VIM-1 metallo-beta-lactamase in Acinetobacter baumannii

In 2004 and 2005, 5 metallo-beta-lactamase (MBL)-positive Acinetobacter baumannii isolates were found in 2 Greek hospitals. Isolates were unrelated and carried blaVIM-1 in a class 1 integron; bla(OXA-51-) and bla(OXA-58-like) carbapenemase genes were also detected. VIM-1 MBL in Acinetobacter spp. causes concern, given the increasing resistance of this species.     

Emergence of metallo-β-lactamase IMP-14 and VIM-2 in Pseudomonas aeruginosa clinical isolates from a tertiary-level hospital in Thailand

Seventy-five clinical isolates of Pseudomonas aeruginosa collected in a tertiary teaching hospital in Thailand were investigated for susceptibility to antimicrobials including imipenem. Metallo-β-lactamase (MBL) enzymes were detected by E-test MBL assay and PCR; class 1 integron genes were also detected by PCR. Strains positive for bla(IMP) and bla(VIM) genes were further characterized by DNA sequencing and examined for clonality by pulsed-field gel electrophoresis. High rates of resistance to anti-pseudomonal agents were found. MBL enzymes were found in 13 (17·3%) strains and 24 (32%) carried class 1 integron genes. Twelve of the latter strains harboured the bla(IMP-14) gene and one strain the bla(VIM-2) gene. All of the IMP-14 strains were identical or closely related suggesting clonal dissemination of these genes.     

Long-term evolution of a nosocomial outbreak of Pseudomonas aeruginosa producing VIM-2 metallo-enzyme

From April 1996 to July 2004, an outbreak of metallo-beta-lactamase-positive (MBL) Pseudomonas aeruginosa occurred in the haematology ward at Nantes University Hospital in France. Fifty-nine patients were carriers of VIM-2-positive strains of whom 14 were infected (mostly urinary tract infections and pneumonia). Pulsed-field gel electrophoresis identified related isolates demonstrating resistance to all beta-lactams, aminoglycosides, fluoroquinolones, fosfomycin, rifampicin but not colistin. The bla(VIM-2) gene responsible for VIM-2 MBL was not plasmid-encoded but part of a novel type of class 1 integron. VIM-2-positive strains were mostly from urine samples and clinical data suggest that in the absence of therapeutic guidelines, piperacillin-tazobactam or aztreonam may be a reliable choice for treating infections with MBL-producing strains.     

长沙地区产金属β-内酰胺酶铜绿假单胞菌的分子流行病学特征

目的了解长沙地区多重耐药铜绿假单胞菌（PA）产金属β-内酰胺酶(MBL)菌株基因型和流行情况。方法收集该地区7所综合医院临床分离的PA,对菌株进行鉴定和药敏试验,采用EDTA协同试验、E-test MBL进行MBL表型筛选,聚合酶连反应（PCR）明确其基因型,肠杆菌科基因间重复序列聚合酶链反应(ERIC-PCR)进行同源性分析。结果经EDTA协同和E-test MBL初步筛查,81株PA中仅10株为强阳性;PCR结果显示,18株 PA MBL基因阳性,其中IMP-9型11株,IMP-1型1株和VIM-2型6株,未检测出SIM、SPM、GIM和NDM-1基因型。ERIC-PCR分型结果显示,12株产IMP-PA存在多个型别,而6株产VIM-2菌株为同一型别。结论长沙地区多重耐药PA以IMP-9和VIM-2基因型最常见。     

VIM-2-producing Multidrug-Resistant Pseudomonas aeruginosa ST175 Clone, Spain

A total of 183 patients were colonized or infected with multidrug-resistant Pseudomonas aeruginosa isolates at a hospital in Spain during 2007-2010; prevalence increased over this period from 2.8% to 15.3%. To characterize these isolates, we performed molecular epidemiologic and drug resistance analysis. Genotyping showed that 104 (56.8%) isolates belonged to a single major clone (clone B), which was identified by multilocus sequence typing as sequence type (ST) 175. This clone was initially isolated from 5 patients in 2008, and then isolated from 23 patients in 2009 and 76 patients in 2010. PCR analysis of clone B isolates identified the bla(VIM-2) gene in all but 1 isolate, which harbored bla(IMP-22). ST175 isolates were susceptible to only amikacin (75%) and colistin (100%). Emergence of the ST175 clone represents a major health problem because it compromises therapy for treatment of P. aeruginosa nosocomial infections.     

High prevalence of metallo-beta-lactamase-mediated resistance challenging antimicrobial therapy against Pseudomonas aeruginosa in a Brazilian teaching hospital

The prevalence of metallo-beta-lactamase (MBL) production among Pseudomonas aeruginosa nosocomial isolates from a Brazilian teaching hospital was determined. A total of 512 P. aeruginosa isolates were recovered from 245 patients during a 10-month period. Ninety-four (38.4%, 95% CI 32.2-44.8%) isolates were MBL producers. Most resistance to beta-lactams was mediated by MBL. Forty-one (16.7%) were resistant to all drugs except polymyxin B and 33 (80.5%) of these were MBL producers. Clonal dissemination, documented by DNA macrorestriction, played a major role for the spread of MBL isolates. The blaSPM-1 gene was demonstrated by PCR in 14 randomly selected MBL isolates. The extremely high prevalence of MBL production found challenges the choice of therapeutics for P. aeruginosa, and measures to control horizontal dissemination of MBL producers are urgently required.     

Dissemination of IMP-1 metallo- beta -lactamase-producing Acinetobacter species in a Brazilian teaching hospital.

OBJECTIVE: To evaluate the emergence and dissemination of metallo- beta -lactamase (MBL)-producing Acinetobacter species. DESIGN: All carbapenem-resistant Acinetobacter strains (1 strain per patient) collected during the period 1993-2001 were evaluated. SETTING: A Brazilian tertiary care teaching hospital (Hospital Sao Paulo, Sao Paulo). METHODS: Seventy-three strains of carbapenem-resistant Acinetobacter species were recovered from the organism bank of the hospital. All isolates were tested for antimicrobial susceptibility by broth microdilution methods, and the production of MBL was initially assessed by phenotypic tests (MBL Etest strip and a disk approximation test). The MBL enzymes were identified by polymerase chain reaction using primers for bla(IMP), bla(VIM), and bla(SPM), followed by gene sequencing. Genetic similarity among the carbapenem-resistant strains was evaluated by automated ribotyping. RESULTS: Only colistin and ampicillin-sulbactam showed reasonable in vitro activity against carbapenem-resistant isolates (97% and 74% of isolates susceptible, respectively). More than half of the isolates (55%) had a positive MBL phenotypic test result and a positive polymerase chain reaction result for bla(IMP-1). The proportion of IMP-1-producing Acinetobacter isolates among carbapenem-resistant strains increased from 0% in the 1993-1997 period to 29% in 1998 and 100% in the 1999-2001 period. No carbapenem-resistant Acinetobacter isolates that harbored bla(VIM) or bla(SPM) were detected. Molecular typing results revealed 20 ribogroups among carbapenem-resistant isolates. During the study period of 1994-2001, we identified 2 major ribogroups, 52-1 (MBL-negative and MBL-positive strains) and 60-7 (MBL-positive strains), that had a coefficient of similarity of 0.85 or higher. CONCLUSIONS: Our results indicate that IMP-1-producing strains of Acinetobacter emerged in our institution in 1998. Since then, production of this MBL was detected not only in the major ribogroups of carbapenem-resistant Acinetobacter species but also among isolates that belonged to 17 distinct ribogroups, indicating that this important mechanism of antimicrobial resistance was disseminated among distinct clones.     

An in silico approach for understanding the molecular evolution of clinically important metallo-beta-lactamases

Resistance to carbapenem, considered to be the drug of last resort for treating serious enterobacterial infections, is a growing problem. Metallo-beta-lactamase (MBL) mediated carbapenem resistance is considered to be clinically most important, since no traditional inhibitors work against them. Hence, we have investigated the changes in drug profile, affinity and binding stability of meropenem with clinically important MBLs viz., IMP, VIM and NDM during the course of molecular evolution. Phylogenetic trees were constructed and amino acids positions, presumed to be exposed to positive selection pressure were analyzed. Based on sequence diversity and selection pressure, IMP-1, IMP-8, IMP-9, IMP-21, IMP-27, IMP-20 and IMP-26 among IMP genes; VIM-1, VIM-2, VIM-13, VIM-29, VIM-18 and VIM-7 among VIM genes and NDM-1 had been selected for in silico analysis. Mode of interaction of selected MBL variants with meropenem were analyzed by Autodock4.0 and LIGPLOT analysis. In all the trajectories, we had found an increase in mouth opening/solvent accessible volume/area of the catalytic pocket and decrease in Gibbs’ free energy (ΔG°) for binding with meropenem and Michealis–Menten constant (K_m) – indicating an increase in choice of drugs, binding stability and meropenem affinity from primitive to advanced MBL variants, with exceptions of IMP-20, IMP-26, VIM-13 and VIM-18 which might be due to sign epistasis. Intergenic comparison revealed NDM-1 might have greater drug profile and catalytic efficiency than IMP-1 and VIM-2 due to largest pocket opening and least distance between the Zn-I ion and β-lactam oxygen of meropenem.     

Emergence of ceftriaxone-resistant Salmonella isolates and rapid spread of plasmid-encoded CMY-2-like cephalosporinase,Taiwan

Of 384 Salmonella isolates collected from 1997 to 2000 in a university hospital in Taiwan, six ceftriaxone-resistant isolates of Salmonella enterica serovar Typhimurium were found in two patients in 2000. The resistance determinants were on conjugative plasmids that encoded a CMY-2-like cephalosporinase. During the study period, the proportion of CMY-2-like enzyme producers among Escherichia coli increased rapidly from 0.2% in early 1999 to >4.0% in late 2000. Klebsiella pneumoniae isolates producing a CMY-2-like beta-lactamase did not emerge until 2000. The presence of bla(CMY)-containing plasmids with an identical restriction pattern from Salmonella, E. coli, and K. pneumoniae isolates was found, which suggests interspecies spread and horizontal transfer of the resistance determinant. Various nosocomial and community-acquired infections were associated with the CMY-2-like enzyme producers. Our study suggests that the spread of plasmid-mediated CMY-2-like beta-lactamases is an emerging threat to hospitalized patients and the public in Taiwan.     

Phylogenetic analysis of carbapenem-resistant Acinetobacter baumannii isolated from different sources using Multilocus Sequence Typing Scheme

The emergence and worldwide distribution of carbapenem-resistant Acinetobacter baumannii strains has become a major public health threat. The objective of this study was to investigate the clonal relatedness of A. baumannii isolates collected from clinical and extra-hospital environments in Mthatha, South Africa. Forty carbapenem-resistant isolates comprising of clinical (20) and extra-hospital (20) were identified and tested for antimicrobial susceptibility. Detection of carbapenemase encoding genes was performed by Real-time PCR. The clonal relationship of clinical isolates relative to extra-hospital isolates was determined via multilocus sequence typing (MLST). All isolates (clinical and extra-hospital) were resistant to most common antibiotics including carbapenems (imipenem; MIC ≥32 μg/mL and meropenem; MIC ≥32 μg/mL) with the only exception being amikacin (with 3 isolates susceptible), tigecycline (14 isolates susceptible) and colistin (all isolates susceptible). The bla OXA-23-like and the intrinsic bla OXA-51 -like genes were detected in all the isolates tested. The bla OXA-58-like and bla IMP-type genes were detected in 2 clinical isolates whilst the bla OXA-24-like, bla VIM-type, bla NDM-1, bla SIM, and bla AmpC were not detected. The bla OXA-24-like, bla OXA-58-like, bla IMP-type, bla VIM-type, bla NDM-1, bla SIM, and bla AmpC were negative in the extra-hospital isolates. Co-occurrence of bla OXA-23 -like, bla OXA-58-like and bla IMP-type was observed in 2 clinical isolates. The MLST performed on 33 isolates identified 5 existing sequence types (ST) (ST1, ST2, ST25, ST85 and ST215) in clinical isolates and 2 existing STs (ST1 and ST2) in extra-hospital isolates. The most dominant ST was ST2 accounting for 68.8% of the clinical isolates and 82.4% of the extra-hospital isolates. The study demonstrated high prevalence and potential clonal spread of globally-disseminated clonal complex 2 carrying bla OXA-23-like within our local settings. However, ST25 might be an emerging lineage carrying the bla OXA-23-like . Continuous monitoring is important in limiting the spread of these strains in other healthcare settings and the community.     

Clinical surveillance of surgical imipenem-resistant Pseudomonas aeruginosa infection in a Japanese hospital

In order to elucidate any changes in imipenem-resistant Pseudomonas aeruginosa (IRPA) infections in Japan, we examined 511 P. aeruginosa stains isolated from our surgical ward between 1987 and 2001. These isolates were subjected to susceptibility testing against various antipseudomonal agents including imipenem, meropenem, ceftazidime, gentamicin and ciprofloxacin. They were serotyped with the slide agglutination test and genotyped using pulsed-field gel electrophoresis (PFGE). The annual incidences of IRPA infections were particularly high in the early 1990s. Epidemiological investigations revealed that these outbreaks were due to dissemination of hospital-acquired IRPA isolates. Intensive use of imipenem promoted the selection of highly resistant strains. Further study of resistance mechanisms revealed that none of the 110 IRPA strains were metallo-beta-lactamase (MBL) producers. Polymerase chain reaction (PCR) analysis using bla(IMP) specific primers confirmed that no IMP-1 type MBL gene-positive strains were detected from our ward. Susceptibilities of those IRPA strains against other antipseudomonal agents showed relatively low levels, suggesting that imipenem resistance was mainly due to impermeability of the OprD porin. In conclusion, hospital-acquired outbreaks of IRPA were recently reduced by guidelines for, and surveillance of, appropriate use of antimicrobial agents. When the rate of IRPA isolation increases, serotyping should be performed initially and PFGE is required to confirm outbreaks. A computer-assisted genotyping technique is available to perform epidemiological studies of IRPA isolates.     

Genetic analysis of antibiotic-resistance determinants in multidrug-resistant Shigella strains isolated from Chilean children

A total of 162 clinical isolates of Shigella collected from children in a semi-rural community of Chile were examined for the presence of genetic determinants of resistance to ampicillin, chloramphenicol, tetracycline, and trimethoprim. Ampicillin resistance was most frequently associated with the presence of bla(OXA) in S. flexneri and with bla(TEM) in S. sonnei. The bla(OXA) gene but not bla(TEM) was located in class 1 integrons. The dhfrIa gene encoding for resistance to trimethoprim was associated to class 2 integrons and detected exclusively in S. flexneri, whereas dhfrIIIc was found in all S. sonnei strains and in 10% of the S. flexneri isolates. Cat, coding for choramphenicol resistance, and bla(OXA) genes were located in the chromosome in all cases, whereas tetA gene, coding for tetracycline resistance, and bla(TEM), dhfrIa and dhfrIIIc genes were found either in the chromosome or in conjugative plasmids. Our results show a heterogenous distribution of antibiotic-resistance determinants between S. flexneri and S. sonnei.     

重症监护病房耐亚胺培南铜绿假单胞菌耐药机制和分子分型的研究

目的 分析重症监护病房(intensive care unit,ICU)耐亚胺培南铜绿假单胞菌的耐药特征及其耐药机制. 方法 收集2018年1月-2019年9月广州市某医院ICU分离的37株耐亚胺培南铜绿假单胞菌(imipenem resistant Pseudomonas aeruginosa,IRPA),使用微生物...     

多药耐药鲍氏不动杆菌与铜绿假单胞菌对头孢哌酮/舒巴坦的敏感性研究

目的 比较多药耐药鲍氏不动杆菌与多药耐药铜绿假单胞菌产金属β-内酰胺酶的情况和对头孢哌酮/舒巴坦的耐药性,为临床治疗该细菌感染提供实验室依据.方法 用VITEK-32、GNS-132系统常规检测非重复鲍氏不动杆菌42株、铜绿假单胞菌95株,同时用Etest检测金属β-内酰胺酶,用纸片扩散法(K-B法)检测头孢哌酮/舒巴坦和多黏菌素E的药敏试验,分析二者不同耐药表型对头孢哌酮/舒巴坦的敏感性差异.结果 鲍氏不动杆菌、铜绿假单胞菌对亚胺培南耐药率分别为66.7%、31.6%;多药耐药鲍氏不动杆菌、铜绿假单胞菌均有较‘多产金属β-内酰胺酶株;头孢哌酮/舒巴坦耐对亚胺培南非产金属β-内酰胺酶的敏感性差异无统计学意义,头孢哌酮/舒巴坦对耐亚胺培南产金属β-内酰胺酶的敏感性差异有统计学意义(P<0.05).结论 多药耐药鲍氏不动杆菌对头孢哌酮/舒巴坦敏感性高于多药耐药铜绿假单胞菌,治疗耐亚胺堵南的多药耐药鲍氏不动杆菌可选用头孢哌酮/舒巴坦,治疗耐亚胺培南的多药耐药铜绿假单胞菌特别是MBLs应选用多黏菌素E或联合用药.     

老年住院患者产金属酶耐亚胺培南铜绿假单胞菌的耐药监测

目的分析老年住院患者感染产金属酶的铜绿假单胞菌耐药现状,指导临床合理使用抗菌药物。方法采用法国生物梅里埃公司的API微生物自动鉴定系统,对医院2008-2010年,老年病房住院患者的各类临床标本进行细菌分离,药敏试验采用纸片扩散法进行检测;用双纸片协同法检测铜绿假单胞菌产金属酶。结果分离的1056株铜绿假单胞菌中耐亚胺培南的菌株有205株,检出率为19.4%,其中产金属酶的有57株,阳性率为27.8%;耐亚胺培南铜绿假单胞菌主要分布于老年重症病房、老年呼吸内科、老年神经内科,耐亚胺培南铜绿假单胞菌与亚胺培南敏感铜绿假单胞菌耐药性有明显差异。结论加强金属β-内酰胺酶检测,按照药敏试验选择治疗药物,以防止菌株扩散是非常重要的。     

耐亚胺培南铜绿假单胞菌的耐药机制及亲缘性研究

目的 探讨广东省中医院4所分院临床分离的72株耐亚胺培南铜绿假单胞菌的多药耐药性、主要耐药机制和亲缘性.方法 采用聚合酶链反应检测Ⅰ类整合子遗传标记intⅠ1,外膜通道蛋白基因oprD2,β-内酰胺酶GES、KPC、IMP-1、IMP-9 、VIM-1 、VIM-2、SIM、SPM、GIM、AIM、NDM、KHM、DHA、PDC、OXA-40、OXA-23等18种基因,并对所获得的结果进行多基因聚类分析.结果 72株铜绿假单胞菌中IMP-9、PDC、oprD2、intⅠ1基因阳性率较高,分别为70.8％、98.6％、86.1％、100.0％,KPC、IMP-1、VIM-2、DHA基因阳性率较低,分别为2.8％、9.7％、13.9％、18.1％,GES、VIM-1、SIM、SPM、GIM、AIM、NDM、KHM、OXA- 40、OXA- 23基因均为阴性;72株铜绿假单胞菌有52株产金属酶;多基因聚类分析结果显示,72株铜绿假单胞菌可分3群,每群均存在克隆传播.结论 产生金属酶是72株铜绿假单胞菌对亚胺培南耐药的最主要原因;多基因聚类分析法对临床治疗和医院感染的预防控制具有指导意义.     

A survey of β-lactamase and 16S rRNA methylase genes among fluoroquinolone-resistant Escherichia coli isolates and their horizontal transmission in Shandong, China

The prevalence of β-lactamase, 16S rRNA methylase genes, and plasmid-mediated fluoroquinolone-resistance (PMQR) determinants (qnrC and qnrD) was determined by polymerase chain reaction in fluoroquinolone-resistant Escherichia coli isolated from a chicken farm, a pig farm, and a hospital in Shandong, China in 2007. The bla(TEM) and bla(CTX-M) were the most prevalent β-lactamase genes in isolates from chickens (88.4%, 175/198 and 81.3%, 161/198) and hospitalized patients (87.8%, 122/139 and 69.1%, 96/139). The bla(TEM) was the most prevalent β-lactamase gene observed in isolates from pigs (98.5%, 135/137). The gene bla(CMY-2) was also predominant among isolates from chickens (20.2%, 40/198). The bla(LAP-1) gene was first detected in one strain from chickens and humans (pig farm workers) in China. Only one strain from hospitalized patients was found to possess bla(SHV). The rmtB was the most prevalent 16S rRNA methylase gene detected in isolates from chickens (19.7%, 39/198) and hospitalized patients (15.8%, 22/139). To our knowledge, this is the first report of the detection of the qnrD gene in E. coli from chickens and pigs in China. The qnrC and bla(KPC) genes were not detected in any of the isolates. Results of southern hybridization revealed that PMQR determinants, β-lactamases, and 16S rRNA methylase genes were located on the same plasmid in E. coli strains derived from patients. Also, PMQR determinants and β-lactamase genes were localized on the same plasmid in an E. coli strain of animal origin. Results of conjugation experiments revealed that all of these plasmid-based resistance genes can be transferred by conjugation through horizontal transmission.     

Molecular characterization of class b carbapenemases in advanced stage of dissemination and emergence of class d carbapenemases in Enterobacteriaceae from Croatia

Carbapenemases involved in acquired carbapenem resistance in Enterobacteriaceae belong to Ambler class A serin β-lactamases, class B metallo-β-lactamases (MBL) or class D OXA-48-like β-lactamases. The aim of the present study was to analyse the molecular epidemiology and the mechanisms and routes of spread of class B and class D carbapenemases in Croatia.In total 68 isolates were analyzed. Antibiotic susceptibility was determined by broth microdilution method. PCR was used to detect antibiotic-resistance genes. Genotyping was performed by rep-PCR and MLST.Sixty-five isolates were found to harbour VIM-1 carbapenemase, seven of which were positive also for NDM-1, while two strains harboured only NDM-1. OXA-48 was detected in three isolates, two of which coproduced VIM-1. Thirty-six strains possessed additional CTX-M-15 β-lactamase whereas 64 were positive for TEM-1. CMY was found in 18 Citrobacter freundii isolates and DHA-1 in one Enterobacter cloacae isolate. Four different plasmid-incompatibility groups were found: A/C, L/M, N and FIIAs. Unlike C. freundii and E. cloacae, Klebsiella pneumoniae showed high diversity of rep-PCR patterns. E. cloacae and C. freundii predominantly belonged to one large clone which was allocated to ST105 and ST24, respectively.Three different types of carbapenemases were identified showing the complexity of CRE in Croatia.     

泛耐药铜绿假单胞菌体外耐药模型构建及耐药机制的研究

目的 通过多种抗菌药物体外诱导构建泛耐药铜绿假单胞菌(PDRPA)体外模型,检测其相关的耐药机制;探讨联合抗菌药物体外诱导对铜绿假单胞菌( PAE)耐药性的影响.方法 采用梯度浓度琼脂平皿传代诱导法,将4株细菌接种于梯度浓度的药物平皿,直至对该药产生耐药,诱导前后测定细菌的敏感性,采用E-test法对诱导前的敏感株、诱导的泛耐药株以及临床分离的泛耐药菌株进行MBL检测；采用Real-time PCR技术,对诱导前的4株敏感PAE和诱导后的4株PDRPA以及4株临床分离的PDRPA进行主动外排系统的检测,包括:MexAB-OprM、MexCD-OprJ、MexEF-OprN、MexXY-OprM.结果 经诱导后4株敏感PAE对抗菌药物的敏感性下降,MIC明显升高,按照CLSI耐药折点4株菌株全部转变为泛耐药菌株,4株临床分离的敏感PAE株诱导前后MBL检测均为阴性;临床分离的4株PDRPA株MBL检测有2株为阳性,诱导前的4株敏感PAE,4个外排系统均无高表达；相对于诱导前,经过诱导成为泛耐药菌株之后,4株耐药诱导株均存在MexA-OprM外排系统高表达,有1株存在MexCD-OprJ和1株MexEF-OprN高表达,全部菌株均无MexXY-OprM高表达；在临床分离的PDRPA中,有1株所有4个主动外排系统均高表达,有1株只有MexAB-OprM高表达,另外3个外排系统无高表达,另外2株4个外排系统均无高表达.结论 在体外抗菌药物的压力下,敏感的PAE可以经诱导变为PDRPA,耐药性稳定；体外诱导的PDRPA非产金属β-内酰胺酶,其耐药性的产生可能与主动外排泵有关,尤其是mexAB-OprM.