碳青霉烯类耐药肠杆菌科细菌实验室检测的研究

碳青霉烯类耐药肠杆菌科细菌（CRE）已经成为全球性公共卫生问题,这类细菌往往伴随高致病率、高致残率、高死亡率,为临床治疗带来了极大的挑战。CRE的耐药机制主要是产生碳青霉烯酶。快速、准确地检测产碳青霉烯酶的肠杆菌科细菌,对于合理使用抗生素,预防和控制产酶菌株的传播具有重要意义。本文就实验室检测肠杆菌科细菌产碳青霉烯酶的研究方法进展作一综述。     

肠杆菌科细菌产碳青霉烯酶研究进展

碳青霉烯类抗生素抗菌谱广,抗菌活性强,杀菌作用快,尤其适用于治疗由产超广谱β内酰胺酶(ESBLs)或(和)头孢菌素酶(AmpC)细菌所引起的严重感染.既往临床上常见的耐碳青霉烯类抗生素菌株为以绿脓假单胞菌和不动杆菌属细菌为代表的非发酵细菌,但随着碳青霉烯类抗生素在临床上的广泛使用,耐碳青霉烯类抗生素的肠杆菌科菌株逐渐增加,给临床治疗带来了极大困难.肠杆菌科细菌对碳青霉烯类抗生素耐药的主要机制为产生碳青霉烯酶,现回顾近年的相关研究,就肠杆菌科细菌产碳青霉烯酶的研究进展及其检测方法、耐药菌株的治疗等做一综述.     

碳青霉烯耐药肠杆菌目细菌耐药性及耐药传播机制研究

目的:探讨碳青霉烯耐药肠杆菌目细菌(CRE)的耐药性及耐药传播机制,为CRE感染的治疗和防控提供科学依据。方法:收集绍兴第二医院2016年5月—2018年8月临床分离的CRE 76株。采用基质辅助激光解吸电离飞行时间质谱(MALDI-TOF MS)进行菌种鉴定;微量肉汤稀释法或琼脂稀释法测定多黏菌素、替加环素、头孢他啶...     

我院耐碳青霉烯类肠杆菌科细菌碳青霉烯酶不同检测方法的比较

目的 探讨评价碳青霉烯酶的不同检测方法,为实验室检测及临床诊疗提供依据。方法 选用EDTA碳青霉烯灭活方法(mCIM/eCIM)检测耐碳青霉烯类肠杆菌(carbapenem-resistant Enterobacteriaceae, CRE)菌株耐药表型,以两种免疫层析碳青霉烯酶检测试剂盒、荧光定量PCR法检测CRE菌株基因型。统计学方法采用Kappa一致性检验,比较各种检测方法的临床可操作性。结果 经荧光定量PCR扩增后基因测序显示73株肺炎克雷伯菌中71株携带KPC基因,2株携带NDM基因;7株大肠埃希菌中6株携带NDM基因,1株携带KPC基因;mCIM联合eCIM检测KPC的灵敏度为68.1%(49/72),检测NDM的灵敏度为100.0%(8/8);NG-test Carba 5和免疫显色试剂盒检测KPC和NDM灵敏度为100.0%、特异性为100.0%。结论 NG-test Carba 5和免疫显色试剂盒检测可以成为检测我国最常见碳青霉烯酶的快速、方便的诊断工具,从而有助于控制产碳青霉烯酶的分离株在医院间的传播,为院感防控工作起到至关重要的意义。     

耐碳青霉烯类革兰阴性菌对头孢他啶/阿维巴坦的耐药性及产酶表型分析

目的 分析耐碳青霉烯类革兰阴性菌(CRO)对头孢他啶/阿维巴坦(CAZ/AVI)的耐药特点及产酶表型,为临床合理用药及感染防控提供指导。方法 收集2020年1月至2021年9月临床分离的CRO 206株,采用酶抑制剂增强试验和酶免疫层析法检测碳青霉烯酶分型,纸片扩散法(K-B法)检测CAZ/AVI敏感性,并用E-test法对抑菌圈直径为20～22mm的菌株进行复核。结果 206株CRO菌株经鉴定为耐碳青霉烯类肠杆菌131株和铜绿假单胞菌75株,主要来源于神经内科、急诊EICU、神经外科等临床科室。经耐药表型检出,CRO菌株以产A类丝氨酸酶为主。对CAZ/AVI总的耐药率为17.5%(36/206),产丝氨酸酶菌株对CAZ/AVI的耐药率为5.7%(6/106),产金属酶菌株对CAZ/AVI 100%(21/21)耐药。其中肺炎克雷伯菌、铜绿假单胞菌、大肠埃希菌、阴沟肠杆菌对CAZ/AVI的耐药率分别为8.7%(9/104)、13.3%(10/75)、91.7%(11/12)、100%(6/6),产气克雷伯菌、奇异变形杆菌、产酸克雷伯菌对CAZ/AVI均100%敏感。有30株CAZ/AV...     

儿科对碳青霉烯类耐药铜绿假单胞菌产金属酶的研究

目的 了解目前儿科对碳青霉烯类抗生素耐药铜绿假单胞菌产金属酶的情况。方法 本研究收集了2003年12月至2005年11月，北京儿童医院住院患儿中分离出对碳青霉烯类抗生素耐药的铜绿假单胞菌59株。使用E试验法检测金属酶的耐药表型，PCR技术检测编码金属酶的IMP、VIM、SPM和GIM4种基因型。结果 本研究59株对碳青霉烯类抗生素耐药铜绿假单胞菌中，29株金属酶耐药表型结果阳性，占49．2％。39株金属酶基因型阳性，占66．1％，其中扩增出IMP型阳性35株，占89．7％，VIM型阳性4株，占10．3％。未检测出SPM和GIM型金属酶。对哌拉西林、哌拉西林-他唑巴坦产金属酶不敏感率高于非产金属酶菌株，差异有统计学意义（P〈0．05）。对产金属酶菌株β-内酰胺类抗生素头孢噻肟、头孢哌酮、头孢他啶、头孢吡肟、头孢哌酮-舒巴坦的MIC90均达到256μg／ml，MIC90均大于256μg／ml，高于非产金属酶菌株。结论 从本研究分离的菌株显示儿科铜绿假单胞菌产金属酶率高于成人报道，产金属酶是儿科分离对碳青霉烯类抗生素耐药铜绿假单胞菌的重要耐药机制。且以产IMP型金属酶为主，少部分产VIM型金属酶。产金属酶菌株对β-内酰胺类抗生素耐药比非产金属酶菌株更严重，尤其对哌拉西林和哌拉西林-他唑巴坦耐药性高。E试验法易用于铜绿假单胞菌产金属酶的初步筛选，但不能单独作为检测铜绿假单胞菌产生金属酶的确证性试验。     

Carba NP试验在产碳青霉烯酶肠杆菌科细菌检测价值的meta分析

目的:系统评价Carba NP试验对产碳青霉烯酶菌株的诊断价值.方法:检索PubMed数据库、EMBASE数据库、Cochrane图书馆、维普中文科技期刊数据库、中国知网、万方数据库、中国生物医学文献数据库(CBM),并辅以文献追溯、手工检索,检索时间为2001年1月1日至2016年6月30日.严格按照纳入和排除标准进行文献筛选.参照诊断准确性研究质量评价工具(Quality Assessment of Diagnostic Accuracy Studies 2,UADAS-2)对纳入文献进行质量评价.应用STATA14.0软件进行数据提取及质量评价、统计和数据分析,绘制森林图分析结果,用漏斗图和Deek检验评价纳入文献的发表偏倚情况.结果:纳入的29项研究的合并敏感性和特异性分别是0.97(95％CI:0.93～0.98)和1.0(95％CI:1.0～1.0),说明敏感性及特异性较高.经异质性分析检验,敏感性和特异性的I2分别是96.49(95％CI:95.79～97.18)和69.88(95％CI:58.69～81.08),说明纳入数据存在异质性.结论:Carba NP试验对于产碳青霉烯酶肠杆菌科细菌(Carbapenem-resistant Enterobacteriaceae,CRE)的检测具有很高的敏感性和特异性.     

对碳青霉烯类耐药的铜绿假单胞菌的金属β-内酰胺酶研究

目的：研究耐碳青霉烯类的铜绿假单胞菌产金属β-内酰胺酶的情况及铜绿假单胞菌的耐药机制。方法：微量二倍稀释法测定亚安培南及美洛培南对铜绿假单胞菌的MIC值；DETA纸片法检测产金属β-内酰胺酶的铜绿假单胞菌。结果：从62株临床分离的铜绿假单胞菌中共检测到28株对亚胺培南耐药，耐药率为45％，18株对美洛培南耐药，耐药率为29．03％。从18株同时对亚胺培南及美洛培南耐药株中共检测到16株产金属β-内酰胺酶，占同期分离铜绿假单胞菌的25．8％。结论：产生金属β-内酰胺酶是铜绿假单胞菌对碳青霉烯类药物耐药的机制之一。     

大肠埃希菌对碳青霉烯类药物的耐药机制研究

目的 研究我院普外科重症监护病房(ICU)出现的对碳青霉烯类抗菌药物耐药的大肠埃希菌分子流行病学特征和碳青霉烯耐药机制.方法 采用琼脂稀释法检测分离株的抗菌药物敏感性,采用脉冲场琼脂糖凝胶电泳(PFGE)研究碳青霉烯类耐药的大肠埃希菌分子流行病学机制,采用特异性PCR、DNA测序分析、接合试验、质粒提取和质粒转化试验、外膜蛋白分析等技术研究碳青霉烯耐药的分子机制.结果 我院分离的14株碳青霉烯耐药菌分别属于10个流行克隆型,均表现对包括亚胺培南和美罗培南在内多种抗菌药物耐药,碳青霉烯类耐药基因扩增显示均携带KPC-2型碳青霉烯酶基因,质粒提取与转化试验显示KPC-2定位于约56 kb大小的质粒上,SDS-PAGE分析发现耐药株多出现外膜蛋白的改变.结论 我院出现多个流行克隆型的碳青霉烯类耐药的大肠埃希菌,质粒型碳青霉烯酶KPC-2是我院泛耐型大肠埃希菌介导对碳青霉烯类耐药的主要原因,外膜孔蛋白改变参与介导大肠埃希菌对碳青霉烯类高度耐药.     

北京康复医院耐碳青霉烯类肺炎克雷伯菌临床分布及耐药性分析

目的 分析该院耐碳青霉烯类肺炎克雷伯菌(CR-KP)的临床分布特点及药敏检测结果,为临床合理有效治疗CR-KP感染提供理论依据。方法 分析2021年全年该院检出的非重复分离CR-KP菌株。利用VITEK 2 Compact全自动微生物分析系统进行菌株鉴定和药敏检测试验。药敏结果参照美国临床实验室标准化协会(CLSI M100)标准判读。所有数据使用WHONET 5.6软件进行统计分析。结果 2021年全年该院共分离非重复的肠杆菌科细菌942株,其中CR-KP菌株295株,检出率高达31.3%。CR-KP菌株的标本来源主要是尿液、痰液、血液标本。主要分布科室为神经康复中心、老年康复中心、呼吸康复中心。CR-KP菌株对大部分临床常用的抗菌药物呈现高度耐药性,除对替加环素较为敏感(耐药率为13.5%),对阿米卡星和复方新诺明耐药率在60%以下,对其余检测的抗菌药物耐药率均大于90.0%。结论 该院CR-KP检出占比较高,对大多数抗菌药物耐药情况非常严峻,医护人员应高度重视,合理使用抗菌药物,同时应加强院感防控和手卫生,防止CR-KP暴发流行。     

Identification and screening of carbapenemase-producing Enterobacteriaceae

Carbapenem-hydrolysing β-lactamases are the most powerful β-lactamases, being able to hydrolyse almost all β-lactams. They are mostly of the KPC, VIM, IMP, NDM and OXA-48 types. Their current extensive spread worldwide in Enterobacteriaceae is an important source of concern, as these carbapenemase producers are multidrug-resistant. Detection of infected patients and of carriers are the two main approaches for prevention of their spread. Phenotypic and molecular-based techniques are able to identify these carbapenemase producers, although with variable efficiencies. The detection of carriers still relies mostly on the use of screening culture media.     

Rapid detection of carbapenemase-producing Enterobacteriaceae from blood cultures

The biochemical-based Carba NP test has been evaluated to detect carbapenemase-producing Enterobacteriaceae (n = 193) directly from spiked blood cultures. It was able to rapidly detect KPC (n = 50), IMP (n = 27), VIM (n = 37), NDM (n = 33) and OXA-48-like producers (n = 46) with sensitivity and specificity of 97.9% and 100%, respectively. This cost-effective technique may be implemented in any microbiology laboratory and offers a reliable test for an early identification of carbapenemase-producing Enterobacteriaceae directly from blood culture that could be useful for the management of infected patients.     

儿科产超广谱β-内酰胺酶志贺菌的基因型和耐药性研究

目的 探讨儿科临床分离志贺菌产超广谱β-内酰胺酶(ESBLs)的基因型及其耐药特点.方法 收集2004年1月至2008年12月北京儿童医院细菌性痢疾住院患儿粪便标本中分离出志贺菌共59株,按照美国临床和实验室标准协会推荐的表型确证试验检测ESBLs,用琼脂稀释法进行最低抑菌浓度(MIC)测定,对产ESBLs菌株进行PCR扩增明确其基因型,对扩增产物进行DNA序列分析确定基因亚型.结果 59株志贺菌中共检出产ESBLs者21株,占35.6%.21株产ESBLs志贺菌PCR均扩增到CTX-M型ESBLs,包括CTX-M-1型6株,CTX-M-9型15株,其中有4株同时伴有TEM型酶,6株伴有OXA型酶.DNA序列分析证实6株CTX-M-1型分别为CTX-M-3亚型(1株)、CTX-M-15亚型(2株)、CTX-M-57亚型(3株),15株CTX-M-9型均为CTX-M-14亚型,伴随存在的TEM型及OXA型耐药基因分别为TEM-1型广谱酶、OXA-1型广谱酶.药敏结果显示对产ESBLs菌株敏感性较好的抗生素有亚胺培南、美罗培南、哌拉西林/他唑巴坦、头孢哌酮/舒巴坦和头孢西丁,其耐药率均小于15%.产不同CTX-M基因亚型的菌株对头孢他啶的耐药性不同.结论 本地区儿科分离志贺菌产ESBLs阳性率高,均为CTX-M型,其中以CTX-M-14亚型为主,少部分为CTX-M-3、CTX-M-15和CTX-M-57亚型.大部分产ESBLs菌株呈多重耐药,碳青霉烯类抗生素应作为治疗产ESBLs志贺菌的首选.     

Incidence of carbapenem resistant nonfermenting gram negative bacilli from patients with respiratory infections in the intensive care units

Resistance to carbapenems is commonly seen in nonfermenting gram negative bacilli (NFGNB). We document herein the prevalence of carbapenem resistance in NFGNB isolated from patients with respiratory tract infections in the intensive care units (ICUs). A total of 460 NFGNB were isolated from 606 endotracheal aspirate specimens during January through December 2003, of which 56 (12.2%) were found to be resistant to imipenem and meropenem. Of these, 24 (42.8%) were Pseudomonas aeruginosa , 8 (14.2%) were Acinetobacter spp. and 24 (42.8%) were other NFGNB. Stringent protocols such as antibiotic policies and resistance surveillance programs are mandatory to curb these bacteria in ICU settings     

Evaluation of Rosco Neo-Sensitabs for phenotypic detection and subgrouping of ESBL-, AmpC- and carbapenemase-producing Enterobacteriaceae

The increasing prevalence of ESBL‐, AmpC‐ and carbapenemase‐producing Enterobacteriaceae necessitates reliable phenotypic tests for detection and categorization. The main objective of this study was to evaluate ROSCO Neo‐Sensitabs with different β‐lactam‐β‐lactam inhibitor combinations for phenotypic detection and categorization of β‐lactamases in Enterobacteriaceae. Using standard CLSI/EUCAST methodology, differences in zones of inhibitions between a β‐lactam alone compared with the combination with a β‐lactamase inhibitor as well as subjective synergy observations were determined for 172 well characterized Enterobacteriaceae strains with defined resistance mechanisms. The results showed that for all ESBL‐positive strains (n = 66), combinations of clavulanic acid synergy with cefotaxime, ceftazidime or cefepime, were observed. All acquired AmpC β‐lactamases (n = 17) were detected using cloxacillin combined with cefotaxime and/or ceftazidime (both combinations were required). Carbapenemase producers (n = 59) with the exception of one KPC‐producer were correctly grouped using the combination of meropenem ± aminophenylboronic acid (APBA) or dipicolinic acid (DPA). Ethylene diamine tetraacetic acid (EDTA) also inhibited all metallo‐β‐lactamases, but as with DPA, one false positive result was observed. Based upon these data, we propose a tablet layout for 14 cm agar plates, which could be used as a whole or in a targeted approach for detection and categorizing of relevant acquired β‐lactamases in Enterobacteriaceae.     

Emergence of carbapenem-resistant Enterobacteriaceae in Austria, 2001-2010

We report the emergence of carbapenem-resistant Enterobacteriaceae in Austria. Over a 10-year period, carbapenem-resistant Enterobacteriaceae isolates were obtained from 13 hospitalized patients, with the first isolation in the year 2005 and a remarkable increase in the number of involved patients in 2010. Carbapenem-resistant Enterobacteriaceae comprise eight Klebsiella pneumoniae isolates, four Klebsiella oxytoca isolates, and one Escherichia coli isolate. The detected carbapenemases were the metallo-β-lactamases New Delhi β-lactamase, VIM and IMP, and the serin-β-lactamase Klebsiella pneumoniae carbapenemase.     

Development of carbapenem resistance during therapy for non-typhoid Salmonella infection

Multidrug-resistant Salmonella infection is a global problem, and carbapenems may represent the last therapeutic choice. We report a case of infection caused by ceftriaxone-resistant and ciprofloxacin-resistant Salmonella enterica serotype Typhimurium. A bla_CMY-2-containing Tn6092, located on a self-transferable IncI1 plasmid, was found in all isolates derived from the patient. During ertapenem treatment, the strain developed carbapenem resistance. Apart from the OmpD deficiency found in all isolates, the strain further developed OmpC deficiency through a single gene mutation, and became carbapenem-resistant. Salmonella appears to be very plastic in developing antimicrobial resistance. Care must be taken by physicians when treating multidrug-resistant Salmonella infection.     

铜绿假单胞菌oprD2基因突变及表达量改变与碳青霉烯类耐药的关系

目的 研究铜绿假单胞菌外膜通道蛋白OprD2的表达减弱或缺失,以及OprD2蛋白自身突变是否会影响铜绿假单胞菌对碳青霉烯类药物的耐药性.方法 收集分离自临床对亚胺培南(IPM)的最低抑菌浓度(MIC)值≥8μg/m1的铜绿假单胞菌共101株,采用肉汤稀释法检测菌株对比阿培南(BPM)、美罗培南(MEM)、帕尼培南(PEM)的MIC值;荧光定量RT-PCR检测铜绿假单胞菌膜通道蛋白oprD2基因的表达量情况;针对oprD2相对表达量正常并对亚胺培南耐药的铜绿假单胞菌,采用普通PCR的方法扩增oprD2全长基因并测序.结果 根据铜绿假单胞菌的OprD2蛋白相对表达量结果,将101株铜绿假单胞菌分成两组,组1为OprD2相对表达量降低组;组2为OprD2相对表达量正常组;组1与组2相比,对IPM、BPM、MEM、PEM的MIC值≥16μg/ml的菌株比例差异均有统计学意义(P＜0.01).组1中,28株同时对4种药物的MIC均≥16 μg/ml,其中有25株的OprD2的相对表达量明显减低(＜0.4);外膜孔蛋白OprD2转录水平与4种碳青霉烯类药物MICs之间呈负相关趋势.组2中,有16株9prD2基因发生突变,按照突变的类型主要分成4组;与PAO1相比,这些菌株对IPM、BPM、MEM、PEM的MIC值有不同程度的增加.结论 OprD2外膜蛋白的表达量减少或缺失是铜绿假单胞菌对亚胺培南耐药的主要机制,可能也与其他3种碳青霉烯类药物耐药有密切关系;铜绿假单胞菌的oprD2基因发生突变,可能会降低铜绿假单胞菌对这儿种碳青霉烯类药物的敏感性.     

Prevalence of faecal carriage of Carbapenemase Producing Enterobacteriaceae in healthy Indian subjects from the community

PurposeFaecal carriage of carbapenemase-producing Enterobacterales (CPE) has been extensively investigated in hospitalized patients, but limited data is available on the carriage rate in healthy individuals in India.MethodsA total of 1000 stool samples were screened for CPE from healthy individuals in Chennai (n ?= ?50), Hyderabad (n ?= ?184) and Mumbai (n ?= ?766). Diluted stool samples were cultured on chromID CARBA SMART plates. Growing colonies were screened for CPE by RAPIDEC® CARBA NP Test and minimum inhibitory concentration (MIC) of imipenem by E-Test. PCR was performed for confirmation of CPE genes.ResultsOut of the 1000 stool samples tested, 6.1% were positive for CPE. A total of 64 carbapenem resistant isolates (56 ?E.coli, 4 Klebsiella pneumoniae, 3 Enterobacter cloacae and 1 Citrobacter freundii) were recovered from ChromID CARBA SMART biplate. Carbapenemase production was identified in 57/64 isolates by RAPIDEC® CARBA NP test. PCR analysis showed 28 bla_NDM-1 and 33 bla_OXA48. Three remaining isolates (2 ?E.coli, 1 ?K.pneumoniae) were negative for the tested carbapenemase genes. Interestingly, out of these 61 PCR positive isolates, 49.1% displayed imipenem MIC within the susceptibility range on the basis of CLSI interpretative criteria.ConclusionsFaecal carriage of CPE among healthy individuals was 6.1%. Comprehensive measures to improve the sanitation scenario and implementation of National AMR action plan are needed to prevent further generation and dissemination of carbapenem resistant Enterobacterales (CRE).     

老年人亚胺培南耐药铜绿假单胞菌耐药性研究

目的 明确我院老年病人临床分离铜绿假单胞菌的耐药性、同源性及耐碳青霉烯菌株的基因型。方法 收集我院2006年5月-2009年5月自临床老年病人分离的262株铜绿假单胞菌,纸片扩散法测定其对16种抗菌药物的耐药性;琼脂稀释法和E test法测定耐碳青霉烯菌株对14种抗菌药物的MIC值,PCR扩增及克隆测序分析金属酶基因型。脉冲场凝胶电泳(PFGE)分析携带金属酶基因型菌株的同源性。结果 262株铜绿假单胞菌中筛选到104株耐碳青霉烯。104株耐碳青霉烯铜绿假单胞菌对氨苄西林/舒巴坦、头孢哌酮/舒巴坦两个含舒巴坦制剂药物耐药率分别为78.9％和35.9％,对多黏菌素E耐药率最低为6.0％,对米诺环素耐药率58.3％,其余抗菌药物耐药率均大于70.0％;104株亚胺培南耐药铜绿假单胞菌中12株携带金属酶基因,10株检测到有携带VIM-2基因的1类整合子。PFGE分型中12株菌株属于5个克隆株。结论 在我院流行的亚胺培南耐药铜绿假单胞菌中,金属酶基因不是最主要的基因型,金属β-内酰胺酶均为VIM-2型金属酶,耐药基因盒分布于不同的1类整合子中,整合子播散是最主要的流行方式。