阴沟肠杆菌Ⅰ类整合酶基因及质粒AmpC酶基因检测

目的明确我院临床分离的阴沟肠杆菌中Ⅰ类整合酶基因(intⅠ1)、qacE△1-sulⅠ基因和质粒Am鄄pC酶基因(AmpCMIR、AmpCDHA)存在状况。方法采用ATB药敏试验板微量肉汤法测定临床分离的20株阴沟肠杆菌对20种抗菌药物的敏感性,采用聚合酶链反应(PCR)技术检测耐药基因。结果该20株菌呈现多重耐药,对亚胺培南和美罗培南均敏感,对阿莫西林、阿莫西林/克拉维酸、头孢噻吩和头孢西丁完全耐药,头孢吡肟和复方新诺明的耐药率分别为25.0%和85.0%,对氨基糖苷类抗生素的耐药率在60.0%～90.0%之间,其余的耐药率在80.0%～95.0%之间。intⅠ1、qacE△1-sulⅠ、MIR和DHA基因的阳性株数(%)分别为19株(95.0%)、17株(85.0%)、17株(85.0%)、1株(5.0%)。结论我院临床分离的阴沟肠杆菌多重耐药严重,intⅠ1、qacE△1-sulⅠ和MIR基因携带率很高。在阴沟肠杆菌中检出intⅠ1、qacE△1-sulⅠ以及质粒型AmpCMIR基因在我国大陆均属首次报道。     

探讨对头孢西丁不敏感大肠埃希菌产AmpC酶情况及其耐药性

目的了解对头孢西丁(FOX)不敏感的大肠埃希菌产AmpC酶及其耐药情况,为临床合理用药提供依据。方法收集2009年度不重复临床分离对FOX不敏感的大肠埃希菌123株,三维试验检测产AmpC酶,纸片扩散法(Kirby-Bauer)进行药敏试验。结果 123株对FOX不敏感的大肠埃希菌中AmpC酶检出率为8.1%(10/123),产酶菌对15种抗生素的药敏结果除对亚胺培南和美洛培南显示100%敏感外,对其他抗菌药物的耐药率均高于不产酶株。结论产生AmpC酶是大肠埃希菌对头孢西丁耐药的一个重要原因,对产酶株进行检测和监测有助于临床合理选择抗菌药物以及避免医院内感染的流行和发生,对产AmpC酶株治疗建议以碳青霉烯类抗生素为主。     

94株产气肠杆菌的临床分布与耐药性分析

目的 了解产气肠杆菌的临床分布及耐药性.方法 用常规方法检出可疑菌落,用法国生物梅里埃公司ID 32E条和ATB G-5条做菌种鉴定与药敏试验;分别用复合纸片表型确认试验和FOX琼脂平板试验检测ESBLs和AmpC酶.结果 临床标本主要来自痰液,占52.1％,其次为中段尿,占16.0％;临床科室中呼吸内科和肝胆外科检出最多,分别占43.6％和17.0％;亚胺培南、美罗培南对所有菌株均敏感;头孢吡肟和哌拉西林/他唑巴坦的耐药率＜16.0％;产气肠杆菌对阿莫西林、头孢噻吩、头孢西丁的耐药率均＞95.0％,对其余β-内酰胺类抗菌药物均有一定程度的耐药,对氨基糖苷类、喹诺酮类、磺胺甲噁唑/甲氧苄啶耐药率较低;ESBLs和AmpC酶的检出率分别为16.0％和11.7％.结论 产气肠杆菌临床主要来自痰液标本及呼吸内科;所有菌株均对亚胺培南、美罗培南敏感,对阿莫西林、头孢噻吩、头孢西丁的耐药率最高;产ESBLs和AmpC酶菌株已占一定比例.     

Comparison of disk diffusion,Etest and VITEK2 for detection of carbapenemase‐producing Klebsiella pneumoniae with the EUCAST and CLSI breakpoint systems

The aim of this study was to compare CLSI and EUCAST MIC and disk diffusion carbapenem breakpoints for the detection of carbapenemase‐producing Klebsiella pneumoniae. K. pneumoniae strains with known KPC (n = 31) or VIM (n = 20) carbapenemases were characterized by disk diffusion (Oxoid) and Etest (bioMérieux) vs. imipenem, meropenem and ertapenem, and with VITEK2 (bioMérieux, five different cards). Extended‐spectrum β‐lactamase (ESBL) testing was performed with VITEK2 (bioMérieux), ESBL combination disks (Becton Dickinson) and the ESBL Etest (bioMérieux). With CLSI and EUCAST MIC breakpoints, respectively, 11 and seven of the strains were susceptible to imipenem, 12 and eight to meropenem, and seven and none to ertapenem. The EUCAST epidemiological cut‐off (ECOFF) values for meropenem and ertapenem identified all carbapenemase producers, whereas the imipenem ECOFF failed in five strains. All carbapenemase producers were detected with EUCAST disk diffusion breakpoints for ertapenem and meropenem, and four strains were susceptible to imipenem. CLSI disk diffusion breakpoints characterized 18 (imipenem), 14 (meropenem) and three (ertapenem) isolates as susceptible. When cards with a single carbapenem were used, detection failures with VITEK2 were four for imipenem, none for meropenem and one for ertapenem. Cards containing all three carbapenems had one to two failures. With ESBL combination disks, 21/31 KPC producers and 2/20 VIM producers were positive. With VITEK2, no VIM producers and between none and seven KPC producers were ESBL‐positive. All carbapenemase producers were detected with the meropenem MIC ECOFF, or the clinical EUCAST breakpoint for ertapenem. EUCAST disk diffusion breakpoints for meropenem and ertapenem detected all carbapenemase producers. VITEK2 had between none and four failures in detecting carbapenemase producers, depending on the antibiotic card.     

儿童感染人苍白杆菌的耐药性及DHA-1基因检出

目的初步探讨败血症患儿血标本分离的人苍白杆菌耐药机制。方法采用VITEK2compact全自动微生物鉴定药敏系统对败血症患儿分离的病原菌进行鉴定和药敏试验;聚合酶链反应(PCR)法对人苍白杆菌受试株进行AmpC、AmpR、DHA-1、AcrA、RamA、OprD、TEM、SHV等多重耐药基因检测,扩增产物经纯化后测序并进行序列分析。结果从败血症患儿的血标本中检出155株人苍白杆菌,从中随机选取30株进行检测。30株试验菌株中,有20株耐哌拉西林/他唑巴坦,其中16株(80.00%,16/20)扩增出人苍白杆菌AmpC/R基因,2株(10.00%,2/20)扩增出DHA-1基因。30株受试菌均未扩增出AcrA、RamA、OprD、TEM、SHV等基因。将155株人苍白杆菌根据其是否耐哌拉西林/他唑巴坦,分成耐药组(126株)和非耐药组(29株),其中耐药组菌株对氨苄西林、第一～三代头孢菌素、氨曲南等几乎100%耐药,但对左氧氟沙星、亚胺培南、美罗培南、环丙沙星、阿米卡星、庆大霉素等耐药率(0.00%～1.59%)低。结论产AmpC/R是儿童感染人苍白杆菌耐多种抗菌药物的机制,重症患儿可选用亚胺培南。应加强对人苍白杆菌耐药性的监测,防止DHA基因在革兰阴性杆菌中流行。     

铜绿假单胞菌超广谱β-内酰胺酶、质粒介导AmpC酶基因分布及流行特征分析

目的研究临床分离铜绿假单胞菌超广谱β-内酰胺酶(ESBLs)和质粒介导AmpC酶的基因分布及流行特性,为细菌耐药性的监控提供依据。方法回顾性调查5年间临床分离的铜绿假单胞菌的分布和耐药情况。选取每年同一时间段的临床连续分离株共169株,用聚合酶链反应(PCR)检测TEM、SHV、VEB、PER、GES、TLA、IBC、CTX-M、OXA等β-内酰胺酶基因和AmpC酶基因。使用脉冲场凝胶电泳技术(PFGE)分析菌株间的同源性。结果铜绿假单胞菌主要分离自呼吸道(痰标本),占75.7%;对复方磺胺甲噁唑和头孢曲松耐药情况严重,耐药率分别为91.9%和91.7%;对其他主要抗菌药物的耐药率分别为头孢他啶46.7%、亚胺培南38.2%、左旋氧氟沙星30.2%、环丙沙星28.3%、阿米卡星5.0%。169株铜绿假单胞菌中产TEM、CTX-M、OXA和GES型β-内酰胺酶菌株84株(49.7%);质粒介导AmpC酶23株(13.6%)。PFGE结果显示铜绿假单胞菌仅3株具有同源性,呈散发流行。结论铜绿假单胞菌临床感染率高,呈多重耐药且散发流行趋势。     

产质粒介导AmpC酶大肠埃希菌的流行病学调查及其酶的表达调控

目的研究产质粒介导AmpC酶大肠埃希菌的流行病学状况及其酶的表达调控。方法选择临床中分离到的疑似产质粒介导AmpC酶的大肠埃希菌30株,对产质粒介导AmpC酶大肠埃希菌的流行病学特征进行调查分析,并行转移接合试验。用6组引物对提取的细菌质粒DNA进行多重PCR扩增,从而对质粒介导的AmpC酶的表达调控进行分析。结果流行病学调查,共检出20株产p-内酰胺酶菌株,其中有5株菌株同时产AmpCp-内酰胺酶和超广谱p-内酰胺酶,12株只产超广谱p-内酰胺酶,3株只产AmpCp-内酰胺酶,5株转移接合成功。PCR扩增试验后,与Genbank数据库进行序列比对分析,发现有6株扩增产生质粒介导的AmpC基因,其中CIT型4株,DHA型2株。结论产质粒AmpC酶大肠埃希菌在临床上广泛传播,把握其持续高表达的调控机制,有助于指导临床合理使用抗牛素。     

小檗碱诱导产ACT-1型AmpC酶大肠埃希菌蛋白质组学研究

目的研究小檗碱诱导前后产AmpC酶大肠埃希菌的蛋白质谱变化,找出有价值的靶位。方法提取经小檗碱诱导前后的产ACT-1型AmpC酶国内以大肠埃希菌的全菌蛋白,经双向凝胶电泳技术分析、Bluesilver法染色、凝胶图像分析．MALDI-TOF-MS质谱比较等步骤,找出诱导前后的差异蛋白质。结果诱导前后共有8个差异性蛋白,其中外膜蛋白lI、AmpC—β-内酰胺酶、转座酶的表达明显上调,而磷酸转移酶系统的葡萄糖特异性组分ⅡA蛋白、甘油磷酰基二酯酶、抗碲酸盐蛋白、硫醇过氧化物酶、细胞分裂蛋白nsZ等蛋白表达下调。结论该研究筛选出的差异性蛋白与产ACT—1型AmpC酶大肠埃希菌机制相关,可为医学界研发和选择抗菌药物提供有价值的依据。     

大肠埃希菌ESBLs、AmpC酶的检测及耐药特性

目的 分析大肠埃希菌产超广谱β-内酰胺酶（ESBLs）、头孢菌素（AmpC）酶的情况及耐药特性,指导临床合理用药.方法 收集2011年1月至2012年6月我院临床分离的大肠埃希菌287株.用双纸片确诊法检测ESBLs,三维试验检测AmpC酶,KB法进行药敏试验.结果 287株大肠埃希菌中,产ESBLs 119株（41.5％）,产AmpC酶47株（16.4％）,同时检出产ESBLs和AmpC酶16株（5.6％）.单一产ESBLs或AmpC酶的菌株有较高的耐药性,同时检出ESBLs和AmpC酶的菌株对多种抗菌药耐药,所有菌株对亚胺培南均敏感.结论 大肠埃希菌ESBLs和AmpC酶的分离率及耐药率都较高,同时检出ESBLs和AmpC酶的菌株具多重耐药性;亚胺培南具有良好的抗菌活性.临床应加强ESBLs和AmpC酶的检测及耐药监测,采取有效措施防止耐药菌株产生.     

Detection of extended-spectrum ß-lactamase-producing Enterobacteriaceae in rectal swabs with the Mastdiscs™ ID AmpC ßLSE detection set

Objectives

The double-disk synergy test was compared to the Mastdiscs™ ID AmpC and ESßL method for detection of ESßL production in rectal swab.

Methods

Two hundred and forty-nine rectal swabs were directly inoculated onto Mueller-Hinton plates and analyzed according to both methods.

Results

A total of 41 (16%) and 208 (84%) were positive and negative for ESßL, respectively. Twelve (29%) and 20 (49%) of the 41 rectal swabs positive for ESßL were detected after 24 h of incubation with the double-disk synergy test and the Mastdiscs™ method, respectively (P = 0.013). One hundred fifty-eight (76%) et 183 (88%) of the 208 rectal swabs were detected negative for ESßL after 24 h of incubation with the double-disk synergy test and the Mastdiscs™ method, respectively (P < 0.001). Finally, 79 (32%) and 46 (18%) rectal swabs respectively inoculated according to the double-disk synergy test and the Mastdiscs™ method were inconclusive after 24 h of incubation. The better performance of the Mastdiscs™ method was due to an easier detection of cephalosporinase producing bacteria.

Conclusions

The Mastdiscs™ method is a simple phenotypic method that detects more easily ESßL and non-ESßL producing bacteria in rectal swab.