皖北地区耐碳青霉烯鲍曼不动杆菌产金属β-内酰胺酶检测

目的了解皖北地区ICU病房内耐碳青霉烯鲍曼不动杆菌产金属β-内酰胺酶（metallo-β-lactamase,MBL）情况。方法收集皖北地区3家教学医院ICU病房中不动杆菌,K-B纸片扩散法检测其对碳青霉烯类抗生素耐药情况;PCR法检测blaIMP、blaVIM等MBL基因。结果共收集35株耐亚胺培南、美罗培南非重复分离鲍曼不动杆菌菌株,其中9株（25.7%）金属酶初筛阳性;PCR检测5株（5/35,14.3%）产IMP型MBL,1株（1/35,2.9%）VIM型MBL。结论在本地区耐碳青霉烯鲍曼不动杆菌中首次发现IMP和VIM型金属酶,提示产金属酶机制参与本地区鲍曼不动杆菌对碳青霉烯耐药,值得进一步关注。     

耐亚胺培南铜绿假单胞菌相关耐药基因的检测

目的对耐亚胺培南（IMP）的铜绿假单胞菌（IRPa）相关耐药基因进行检测。方法 2003年至2009年从临床标本中分离到（P.aeruginosa）共220株,采用三维试验筛选产β-内酰胺酶的铜绿假单胞菌,应用普通PCR和多重PCR分别检测碳青霉烯酶基因和质粒携带的C类头孢菌素酶（AmpC酶）耐药基因,应用荧光定量RT-PCR的方法检测oprD2基因的表达情况。结果共检出43株产β-内酰胺酶的菌株,其中产AmpC酶、超广谱β-内酰胺酶（ESBLs）、金属β-内酰胺酶（MBLs）和未知酶菌株的构成比分别58.14%（25/43）、18.60%（8/43）、4.65%（2/43）和16.28%（7/43）。74株耐亚胺培南的铜绿假单胞菌中,有2株菌携带IMP-9基因,1株菌携带DHA质粒型AmpC酶基因,其他碳青霉烯酶基因检测为阴性。40株菌株oprD2基因表达蛋白量降低,34株oprD2基因表达蛋白量正常。结论 oprD2基因的突变或蛋白表达量降低是IRPa对亚胺培南耐药的主要原因,AmpC酶可水解亚胺培南可能与铜绿假单胞菌对亚胺培南的耐药有一定的关系,而KPC-1酶和MBLs在铜绿假单胞菌对亚胺培南耐药机制中不是主要因素。     

鲍曼不动杆菌中OXA碳青霉烯酶的表型筛选与PCR检测

摘要：目的 了解OXA碳青霉烯酶在暨南大学附属第一医院耐亚胺培南鲍曼不动杆菌中的流行状况,完善OXA碳青霉烯酶的分子流行病学资料。方法 采用改良Hodge试验筛选产碳青霉烯酶菌株,多重PCR法检测OXA碳青霉烯酶的编码基因（blaOXA-23-like、blaOXA-24-like、blaOXA-58-like和blaOXA-143）,利用生物信息学的方法对OXA亚型进行比对分析并制作分子进化树。结果 在157株耐亚胺培南鲍曼不动杆菌中Hodge试验筛选出碳青霉烯酶表型阳性菌株141株,PCR检测结果显示有132株携带OXA-23编码基因,未检测到OXA-24-like、OXA-58-like和OXA-143亚型。结论 产OXA-23碳青霉烯酶是该院鲍曼不动杆菌对亚胺培南耐药的主要机制之一。     

一株洛菲不动杆菌对碳青霉烯类抗生素耐药机制的研究

目的研究洛菲不动杆菌对亚胺培南、美洛培南耐药的分子机制。方法K-B纸片琼脂扩散法检测洛菲不动杆菌B69对头孢他啶、头孢曲松、环丙沙星、阿米卡星的耐药性,琼脂对倍稀释法检测B69对亚胺培南、美洛培南的最低抑菌浓度;PCR扩增OXA、IMP、VIM型碳青霉烯酶基因,测序确定耐药基因型别;粗提酶水解亚胺培南纸片试验检测酶活性。结果洛菲不动杆菌B69具有多重耐药性;PCR扩增IMP基因阳性,经测序为IMP-8;粗提酶水解亚胺培南。结论产IMP-8型金属β-内酰胺酶是洛菲不动杆菌B69对碳青霉烯耐药的重要机制。     

摩根摩根菌β-内酰胺酶检测及耐药性分析

目的了解摩根摩根菌临床分离株产超广谱β-内酰胺酶（ESBLs）、头孢菌素酶（AmpC）、金属酶（MBLs）、碳青霉烯酶（KPC）情况,并分析其对17种常见抗菌药物的耐药性。方法 ESBLs和AmpC及MBLs采用三维试验检测,碳青霉烯酶采用改良Hodge试验进行检测,并以K-B法测定17种常见抗菌药物的耐药性。结果 102株摩根摩根菌单产ESBLs 15株,检出率为14.71%;单产AmpC 8株,检出率为7.84%;单产金属β-内酰胺酶（MBLs）3株,检出率为2.94%;所有菌株中未检出碳青霉烯酶（KPC）;同产ESBLs及AmpC 6株,检出率为5.88%;未发现其他双产酶菌株。摩根摩根菌非产酶分离株对17种抗生素的耐药率均低于50.0%;摩根摩根菌产酶分离株对亚胺培南、美罗培南培南的耐药率低于15.0%,与非产酶菌株相比,差异无统计学意义（P〉0.05）;对其余抗生素的耐药率均明显高于非产酶菌株（P〈0.05）。同产ESBLs＋AmpC与耐亚胺培南摩根摩根菌分离株呈多重耐药。结论我院摩根摩根菌分离株产生多种β-内酰胺酶,且对常用抗生素耐药性比较严重,建议临床医师合理使用抗生素,以免耐药菌株的产生。     

呼吸科住院患者耐碳青霉烯鲍曼不动杆菌抗菌药物敏感性及其分子特征

目的调查深圳市人民医院呼吸科住院患者耐碳青霉烯鲍曼不动杆菌（CRAB）的抗菌药物敏感性和耐药分子机制,以及克隆流行情况。方法收集2010年深圳市人民医院呼吸科住院患者临床分离CRAB29株,琼脂稀释法测定亚胺培南等15种抗菌药对CRAB的最低抑菌浓度（MIC）,PCR和DNA测序分析CRAB碳青霉烯酶基因型,脉冲场凝胶电泳（PFGE）分析菌株同源性。结果多粘菌素B对29株CRAB抗菌活性最强,敏感性100％,MIC50／MIC90为1／1μg/mL,其次米诺环素,敏感性96．6％,MIC50／MIC90为4／4μg/mL,替加环素中介率高达96．6％,MIC50／MIC90为4／4μg/mL。96．6％（28／29）CRAB携带ISAba1—blaOXA-23-like,对亚胺培南和美罗培南高度耐药,亚胺培南和美罗培南的MIC集中分布在32—64μg／mL;1株携带ISAba1—blaOXA-51-like CRAB,对亚胺培南和美罗培南中度耐药,亚胺培南和美罗培南的MIC分别为4μg/mL和8μg/mL。29株CRAB未发现blaOXA-24-like、blaOXA-143-like、金属酶基因及KPC酶基因。29株CRAB经PFGE分型共分2型,以A型28株（96．6％）为主要流行克隆,均携带ISAba1-blaOXA-23-like。结论2010年我院呼吸科临床分离CRAB主要携带ISAba1—blaOXA-23-like基因,并以克隆播散流行。     

一株广泛耐药恶臭假单胞菌耐药机制研究

目的研究一株临床分离广泛耐药恶臭假单胞菌(extensively drug resistant Pseudomonas putida,XDR-PP)的耐药机制,以期指导临床合理使用抗菌药物。方法采用VITEK-2compact全自动微生物分析系统对菌株进行鉴定及药敏试验;改良Hodge试验初筛碳青霉烯酶;EDTA-亚胺培南协同试验和亚胺培南双纸片增效法初筛金属酶;PCR检测16SrRNA甲基化酶基因、金属β-内酰胺酶基因及可移动性遗传元件基因,产物测序,结果经BLAST软件比对分析。结果该菌株对所检测的抗菌药物均耐药。改良Hodge试验、EDTA-亚胺培南协同试验和亚胺培南双纸片增效法结果均为阳性。基因检测结果显示,金属β-内酰胺酶基因阳性的有blaVIM-2和blaIMP-4两种基因,16SrRNA甲基化酶基因阳性的有armA基因,13种可移动性遗传元件中,intI、tnpU和merA基因阳性,其余均为阴性。结论blaVIM-2、blaIMP-4、armA、intI、tnpU以及merA基因是导致其广泛耐药的重要机制。恶臭假单胞菌中同时检出blaVIM-2和blaIMP-4两种金属β-内酰胺酶基因及armA基因为国内首次报道。     

426株鲍曼不动杆菌的耐药性分析与OXA-23型碳青霉烯酶检测

目的 了解暨南大学附属第一医院2009年至2011年鲍曼不动杆菌的分布特点和耐药状况,为临床合理使用抗生素提供依据,同时研究广州地区亚胺培南耐药鲍曼不动杆菌中OXA-23型碳青霉烯酶的分子流行病学特征.方法 利用梅里埃VITEK-2 Compact微生物分析仪鉴定细菌,K-B法进行药敏试验,采用WHONET 5.6软件统计分析药敏结果,PCR检测OXA-23型碳青霉烯酶的编码基因.结果 2009年至2011年临床分离出426株鲍曼不动杆菌,主要分离自呼吸科病房(119株,占27.9％)和ICU病房(107株,占25.1％),标本类型以痰液为主(361株,占84.7％),鲍曼不动杆菌对12种抗生素的耐药率总体呈逐年上升趋势,对亚胺培南的耐药率从13.2％上升到39.3％,在124株亚胺培南耐药鲍曼不动杆菌中PCR检出98株OXA-23型碳青霉烯酶阳性菌株.结论 鲍曼不动杆菌对各种抗菌药物的耐药性逐年增强,产OXA-23型碳青霉烯酶是其对亚胺培南耐药的主要机制之一.     

铜绿假单胞菌对碳青霉烯类药物的耐药机制研究

目的 探讨铜绿假单胞菌对碳青霉烯类药物的耐药机制.方法 收集2008年11月至2009年4月我院临床分离的铜绿假单胞菌31株,根据药敏结果分为碳青霉烯类耐药组(21株)和碳青霉烯类敏感组(10株).另设1株标准株ATCC 27853,用亚胺培南-EDTA(乙二胺四乙酸)抑制试验检测菌株是否产生金屑酶,采用PCR法检测各菌株的外膜孔道蛋白oprD2基因,探讨铜绿假单胞菌对碳青霉烯类抗生素耐药机制.结果 21株耐药株有7株产生金属酶;21株耐药株经oprD2基因扩增,15株阴性,6株阳性,10株敏感株全部阳性.统计学检验结果表明,碳青霉烯类耐药组与敏感组oprD2基因阳性率的差异有极显著性(P＜0.01).结论 oprD2基因缺失和金属酶是本院铜绿假单胞菌对碳青霉烯类抗生素耐药的重要机制.     

亚胺培南不敏感的阴沟肠杆菌碳青霉烯酶基因检测

为了解亚胺培南不敏感的阴沟肠杆菌碳青霉烯酶的主要基因型及其流行情况,收集哈尔滨医科大学附属第一医院临床分离出的亚胺培南不敏感的阴沟肠杆菌24株,采用Vitek-2 Compact进行细菌鉴定、药敏试验,聚合酶链反应（PCR）扩增,DNA测序确定菌株产碳青霉烯酶基因型情况。结果显示,24株阴沟肠杆菌均表现为多重耐药,20株菌扩增出KPC-2条带,经测序证实为KPC-2型碳青霉烯酶基因。该院亚胺培南不敏感的阴沟肠杆菌产碳青霉烯酶的主要基因型别为KPC-2型,临床与实验室应加强监测和控制。     

弗劳地枸橼酸杆菌的临床分布及耐药特性分析

目的了解弗劳地枸橼酸杆菌临床分离株的分布特点,探讨其耐药规律,为临床防治其感染提供帮助。方法常规方法进行菌株分离,VITEK-32型或VITEK-2全自动微生物分析仪进行菌株鉴定,K-B法进行药物敏感性试验,采用WHONET 5.5软件分析数据。结果 2007年6月至2009年12月从各种感染性标本中共分离到47株弗劳地枸橼酸杆菌,呼吸道标本占绝大多数,达51.1%(24/47),其次为脓液和尿液,分别为23.4%(11/47)和12.8%(6/47)。该菌种对氨苄西林和头孢唑啉的耐药性最严重,耐药率分别为92.6%和91.9%,其次是氨苄西林/舒巴坦、头孢西丁、头孢噻肟、左氧氟沙星及复方磺胺甲口恶唑,耐药率均45%,耐药率较低的有亚胺培南和阿米卡星,耐药率分别为8.3%和8.6%。结论弗劳地枸橼酸杆菌可引起患者多部位感染,下呼吸道是其主要感染部位,该菌种耐药性较严重,亚胺培南和阿米卡星对其有较强的抗菌活性。     

CMY-39型AmpC酶新基因亚型的序列分析与原核表达

目的对弗劳地枸橼酸杆菌所产CMY-39型AmpC酶新基因亚型进行基因克隆和重组表达。方法以产CMY-39型AmpC酶新基因亚型的弗劳地枸橼酸杆菌总基因组DNA为模板,PCR扩增CMY-39,将其克隆入pGEM-T载体后测定该核苷酸序列,再将CMY-39基因克隆到pET-32 a（＋）系统进行重组,重组菌在大肠埃希菌BL21中表达,SDS-PAGE电泳鉴定酶蛋白的表达。结果 PCR扩增出大小为1 146 bp的基因片段,与GenBank上CMY-39的基因序列同源性为99%。大肠埃希菌BL21转化pET-32 a（＋）/CMY-39重组质粒后,AmpC酶三维试验为阳性。此基因能在大肠埃希菌中大量表达,SDS-PAGE电泳显示,蛋白分子质量大约为60 kD。结论此基因为CMY-39新基因亚型,登陆号为HM565135;成功表达重组的CMY-39型酶,为进一步做酶动力学及酶的其他分子生物学特性研究奠定了基础。     

Identification of New Delhi metallo-β-lactamase gene (NDM-1) from a clinical isolate of Acinetobacter junii in China

New Delhi metallo-β-lactamase-1 (NDM-1) is a novel type of metallo-β-lactamase (MBL) responsible for bacterial resistance to β-lactam antibiotics. Acinetobacter junii was previously shown to possess a MBL phenotype; however, the genes responsible for this phenotype were not identified. In this study, we reported the identification of NDM-1 gene in a clinical isolate of A. junii from a child patient in China, which was resistant to all β-lactams except aztreonam but sensitive to aminoglycosides and quinolones. The cloned NDM-1 gene contained an open reading frame of 813 bp and had a nucleotide sequence 99.9% identical (812/813) to reported NDM-1 genes carried by Acinetobacter baumannii , Enterococcus faecium , Escherichia coli , and Klebsiella pneumoniae . Recombinant NDM-1 protein was successfully expressed in E.?coli BL21, and antibiotic sensitivities of the NDM-1-producing E.?coli were largely similar to the A.?junii 1454 isolate. The findings of this study raise attention to the emergence and spread of NDM-1-carrying bacteria in China.     

耐亚胺培南铜绿假单胞菌金属酶的筛选及基因型研究

目的研究重庆医科大学附属第一医院分离的29株耐亚胺培南铜绿假单胞菌中金属酶（Metallo-β-Lactamase,MBL）的基因型分布情况。方法用亚胺培南-EDTA纸片法筛选29株铜绿假单胞菌中产MBL的铜绿假单胞菌,用PCR扩增法检测29株菌中金属酶VIM和IMP基因。结果29株耐亚胺培南的铜绿假单胞菌中,亚胺培南-EDTA纸片法筛选出MBL阳性菌株5株,阳性率为17％。PCR扩增出IMP基因型有4株,阳性率为14％,均为金属酶IMP-9型,未扩增出VIM基因。以PCR法结果为判定标准,亚胺培南-EDTA纸片法敏感性100％,特异性96％。结论IMP-9型为该院分离的这29株耐亚胺培南的铜绿假单胞菌中主要MBL基因型。本实验中所用的亚胺培南一EDTA纸片法能简单有效的筛选出产MBL的铜绿假单胞菌。     

One origin for metallo-β-lactamase activity,or two? An investigation assessing a diverse set of reconstructed ancestral sequences based on a sample of phylogenetic trees

Bacteria use metallo-β-lactamase enzymes to hydrolyse lactam rings found in many antibiotics, rendering them ineffective. Metallo-β-lactamase activity is thought to be polyphyletic, having arisen on more than one occasion within a single functionally diverse homologous superfamily. Since discovery of multiple origins of enzymatic activity conferring antibiotic resistance has broad implications for the continued clinical use of antibiotics, we test the hypothesis of polyphyly further; if lactamase function has arisen twice independently, the most recent common ancestor (MRCA) is not expected to possess lactam-hydrolysing activity. Two major problems present themselves. Firstly, even with a perfectly known phylogeny, ancestral sequence reconstruction is error prone. Secondly, the phylogeny is not known, and in fact reconstructing a single, unambiguous phylogeny for the superfamily has proven impossible. To obtain a more statistical view of the strength of evidence for or against MRCA lactamase function, we reconstructed a sample of 98 MRCAs of the metallo-β-lactamases, each based on a different tree in a bootstrap sample of reconstructed phylogenies. InterPro sequence signatures and homology modelling were then used to assess our sample of MRCAs for lactamase functionality. Only 5 % of these models conform to our criteria for metallo-β-lactamase functionality, suggesting that the ancestor was unlikely to have been a metallo-β-lactamase. On the other hand, given that ancestral proteins may have had metallo-β-lactamase functionality with variation in sequence and structural properties compared with extant enzymes, our criteria are conservative, estimating a lower bound of evidence for metallo-β-lactamase functionality but not an upper bound.     

Complete 1H, 15N and 13C resonance assignments of Bacillus cereus metallo-β-lactamase and its complex with the inhibitor R-thiomandelic acid

β-Lactamases inactivate β-lactam antibiotics by hydrolysis of their endocyclic β-lactam bond and are a major cause of antibiotic resistance in pathogenic bacteria. The zinc dependent metallo-β-lactamase enzymes are of particular concern since they are located on highly transmissible plasmids and have a broad spectrum of activity against almost all β-lactam antibiotics. We present here essentially complete (>96 %) backbone and sidechain sequence-specific NMR resonance assignments for the Bacillus cereus subclass B1 metallo-β-lactamase, BcII, and for its complex with R-thiomandelic acid, a broad spectrum inhibitor of metallo-β-lactamases. These assignments have been used as the basis for determination of the solution structures of the enzyme and its inhibitor complex and can also be used in a rapid screen for other metallo-β-lactamase inhibitors.     

Cloning and sequencing of the beta-lactamase gene and surrounding DNA sequences of Citrobacter braakii,Citrobacter murliniae,Citrobacter werkmanii,Escherichia fergusonii and Enterobacter cancerogenus

To further identify the origins of plasmid-mediated cephalosporinases that are currently spreading worldwide, the chromosomal beta-lactamase genes of Citrobacter braakii, Citrobacter murliniae, Citrobacter werkmanii reference strains and of Escherichia fergusonii and Enterobacter cancerogenus clinical isolates were cloned and expressed into Escherichia coli and sequenced. These beta-lactamases had all a single pI value >8 and conferred a typical AmpC-type resistance pattern in E. coli recombinant strains. The cloned inserts obtained from genomic DNAs of each strain encoded Ambler class C beta-lactamases. The AmpC-type enzymes of C. murliniae, C. braakii and C. werkmanii shared 99%, 96% and 95% amino acid sequence identity, respectively, with chromosomal AmpC beta-lactamases from Citrobacter freundii. The AmpC-type enzyme of E. cancerogenus shared 85% amino acid sequence identity with the chromosomal AmpC beta-lactamase of Enterobacter cloacae OUDhyp and the AmpC-type enzyme of E. fergusonii shared 96% amino acid sequence identity with that of E. coli K12. The ampC genes, except for E. fergusonii, were associated with genes homologous to regulatory ampR genes of other chromosomal class C beta-lactamases that explain inducibility of beta-lactamase expression in these strains. This work provides further evidence of the molecular heterogeneity of class C beta-lactamases.