrons play a major role in multidrug resistance in Acinetobacter.     

Molecular characterization of metallo-beta-lactamase-producing Acinetobacter baumannii and Acinetobacter genomospecies 3 from Korea: identification of two new integrons carrying the bla(VIM-2) gene cassettes

Carbapenem-resistant Acinetobacter spp. used to be rare, but are increasingly isolated in Korea. Among 28 isolates of imipenem-resistant Acinetobacter spp. found in a Korean hospital in 1998 and 1999, 14 produced metallo-beta-lactamases. The bla(VIM-2) gene was detected, by PCR, in 11 and two isolates of Acinetobacter baumannii and Acinetobacter genomospecies 3, respectively, and bla(IMP-1) in one isolate of A. baumannii. The MICs of imipenem for the isolates were 8-32 mg/L. PFGE analysis of SmaI-digested genomic DNA gave identical patterns in eight of 11 bla(VIM-2)-positive A. baumannii isolates from respiratory specimens of ICU patients. The bla(VIM-2) gene cassettes in the isolates are identical to those from Pseudomonas aeruginosa isolates in Europe, but are inserted into new class I integrons In105 and In106. The attC site of the last cassette of the array in In106 is interrupted by the insertion of a putative class II intron. This is the first report of VIM-2 beta-lactamase-producing A. baumannii and Acinetobacter genomospecies 3. Production of the VIM-2 enzyme presents an emerging threat of carbapenem resistance among Acinetobacter spp. in Korea.     

Occurrence and characteristics of class 1, 2 and 3 integrons in Escherichia coli, Salmonella and Campylobacter spp. in the Netherlands

OBJECTIVES: To determine the occurrence and transmission of class 1, 2 and 3 integrons in multidrug-resistant or sulfamethoxazole-resistant Salmonella from human and animal sources and in Campylobacter spp. and Escherichia coli from broilers isolated in the Netherlands in 2004. METHODS: PCR, restriction fragment length polymorphism (RFLP) and DNA sequencing were used to detect integrase genes and gene cassettes within 234 E. coli isolates, 40 Campylobacter isolates and 228 Salmonella isolates. RESULTS: Class 1 integrons were found in 76% of the E. coli and in 43% of the Salmonella isolates. Class 2 integrons were found in 11% of the E. coli and 1% of the Salmonella isolates. No class 1 or 2 integrons were detected in the Campylobacter isolates, and no class 3 integrons were detected in any of the bacterial species examined. The 22 different integrons detected harboured 20 different gene cassettes. The cassette arrays dfrA1-aadA1 and dfrA1-sat2-aadA1 were most frequently associated with class 1 and 2 integrons, respectively. For the first time linF was found to be associated with a class 2 integron as part of the linF-sat2-aadA1 cassette. The gene cassettes found within the integrons explain only a part of the resistance profile of the isolates. Conjugation experiments demonstrated transfer of class 1 and 2 integrons. CONCLUSIONS: Our data demonstrate the importance of integrons for the occurrence and transmission of multidrug resistance. Identical predominant class 1 and 2 integrons in E. coli and Salmonella serovars indicate horizontal transfer between these species.     

痰标本分离革兰阴性杆菌整合子分布及分型研究

目的研究临床痰标本分离革兰阴性杆菌中整合子的分布与分型。方法用Ⅰ类、Ⅱ类和Ⅲ类3种整合酶基因通用引物扩增398株痰标本分离革兰阴性杆菌的相应基因;用琼脂对倍稀释法检测临床菌株的MIC。结果Ⅰ类整合酶基因总阳性率为48.7%,Ⅱ类整合酶基因总阳性率为2.3%,未检出Ⅲ类整合酶基因阳性菌株,Ⅰ类和Ⅱ类整合子同时阳性率为1.5%。肠杆菌科细菌Ⅰ类整合酶基因阳性率为69%,Ⅱ类整合子阳性率为2.7%;非发酵菌中Ⅰ类整合子阳性率为34%,Ⅱ类整合子阳性率为1.8%。在肠杆菌科细菌中,Ⅰ类整合酶基因阳性菌株对多种抗菌药物的耐药率均明显高于阴性菌株。结论在痰标本分离的革兰阴性杆菌中,肠杆菌科细菌Ⅰ类整合子的携带率高于非发酵菌,Ⅱ类整合子则无明显区别。