Enhanced detection of carbapenemase-producing Enterobacteriaceae by an optimized phenol red assay

Screening for the detection of carbapenemase-producing bacteria still encounters issues related to workflow, limit of detection, or qualitative interpretation. We developed a spectrophotometry-based version of the Carba NP phenol red assay (Nordmann et al., 2012) in a microtiter plate format, compatible with low bacterial cell counts. We were able to detect highly active carbapenemases such as KPC and IMP in 30 min. A wider range of carbapenemases including OXA-48 were detected using higher inocula, still being competitive compared with currently available phenol red assays. Validation experiments of our test with a panel of 81 Enterobacteriaceae showed good performance with 93% of sensitivity and 92% of specificity. The compatibility of our routine-friendly protocol with automation offers great perspectives for high throughput screening in outbreak situations and/or in big laboratories.     

KPC型碳青霉烯酶分子进化及与底物结合自由能分析

目的 分析KPC-2、KPC-5和KPC-10型碳青霉烯酶的分子进化及与10种β-内酰胺类药物的结合自由能.方法 用MEGA 4.1软件中的Minimum Evolution法分析KPC-2、KPC-5和KPC-10型碳青霉烯酶的分子进化,用ArgusLab 4.1软件中的Dock模块作这3种酶与10种β-内酰胺类药物的分子对接,并计算酶与底物的结合自由能（△G）.结果 有碳青霉烯酶活性的A类β-内酰胺酶在同一簇且保守性较好,无碳青霉烯酶活性的普通A类β-内酰胺酶则在另一簇.KPC-2、KPC-5和KPC-10型碳青霉烯酶与碳青霉烯类药物结合自由能均下降,且降幅居前,它们的结合自由能比第三代头孢类抗生素更低.结合自由能较高的为氨曲南和克拉维酸.结论 KPC型碳青霉烯酶对碳青霉烯类药物的催化能力高于对第三代头孢类抗生素的催化能力,对氨曲南和克拉维酸的催化活性最低.     

细菌NDM-1质粒耐药表型与其表达的相关性研究

目的 探讨NDM-1质粒携带菌株耐药表型与其基因表达的相关性,为临床抗菌药物选择提供理论参考。方法 携带NDM-1的质粒电转化入大肠埃希菌TOP10,采用琼脂平板倍比稀释法测定碳青霉烯类药物对野生株和电转化子的最低抑菌浓度(minimal inhibitory concentration, MIC),TaqMan RT-PCR检测碳青霉烯类耐药菌株的blaNDM-1基因表达及亚-MIC亚胺培南诱导对blaNDM-1基因表达的影响。结果 12株产NDM-1菌株中共有8株电转化成功,电转化子对碳青霉烯类药物的耐药性与野生菌株基本一致。细菌对碳青霉烯类的耐药性与blaNDM-1基因表达成正比。亚-MIC亚胺培南诱导后菌株对碳青霉烯类药物的MIC值和blaNDM-1基因表达均显著高于野生株。结论 碳青霉烯类耐药菌株耐药表型与blaNDM-1基因表达具有正相关性,TaqMan     

A 5-day course of oral antibiotics followed by faecal transplantation to eradicate carriage of multidrug-resistant Enterobacteriaceae: a randomized clinical trial

ObjectivesIntestinal carriage with extended spectrum β-lactamase Enterobacteriaceae (ESBL-E) and carbapenemase-producing Enterobacteriaceae (CPE) can persist for months. We aimed to evaluate whether oral antibiotics followed by faecal microbiota transplantation (FMT) can eradicate intestinal carriage with ESBL-E/CPE.MethodsRandomized, open-label, superiority trial in four tertiary-care centres (Geneva (G), Paris (P), Utrecht (U), Tel Aviv (T)). Non-immunocompromised adult patients were randomized 1: 1 to either no intervention (control) or a 5-day course of oral antibiotics (colistin sulphate 2 × 10⁶ IU 4×/day; neomycin sulphate 500 mg 4×/day) followed by frozen FMT obtained from unrelated healthy donors. The primary outcome was detectable intestinal carriage of ESBL-E/CPE by stool culture 35–48 days after randomization (V4). ClinicalTrials.gov NCT02472600. The trial was funded by the European Commission (FP7).ResultsThirty-nine patients (G = 14; P = 16; U = 7; T = 2) colonized by ESBL-E (n = 36) and/or CPE (n = 11) were enrolled between February 2016 and June 2017. In the intention-to-treat analysis 9/22 (41%) patients assigned to the intervention arm were negative for ESBL-E/CPE at V4 (1/22 not receiving the intervention imputed as positive) whereas in the control arm 5/17 (29%) patients were negative (one lost to follow up imputed as negative) resulting in an OR for decolonization success of 1.7 (95% CI 0.4–6.4). Study drugs were well tolerated overall but three patients in the intervention group prematurely stopped the study antibiotics because of diarrhoea (all received FMT).ConclusionsNon-absorbable antibiotics followed by FMT slightly decreased ESBL-E/CPE carriage compared with controls; this difference was not statistically significant, potentially due to early trial termination. Further clinical investigations seem warranted.     

检测多药耐药鲍氏不动杆菌的碳青酶烯酶及整合酶基因

目的检测多药耐药鲍氏不动杆菌(MDRAba)的碳青酶烯酶及整合酶基因。方法从70株对耐亚胺培南鲍氏不动杆菌中用PCR方法测定碳青酶烯酶OXA23类、OXA24类、OXA51类和OXA58类基因及Ⅰ类和Ⅱ类整合酶基因,对扩增阳性的碳青酶烯酶基因进行测序分析;抗菌药物敏感试验采用琼脂稀释法。结果获得OXA-23类基因阳性56株,占80.0%,OXA-58类基因阳性1株,仅1株菌OXA51类基因阴性;Ⅰ类整合酶基因阳性61株,占87.1%,未测得OXA24类碳青酶烯酶和Ⅱ类整合酶基因;序列分析提示分离的CRAba为OXA-23型和OXA-58型,所测菌株对环丙沙星、头孢他啶、庆大霉素、哌拉西林/他唑巴坦高度耐药,对多黏菌素B一致性敏感。结论新近出现的CRAba菌株主要是以Ⅰ类整合子携带的OXA-23型碳青酶烯酶。     

鲍曼不动杆菌OXA-23型碳青霉烯酶基因的研究

目的分析鲍曼不动杆菌的耐药性及其产OXA-23型碳青霉烯酶基因的核苷酸序列。方法用自动微生物分析仪测定常用抗菌药的MIC50；对OXA-23型碳青霉烯酶基因进行PCR扩增，产物纯化测序。结果15株产ESBLs鲍曼不动杆菌中有12株携带OXA-23型碳青霉烯酶；PCR产物纯化后测序表明与鲍曼不动杆菌（AY795964．1）blaxxA-23基因序列100％同源。结论携带OXA-23型碳青霉烯酶基因的鲍曼不动杆菌对临床常用抗菌药的耐药率高，其编码基因为blaoxA-23。     

亚胺培南耐药鲍曼不动杆菌分子流行病学及耐药基因研究

目的探讨岳阳市第一人民医院耐碳青霉烯类鲍曼不动杆菌（CRAB）的分子流行病学特征,以及碳青霉烯酶基因在介导CRAB对碳青霉烯类药物耐药性形成中的作用。方法收集岳阳市第一人民医院分离的CILAB共65株,其中对亚胺培南耐药鲍曼不动杆菌（IRAB）38株,亚胺培南敏感鲍曼不动杆菌（ISAB）27株。采用琼情稀释法检测上述细菌对15种抗菌药物的最低抑菌浓度（MIC）,改良Hodge试验筛查碳青霉烯酶;PCR扩增碳肯霉烯酶基因;脉冲场凝胶电泳（PFGE）分析菌株同源性。结果在检测的15种药物中,敏感性最高的药物为多黏蒂素B-IRAB与ISAB组除对头饱两丁、多黏菌素B耐药性较一致外,对其他抗菌药物耐药性差异均有统计学意义（P〈0．05）;改良Hodge试验阳性率为66％（43／65）,其中38株IRAB中,阳性36株,27株ISAB中,阳性4株;38侏IRAB有32株携带OXA-23基因,1SAl3组菌株中均未检测到OXA-23基因;65株菌主要分为6型,A型52株,是主要的流行克隆。结论本院存在CRAB的流行,主要为A型株。OXA-23基因的产生在介导CRAB碳青霉烯药物耐药中发挥作用。     

Regional dissemination of Acinetobacter species harbouring metallo-β-lactamase genes in Japan

Metallo-β-lactamase (MBL) producers have been reported among the various Acinetobacter species worldwide. In this study, the epidemiology and molecular characteristics of carbapenemase-encoding genes and mobile elements were studied to analyse the regional dissemination of MBL genes in Acinetobacter species. From January 2001 to December 2006, 48 Acinetobacter isolates harbouring MBL genes identified from five hospitals in Kyoto and Shiga Prefecture, Japan were collected and analysed. The partial rpoB gene or the 16S-23S ribosomal RNA intergenic spacer region was sequenced to obtain a species-level identification. Molecular typing using pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) was performed. Twenty-five Acinetobacter pittii isolates were divided into eight PFGE types and five sequence types (STs) using MLST. Nine Acinetobacter bereziniae isolates belonged to five PFGE types. Five Acinetobacter nosocomialis isolates were divided into two PFGE types and two STs. Three unclassified Acinetobacter species isolates were divided into two PFGE types. Eighteen of the 25 A. pittii isolates belonged to ST119 and were identified from four hospitals. The bla_IMP-19 gene was detected in 41 of 48 isolates, including all of the A. pittii ST119 isolates. The bla_IMP-1 and bla_IMP-11 genes were detected in four and three isolates, respectively. The MBL genes were all embedded within a class 1 integron as a gene cassette array: bla_IMP-19-aac(6′)-31-bla_OXA-21-aadA1, catB8-like/aacA4-bla_IMP-1 and bla_IMP-11. This study is the first report demonstrating the regional dissemination of MBL-producing Acinetobacter species. A. pittii ST119 harbouring bla_IMP-19 was widely spread throughout the Kyoto-Shiga region.     

The difficult-to-control spread of carbapenemase producers among Enterobacteriaceae worldwide

The spread of carbapenemase producers in Enterobacteriaceae has now been identified worldwide. Three main carbapenemases have been reported; they belong to three classes of β-lactamases, which are KPC, NDM, and OXA-48. The main reservoirs of KPC are Klebsiella pneumoniae in the USA, Israel, Greece, and Italy, those of NDM are K. pneumoniae and Escherichia coli in the Indian subcontinent, and those of OXA-48 are K. pneumoniae and Escherichia coli in North Africa and Turkey. KPC producers have been mostly identified among nosocomial isolates, whereas NDM and OXA-48 producers are both nosocomial and community-acquired pathogens. Control of their spread is still possible in hospital settings, and relies on the use of rapid diagnostic techniques and the strict implemention of hygiene measures.