改良Hodge试验检测产碳青霉烯酶肠杆菌科细菌

摘要：目的：用改良Hodge试验检测产碳青霉烯酶肠杆菌科细菌,初步推测产碳青霉烯酶肠杆菌科细菌的流行情况。 方法：筛选出2009年4月~2010年4月对碳青霉烯类抗生素耐药以及敏感性下降或者碳青霉烯类抗生素敏感而一种以上三代头孢菌素耐药的肠杆菌科细菌23株,用改良Hodge试验检测其碳青霉烯酶。 结果：23株菌中21株（91.3%）呈多重耐药,其中5株菌改良Hodge试验阳性,分别为肺炎克雷伯菌1株、弗劳地枸橼酸杆菌1株、大肠埃希菌1株和霍氏肠杆菌2株。 结论：改良Hodge试验阳性提示所测菌株中有产碳青霉烯酶的肠杆菌科细菌的存在,因此要加强对此类细菌的院内感染管理监测。     

改良Hodge试验检测肠杆菌科细菌中碳青霉烯酶的研究

目的 用改良Hodge试验(MHT)检测肠杆菌科细菌碳青霉烯酶的产生,了解碳青霉烯酶基因分布.方法 从浙江大学医学院附属第二医院、浙江省东阳市人民医院、北京医院和四川大学附属华西医院4家医院收集3 718株肠杆菌科细菌,用纸片法检测细菌对厄他培南的敏感性,用MHT检测碳青霉烯酶的产生情况,并用PCR扩增常见的碳青霉烯酶基因.结果 4家医院的肠杆菌科细菌对厄他培南的不敏感率为3.04％ (113/3718),上述4家医院的不敏感率依次为5.09％、2.15％、2.59％和1.72％.MHT的总阳性率为2.29％ (85/3718),4家医院的阳性率依次为4.73％、1.21％、1.06％和1.58％.113株厄他培南不敏感菌株中,MHT阳性82株,阴性31株.85株MHT阳性菌株中,82株对厄他培南耐药或中介耐药,仅有3株敏感.82株MHT阳性、厄他培南不敏感的菌株中,肺炎克雷伯菌碳青霉烯酶(KPC)基因阳性65株,亚胺培南水解酶(IMP)基因阳性15株,其中2株KPC和IMP同时阳性,其他4株未扩增到常见碳青霉烯酶基因.31株MHT阴性、厄他培南不敏感的菌株和3株MHT阳性、厄他培南敏感的菌株均未扩增到常见碳青霉烯酶基因.结论 MHT可有效地检测肠杆菌科细菌中的碳青霉烯酶,具有敏感性高、假阳性率低的特点.肠杆菌科细菌所产碳青霉烯酶主要为KPC,其次为IMP.     

应用改良Hodge试验检测肠杆菌科细菌产碳青霉烯酶的研究

目的应用改良Hodge试验(MHT)检测肠杆菌科细菌产碳青霉烯酶的应用价值。方法应用MHT检测对碳青霉烯类抗菌药物敏感性降低的24株肠杆菌科细菌产碳青霉烯酶的情况。同时利用聚合酶链反应(PCR)检测碳青霉烯酶的基因,包括KPC、NDM、IMP、SIM和VIM,PCR阳性的产物测序结果与Gen Bank数据库进行比对。综合分析MHT和PCR检测碳青霉烯酶的效率。结果对碳青霉烯酶抗菌药物敏感性降低的24株肠杆菌科细菌应用MHT检测碳青霉烯酶的阳性菌株共为13株,而PCR检测出碳青霉烯酶基因的只有5株。且PCR产物测序结果经比对后证实1株为KPC-2和4株为IMP-4。对比分析MHT和PCR的检测结果可知有4株肠杆菌科细菌的MHT和PCR同时检测出碳青霉烯酶;有9株肠杆菌科细菌的MHT为阳性,但PCR没检测出碳青霉烯酶的基因;有1株肠杆菌科细菌MHT为阴性,但PCR检测出碳青霉烯酶的基因。结论 MHT作为筛查碳青霉烯酶肠杆菌科细菌的准确性值得继续探讨。     

加硼酸或氯唑西林的改良Hodge试验检测肠杆菌科细菌碳青霉烯酶的研究

目的 探讨加硼酸或氯唑西林的改良Hodge试验在碳青霉烯酶类药物敏感性降低的细菌中检测碳青霉烯酶的临床应用价值.方法 收集2009年～2010年南京军区总医院对碳青霉烯酶类药物敏感性降低(亚胺培南、美罗培南或厄他培南的MIC≥2 μg/ml)的肠杆菌科细菌65株;采用琼脂倍比稀释法测定其对亚胺培南、美罗培南和厄他培南的MIC;通过改良Hodge试验和加硼酸或氯唑西林的改良Hodge试验检测碳青霉烯酶;采用PCR检测多种β-内酰胺酶基因,并对PCR阳性产物测序鉴定.结果 65株临床分离菌中,53株菌对亚胺培南不敏感(MIC＞4 μg/ml),51株菌对美罗培南不敏感(MIC＞4 μg/ml),58株菌对厄他培南不敏感(MIC＞2 μg/ml).65株菌中38株改良Hodge试验阳性,包括7株弱阳性和31强阳性;33株加硼酸或氯唑西林的改良Hodge试验阳性,包括改良Hodge试验弱阳性2株和强阳性31株.经过对菌株进行β-内酰胺酶基因PCR和测序,发现加硼酸或氯唑西林的改良Hodge试验阳性菌株均携带blaKPC-2;32株阴性菌株均未携带碳青霉烯酶酶基因,但其中5株改良Hodge试验弱阳性菌株携带blampC/blaESBL.结论 加硼酸或氯唑西林的改良Hodge试验可快速、有效地区分改良Hodge试验的真、假阳性,是检测肠杆菌科细菌碳青霉烯酶的简便可靠方法,适用于临床微生物实验室.     

低产碳青霉烯酶肠杆菌科细菌的筛选及耐药基因检测

目的:了解临床分离碳青霉烯类表型敏感的肠杆菌科细菌碳青霉烯酶耐药基因携带情况。方法:VITEK-2系统进行药物的最低抑菌浓度(MIC)检测;改良Hodge试验(MHT)筛查低产碳青霉烯酶肠杆菌科细菌;EDTA双纸片增效法检测金属β-内酰胺酶(MBL);PCR法检测相关耐药基因。结果:115株实验菌有12株MHT阳性;12株阳性菌中,3株EDTA双纸片增效试验阴性,9株阳性,这9株菌除阿米卡星、多粘菌素B敏感外,其他抗菌药物均呈耐药,含I类整合子,且检出IMP-4(blaIMP-4)和IMP-1(blaIMP-1)型的MBL基因,未检出KPC型碳青霉烯酶基因及其他MBL基因。结论:厄他培南对肠杆科细菌产碳青霉烯酶的筛选比亚胺培南、美洛培南敏感;经MHT筛选阳性的,潜在产碳青霉烯酶的肠杆科细菌,应进一步用基因检测法确认。     

rCIM与mCIM对产碳青霉烯酶肠杆菌科细菌的检测性能比较

目的 比较快速碳青霉烯灭活试验(rapid carbapenem inactivation method,rCIM)与改良碳青霉烯灭活试验(modified carbapenem inactivation method,mCIM)对产碳青霉烯酶肠杆菌科细菌(carbapenemase-producing Enter-o...     

用改良Hodge试验筛查低产碳青霉烯酶肠杆菌科细菌

目的 筛查临床分离的对碳青霉烯类表型敏感但携带碳青霉烯酶耐药基因的肠杆菌科细菌,分析改良Hodge试验(MHT)的筛查效果。 方法 用MHT筛查低产碳青霉烯酶肠杆菌科细菌;M-H琼脂稀释法和纸片扩散法（K-B）检测对第三代头孢菌素和碳青霉烯类药物的敏感度;EDTA 双纸片协同试验检测金属碳青霉烯酶;PCR检测细菌相关耐药基因。 结果 203株表型第三代头孢菌素耐药的碳青霉烯类敏感肠杆菌科细菌中,24株MHT阳性,EDTA双纸片协同试验均阴性;M-H琼脂稀释法结果对头孢他啶（CAZ）2株敏感,2株中介,其余均耐药;对厄他培南（ETP）、亚胺培南（IPM）和美洛培南（MEM）均敏感;碳青霉烯酶、β内酰胺酶基因特异性PCR扩增,4株扩增出KPC基因,2株扩增出CTX-M基因,13株扩增出TEM基因。 结论 经MHT筛检出临床分离株中存在潜在的产KPC型碳青霉烯酶肠杆菌科细菌。阳性结果需用基因检测的方法进一步确认。     

mCIM与eCIM在产碳青霉烯酶肠杆菌科细菌表型筛查中的价值

目的 分析改良碳青霉烯灭活试验(mCIM)与乙二胺四乙酸碳青霉烯灭活试验(eCIM)对产碳青霉烯酶肠杆菌科细菌表型筛查的效能及应用价值.方法 收集碳青霉烯类耐药肠杆菌科细菌(CRE)117株(实验组)、碳青霉烯类敏感肠杆菌科细菌50株(对照组).分别采用改良Hodge试验(MHT)、mCIM和eCIM进行表型筛查,采用...     

肠杆菌科细菌碳青霉烯酶的检测

目的调查本院肠杆菌科细菌产碳青霉烯酶的情况。方法收集2010年1～5月临床分离的肠杆菌科细菌,采用K-B纸片法进行药敏试验,以三代头孢菌素、亚胺培南、美罗培南为检测药物,筛选出可能产碳青霉烯酶的菌株120株,采用改良的Hodge试验进行确证。结果在120株耐一种或多种三代头孢菌素,提示可能产生碳青霉烯酶的肠杆菌科细菌中,通过表型确证试验确证为阳性的细菌有4株,肺炎克雷伯菌2株,大肠埃希菌1株,弗劳地枸橼酸杆菌1株。结论表型确证试验阳性的细菌中,如需准确辨别亚型,最好用分子生物学方法检测基因序列。     

CIM，mCIM 和MHT 筛选肠杆菌科细菌产碳青霉烯酶的方法评价

目的 评估碳青霉烯类抑制法(carbapenem inactivation method,CIM)、改良碳青霉烯类失活法(modified carbapenem inactivation method,mCIM)和改良Hodge试验(modified hodge test, MHT )对肠杆菌科细菌碳青霉烯酶表型筛选能力。方法 以PCR检测碳青霉烯酶基因作为金标准,对120株耐碳青霉烯类肠杆菌科细菌(carbapenem-resistant enterbacteriaceae,CRE)和50株碳青酶烯类敏感的肠杆菌科细菌,分别进行CIM,mCIM和MHT表型筛选试验,评估三种方法的表型筛选能力。结果 120株CRE中,93株携带碳青霉烯酶耐药基因,表型筛选试验显示,CIM,mCIM和MHT的敏感度分别是95.6%,96.8%和95.8%,特异度分别为72.7%,92.3%和50%。三种方法筛选碳青霉烯酶,差异有统计学意义(χ²=12.796,P<0.05)。与PCR结果相比较,CIM,MHT和mCIM的一致率分别为90%,95%和77.4%,Kappa值分别为0.898,0.949和0.771。mCIM和CIM试验与PCR高度一致。50株碳青酶烯类敏感的肠杆菌科细菌PCR检测和三种方法均为阴性。结论 三种表型筛选方法均有较高的敏感度,但mCIM较CIM,MHT具有更高的特异度和一致率,是筛选CRE的有效方法。     

改良Hodge试验检测肠杆菌科细菌碳青霉烯酶的性能评估

目的 评估改良Hodge试验在碳青霉烯类药物敏感性降低的肠杆菌科细菌中检测碳青霉烯酶的效能.方法 收集2004-2008年我国16家教学医院的49株碳青霉烯类药物敏感性降低(亚胺培南、美罗培由或厄他培南的MIC≥2μg/ml)的肠杆菌科细菌;采用对倍琼脂稀释法测定其对亚胺培南、美罗培南和厄他培南的MIC;通过改良Hodge试验检测碳青霉烯酶;利用加苯硼酸或苯唑西林的改良Hodge试验区分碳青霉烯酶或AmpCs/ESBLs导致的阳性结果 ;采用PCR检测包括NDM-1型碳青霉烯酶基因在内的多种β内酰胺酶基因,并对PCR阳性产物测序鉴定.结果 49株菌中,36株菌对亚胺培南不敏感(MIC>4μg/ml),31株菌对美罗培由不敏感(MIC>4μg/ml),47株菌对厄他培南不敏感(MIC>2μg/ml).49株临床分离菌中23株菌改良Hodge试验刚性,包括9株弱阳性和14株强阳性.经过对菌株进行β内酰胺酶基因PCR和测序,发现9株Hodge弱阳性菌株中2株携带blaKPC-2,7株不携带碳青霉烯酶基因,但携带blaampC/blaESBL;14株强阳性菌株中4株携带blaKPC-2,8株携带blaIMP-4,2株携带blaIMP-8;26株改良Hodge试验阴性菌株均未携带碳青霉烯酶基因.所有49株菌均未检测到blaNDM-1.以碳青霉烯酶基因检测为标准,则改良Hodge试验对检测碳青霉烯类药物敏感性降低的肠杆菌科菌中碳青霉烯酶的敏感度为100%,特异度为79%,阳性预测值为70%,阴性预测值为100%,准确性为86%.结论 改良Hodge试验具有很好的敏感性,但存在一些假阳性结果 .利用苯硼酸和苯唑西林可有效区分改良Hodge试验的假阳性和真阳性.     

Spectrophotometry-based detection of carbapenemase producers among Enterobacteriaceae

Carbapenem-hydrolyzing ß-lactamases are the most powerful ß-lactamases being able to hydrolyse almost all ß-lactams. They are mostly of the KPC, VIM, IMP, NDM, and OXA-48 type. A spectrophotometry technique based on analysis of the imipenem hydrolysis has been developed that differentiated carbapenemase- from noncarbapenemase producers. This inexpensive technique adapted to screening of carbapenemase producers may be implemented in any reference laboratory worldwide.     

Molecular epidemiology of Enterobacteriaceae that produce VIMs and IMPs from the SMART surveillance program

A study was designed to characterize 35 non-repeat isolates of VIM- and IMP-producing Enterobacteriaceae obtained from the SMART surveillance program. Characterization was done by polymerase chain reaction, sequencing, and multi-locus sequencing. The VIM-1, -2, -5, -26, -27, -33, and IMP-1 and -26–producing Enterobacteriaceae were obtained from Greece, Italy, Spain, Philippines, Turkey, Australia, Mexico, USA, and India. Plasmids varied in size from 60 to 300 kb and belonged to IncA/C, IncF, IncHI1, IncL/M, IncN, and IncK incompatibility groups. The most common gene cassettes consisted of bla_IMP-26, qacG, aacA4 and bla_VIM, aacA7, dhfrI, and aadA1. Intercountry, interhospital, intrahospital, interspecies, and intraclonal spread of bla_VIM and bla_IMP containing plasmids and sequence types (STs) occurred in Greece, Italy, Spain, and Philippines. ST147 with IncA/C and IncF plasmids is an important drug-resistant ST among Klebsiella pneumoniae with VIMs. Our study highlights the importance of surveillance programs using molecular techniques as powerful tools to identify the transmission of STs with their respective plasmids.     

An evaluation of multiple phenotypic screening methods for Klebsiella pneumoniae carbapenemase (KPC)-producing Enterobacteriaceae

Klebsiella pneumoniae carbapenemase (KPC)-producing Enterobacteriaceae may display MICs to carbapenems within susceptible or intermediate ranges, prompting confirmatory testing. Four phenotypic methods to detect KPC producers were evaluated against a collection of clinical Enterobacteriaceae isolates. Meropenem–phenylboronic acid double disk synergy testing demonstrated the best performance with 100% sensitivity and specificity.     

Clinical laboratory detection of carbapenem-resistant and carbapenemase-producing Enterobacteriaceae

Introduction: Carbapenemases, enzymes that hydrolyze carbapenem-class antimicrobials, pose serious clinical and diagnostic challenges, including their recent rapid spread among members of the Enterobacteriaceae, a family with no inherent carbapenem resistance. Currently there is no one-size-fits-all method for detecting carbapenem-resistant Enterobacteriaceae (CRE) in the laboratory, nor how to differentiate carbapenemase-producers (CP) from isolates that are carbapenem-resistant via other or combined mechanisms.

Areas covered: This article reviews definitions for CRE and CP-CRE, and discusses current phenotypic and molecular methods available to the clinical laboratory for the detection of both CP and non-CP CRE.

Expert commentary: Routine evaluation of carbapenem resistance mechanism by the routine clinical laboratory are not necessary for patient care, as clinical breakpoints best predict response. However, evaluation for carbapenemase is integral to infection control efforts, and laboratories should have the capacity to do such testing, either in house or by submitting isolates to a reference laboratory.     

Evaluation of the modified carbapenem inactivation method for the detection of carbapenemase-producing Enterobacteriaceae

Carbapenemase-producing Enterobacteriaceae (CPE) are increasing worldwide. Rapid and accurate detection of CPE is necessary for appropriate antimicrobial treatment and hospital infection control. However, CPE contains some strains that are difficult to detect depending on genotype and MIC value of carbapenem, and a detection method has not been established. The recently reported modified carbapenem inactivation method (mCIM) has been developed in CLSI M100-S27 as a phenotypic technique for detecting carbapenemase activity. In the present study, we examined mCIM as a new CPE detection method using 207 Enterobacteriaceae isolates in comparison with the three existing screening methods of modified Hodge test, Carba NP test and carbapenem inactivation method and evaluated its performance. Consequently, both the sensitivity and specificity of mCIM were 100%, indicating better results than the conventional screening methods. The mCIM is a useful tool for microbiology laboratories due to its simplicity, clear criteria, cost-effectiveness and availability at any laboratory.