IMPs, VIMs and SPMs: the diversity of metallo-β-lactamases produced by carbapenem-resistant Pseudomonas aeruginosa in a Brazilian hospital

Pseudomonas aeruginosa isolates (n=183), collected from bacteraemic patients hospitalised in Sao Paulo Hospital (Brazil) during 2000-2001, were screened for susceptibility to antimicrobial agents. The polymyxins were the most active compounds (100% susceptibility), followed by amikacin and cefepime (59.0%), meropenem (57.4%), and imipenem and gentamicin (55.2%). Imipenem-resistant isolates were ribotyped and screened for production of metallo-beta-lactamases (MBLs) by PCR with primers for bla(IMP), bla(VIM) and bla(SPM). MBL production was detected in 36 isolates (19.7% of the entire collection; 43.9% of the imipenem-resistant isolates) and the MBLs included SPM-1-like (55.6%), VIM-2-like (30.6%) and IMP-1-like (8.3%) enzymes.     

Outbreak of Infection with Multidrug-Resistant Klebsiella pneumoniae Carrying blaIMP-8 in a University Medical Center in Taiwan

Klebsiella pneumoniae strains with the transferable carbapenem-hydrolyzing metallo-beta-lactamases, which include IMP- and VIM-type enzymes, remain extremely rare. To investigate whether IMP- or VIM-producing K. pneumoniae isolates had spread at a university medical center in Taiwan, a total of 3,458 clinical isolates of K. pneumoniae consecutively collected in 1999 and 2000 were tested by the agar diffusion method, colony hybridization, PCR, and nucleotide sequencing. A total of 40 isolates (1.2%), or 17 nonrepetitive isolates, from 16 patients were found to carry bla(IMP-8), a metallo-beta-lactamase gene recently identified from a K. pneumoniae strain in Taiwan. Carriage of bla(VIM) or other bla(IMP) genes was detected in none of the remaining isolates. Of the 17 nonrepetitive bla(IMP-8)-positive isolates, 15 isolates (88.2%) appeared susceptible to imipenem (MICs, 相似文献   

Spread of integron-associated VIM-type metallo-beta-lactamase genes among imipenem-nonsusceptible Pseudomonas aeruginosa strains in Greek hospitals

Fifty-eight imipenem-nonsusceptible (MIC >or= 8 microg/ml) Pseudomonas aeruginosa strains isolated during May 2001 in 15 Greek hospitals were studied. Thirty-six isolates derived from nine hospitals carried VIM-type metallo-beta-lactamase genes, as found by PCR. In 34 isolates, bla(VIM) was associated with class 1 integrons of various sizes. DNA sequencing indicated the presence of bla(VIM-2) gene cassettes in a variety of integron structures. Random amplified polymorphic DNA typing suggested diversity of the bla(VIM)-positive strains. Synergy between 2-mercaptoacetic acid and imipenem indicated carbapenemase activity in 26 bla(VIM)-positive strains.     

VIM and IMP metallo-β-lactamases and other extended-spectrum β-lactamases in Escherichia coli and Klebsiella pneumoniae from environmental samples in a Tunisian hospital

An extremely drug-resistant Enterobacteriaceae species emerged in Kasserine Hospital, Tunisia between 2009 and 2010 causing a local outbreak. We aimed to characterize extended-spectrum β-lactamase (ESBL) and metallo-β-lactamase (MBL)-producing Enterobacteriaceae from the hospital environment. Swabs were collected from ten different wards from Kasserine Hospital, Tunisia. A total of 46 isolates were cultured onto MacConkey agar supplemented with ceftazidime to select for ESBL-producing Enterobacteriaceae. Identification and susceptibility patterns were performed using Phoenix-automated phenotypic identification criteria. Extended spectrum β-lactamases (ESBLs) were detected using cefepime ESBL E-test. Colony blotting was first used to detect the occurrence of bla(SHV) , bla(CTX-M) , bla(CMY) , bla(IMP) , and bla(VIM) genes. PCR was used to amplify these genes, and the amplicons were sequenced and analyzed. Total DNA was digested with XbaI, and PFGE was used to type the major isolates that produced IMP-1. Among the 46 isolates, 63% were Klebsiella pneumoniae, 13% were Escherichia coli, 8.7% were Proteus mirabilis, 6% were Enterobacter cloaceae, 4.3% were Providencia rettgeri, 2.5% were Serratia marcescens, and 2.5% were Pantoea agglomerans. PCR amplification and DNA sequencing showed that hospital environment isolates produced SHV-125, CTX-M-15, CMY-2 ESBLs, and IMP-1 and VIM-2 MBLs. PFGE typing showed the emergence of IMP-1 MBL-producing K. pneumoniae isolates that were not clonal. In this study, we report the first characterization of IMP-1 and VIM-2 MBL-producing K. pneumoniae and E. coli isolates collected from Kasserine Hospital, Tunisia.     

Specific detection of blaVIM and blaIMP metallo-β-lactamase genes in a single real-time PCR

This study describes the development of a real-time PCR protocol for rapid detection of the most common bla(VIM) (bla(VIM-1), bla(VIM-2), bla(VIM-3), bla(VIM-4), bla(VIM-5), bla(VIM-6), bla(VIM-10), bla(VIM-11), bla(VIM-12)) and bla(IMP) (bla(IMP-1), bla(IMP-2), bla(IMP-6), bla(IMP-8), bla(IMP-10), bla(IMP-15), bla(IMP-19), bla(IMP-20)) genes in a single reaction. The genes were specifically detected and clearly differentiated into four groups, i.e., (i) bla(VIM-1)-like (bla(VIM-1), bla(VIM-4), bla(VIM-5), bla(VIM-12)); (ii) bla(VIM-2)-like (bla(VIM-2), bla(VIM-3), bla(VIM-6), bla(VIM-10), bla(VIM-11)); (iii) bla(IMP-1)-like (bla(IMP-1), bla(IMP-6), bla(IMP-10)); and (iv) bla(IMP-2)-like (bla(IMP-2), bla(IMP-8), bla(IMP-15), bla(IMP-19), bla(IMP-20)), by melting curve analysis of the real-time PCR products. The protocol was used to screen positive bla(VIM-1), bla(VIM-2) and bla(IMP-1) control strains, 70 Gram-negative isolates resistant to carbapenems, and 30 Gram-negative isolates susceptible to carbapenems (negative controls).     

Distinct antimicrobial resistance patterns and antimicrobial resistance-harboring genes according to genomic species of Acinetobacter isolates

Using 58 isolates of Acinetobacter species recovered from a university hospital between August 2004 and March 2005, we performed genomic identification by amplified rRNA gene restriction analysis (ARDRA) and investigated the existence of metallo-beta-lactamase (MBL) producers and extended-spectrum beta-lactamase (ESBL) producers. Genomic species identification of Acinetobacter strains using ARDRA showed that 40 strains were genomic species 2 (Acinetobacter baumannii), 9 were 13 sensu Tjernberg and Ursing (13TU), 5 were Acinetobacter phenon 6/ct 13TU, and 4 were Acinetobacter genospecies 3. Among 58 strains, 13 isolates were MBL producers carrying bla(IMP-1) or bla(VIM-2) and 13 isolates were ESBL producers carrying bla(PER-1). Notably, the MBL producers were mostly 13TU, Acinetobacter phenon 6/ct 13TU, and Acinetobacter genospecies 3, which showed susceptibility to ciprofloxacin and ampicillin-sulbactam. However, 12 of 13 strains carrying bla(PER-1) were A. baumannii, showing multidrug resistance. The data revealed that the antimicrobial resistance patterns and resistance-harboring genes of Acinetobacter species are remarkably distinct according to the genomic species of Acinetobacter isolates.     

PCR typing of genetic determinants for metallo-beta-lactamases and integrases carried by gram-negative bacteria isolated in Japan, with focus on the class 3 integron

From January 2001 to December 2002, 587 strains of gram-negative bacterial isolates demonstrating resistance to ceftazidime and a combination of sulbactam and cefoperazone were subjected to a disk diffusion screening test using sodium mercaptoacetic acid; 431 strains (73.4%) appeared to produce metallo-beta-lactamase (MBL). Of these 431 strains, 357 were found by PCR to carry genes for IMP-1 type MBL (bla(IMP-1)), while only 7 and 67 strains carried the IMP-2 gene (bla(IMP-2)) and the VIM-2 gene (bla(VIM-2)), respectively. Neither VIM-1 nor SPM-1 type MBL genes were found among the strains tested. Of 431 strains, 427 carried the intI1 gene, and 4 strains carrying both the intI1 and intI3 genes were reidentified as Pseudomonas putida harboring bla(IMP-1). Of these four P. putida strains, three strains and one strain, respectively, were separately isolated from two hospitals located in the same prefecture, and the three strains showed very similar pulsed-field gel electrophoresis patterns. Of 357 bla(IMP-1) carriers, 116, 53, 51, 47, and 30 strains were identified as Pseudomonas aeruginosa, Alcaligenes xylosoxidans, P. putida/fluorescens, Serratia marcescens, and Acinetobacter baumannii, respectively. Four strains carrying bla(IMP-2) were reidentified as P. putida. Sixty-three P. aeruginosa strains and four P. putida strains carried bla(VIM-2). Of 427 intI1-positive strains, 180, 53, 51, 47, and 35 were identified as P. aeruginosa, A. xylosoxidans, P. putida/fluorescens, S. marcescens, and A. baumannii, respectively. In the present study, it was confirmed that strains carrying bla(IMP-1) with a class 1 integron are the most prevalent type in Japan, although several intI3 carriers have also been identified sporadically in this country.     

Nosocomial infections caused by multidrug-resistant isolates of pseudomonas putida producing VIM-1 metallo-beta-lactamase

Successful carbapenem-based chemotherapy for the treatment of Pseudomonas infections has been seriously hindered by the recent appearance of IMP- and VIM-type metallo-beta-lactamases, which confer high-level resistance to carbapenems and most other beta-lactams. Recently, multidrug-resistant Pseudomonas putida isolates for which carbapenem MICs were >/=32 micro g/ml were recovered from cultures of urine from three inpatients in the general intensive care unit of the Ospedale di Circolo, Varese, Italy. Enzyme assays revealed production of a metallo-beta-lactamase activity, while molecular analysis detected in each isolate a bla(VIM-1) determinant carried by an apparently identical medium-sized plasmid. Conjugation experiments were unsuccessful in transferring the beta-lactamase determinant to Escherichia coli or Pseudomonas aeruginosa. Macrorestriction analysis by pulsed-field gel electrophoresis demonstrated that the isolates were of clonal origin. PCR mapping and sequencing of the variable region of the plasmid-borne class 1 integron carrying the bla(VIM-1) determinant (named In110) showed that the bla(VIM-1)-containing cassette was identical to that previously found in strains of different species from other Italian hospitals and that the cassette array of In110 was not identical but clearly related to that of In70 (a bla(VIM-1)-containing plasmid-borne integron from an Achromobacter xylosoxidans isolate), pointing to a common origin of this cassette and to a related evolutionary history of their cognate integrons.     

Increasing prevalence of imipenem-resistant Pseudomonas aeruginosa and molecular typing of metallo-beta-lactamase producers in a Korean hospital

The types of metallo-beta-lactamases (MBLs), integrons, and genetic relatedness among Pseudomonas aeruginosa were investigated with a recent high prevalence of imipenem resistance in a Korean hospital. During 2000-2003, a total of 116 non-duplicate imipenem-resistant P. aeruginosa isolates were analyzed by PCR and DNA sequencing to detect of bla (IMP-1), bla (VIM-1), bla (VIM-2), bla (SPM-1), intI 1, intI 2, and intI 3 genes. Among them, MBL-producing isolates were evaluated for genetic relatedness using pulsed-field gel electrophoresis (PFGE) profiles. Of 116 isolates, 21 (18.1%) carried bla (VIM-2) gene with the intI 1 gene. Analysis of VIM-2 procuders by PFGE grouped 21 isolates into eight different clusters. Six of eight cluster I strains, all of four cluster II strains, and all of three cluster III strains were isolated in 2000, 2002, and 2003, respectively. Data concluded that P. aeruginosa carrying bla (VIM-2) with a class 1 integron was the only type among MBLs. A hospital outbreak by VIM-2 producers occurred annually, which could be at least a part of a recent high prevalence of imipenem resistance.     

Detection of Pseudomonas aeruginosa producing metallo-beta-lactamases in a large centralized laboratory

Metallo-beta-lactamases (MBLs) have been increasingly recognized from clinical isolates worldwide, but the laboratory detection of these strains is not well defined. We report a study that developed an EDTA disk screen test and a molecular diagnostic assay for the detection of MBL-producing Pseudomonas aeruginosa. Using NCCLS disk methodology, inhibition zone diameters were determined in tests with imipenem (IPM) and meropenem (MEM) disks alone and in combination with 930 microg of EDTA. This test was compared with the MBL Etest. The duplex PCR assay showed 100% sensitivity and specificity for detecting MBL-producing control strains. Of the 241 clinical strains of IPM-nonsusceptible P. aeruginosa from the Calgary Health Region isolated from 2002 to 2004, 110/241 (46%) were MBL positive using phenotypic methods while 107/241 (45%) were PCR positive for MBL genes: 103/241 (43%) for bla(VIM) and 4/241 (2%) for bla(IMP). The EDTA disk screen test using MEM showed 100% sensitivity and 97% specificity for detecting MBLs in control and clinical strains. The EDTA disk screen test is simple to perform and to interpret and can easily be introduced into the workflow of a clinical laboratory. We recommend that all IPM-nonsusceptible P. aeruginosa isolates be routinely screened for MBL production using the EDTA disk screen test and that PCR confirmation be performed at a regional laboratory.     

Evaluation of Etest MBL for detection of blaIMP-1 and blaVIM-2 allele-positive clinical isolates of Pseudomonas spp. and Acinetobacter spp

The Etest MBL (AB BIODISK, Solna, Sweden) correctly differentiated all 57 isolates of Acinetobacter spp. and Pseudomonas aeruginosa with the bla(IMP-1) allele and 135 of 137 (98.5%) Acinetobacter spp. and Pseudomonas spp. isolates with the bla(VIM-2) allele. The Etest MBL was reliable for detecting the IMP-1- and VIM-2-producing Pseudomonas and Acinetobacter isolates.     

Clonal spread of IMP-1-producing Pseudomonas aeruginosa in two hospitals in Singapore

Thirty-six isolates of carbapenem-resistant Pseudomonas aeruginosa were studied. Pulsed-field gel electrophoresis revealed the presence of two clones. One clone carried a bla(IMP-1) gene identical to that first described in Japan. The other clone carried a bla(IMP-1) variant containing four silent mutations. One isolate with a unique pulsed-field gel electrophoresis pattern contained bla(IMP-7).     

耐亚胺培南铜绿假单胞菌金属β-内酰胺酶和oprD2 基因的检测

目的: 了解我院临床分离下呼吸道感染的耐亚胺培南铜绿假单胞菌(IRPA) oprD2 基因和金属β-内酰胺酶(MBL)的分子流行病学情况。方法: 铜绿假单胞菌的鉴定和药敏采用VITEK-2系统进行药物的最低抑菌浓度(MIC)检测。应用双纸片增效法和聚合酶链反应(PCR)方法检测其产MBL的表型及相关的 oprD2 基因、IMP型和VIM型金属酶基因。结果: 157株亚胺培南铜绿假单胞菌对多种抗菌药物的耐药率均在40%以上,经双纸片增效法筛选出20株MBL阳性(12.7%),经PCR方法检测20株MBL阳性株中有13株IMP型基因阳性, oprD2 基因有67株阳性,其余90株缺失(57.3%),未发现VIM型基因。有10株IRPA既携带IMP型基因同时又有 oprD2 基因缺失。结论: 我院临床分离下呼吸道感染的耐亚胺培南铜绿假单胞菌呈较严重的多重耐药。IMP型基因是我院IRPA产MBL的主要基因型; oprD2 基因缺失可能是铜绿假单胞菌亚胺培南耐药的重要原因之一。     

Outbreak of carbapenem-resistant Pseudomonas aeruginosa producing VIM-8, a novel metallo-beta-lactamase, in a tertiary care center in Cali, Colombia

The prevalence of imipenem resistance among Pseudomonas aeruginosa isolates at a 195-bed tertiary care medical center in Cali, Colombia, rose from 2% in 1996 to 28% in 1997 and to over 40% in 2003. Many isolates showed high-level multiresistance, and phenotypic characterization suggested the spread of a predominant strain with minor variants. Sixty-six resistant isolates collected between February 1999 and July 2003 from hospitalized patients (n = 54) and environmental samples (n = 12) were subjected to a fuller analysis. Genetic fingerprints were compared by pulsed-field gel electrophoresis (PFGE) of SpeI-digested genomic DNA, and bla(IMP) and bla(VIM) genes were sought by PCR. PFGE and serotyping indicated that 52 of the 66 isolates belonged to a single strain, with 82% similarity; the PFGE pattern for this organism was designated pattern A. Two further pairs of isolates represented single strains; the remaining nine isolates were unique, and in the case of one isolate, no satisfactory PFGE profile could be obtained. The pattern A isolates were mostly of serotype O12 and were highly resistant to imipenem (MICs, 32 to >256 microg/ml), with this resistance decreased eightfold or more in the presence of EDTA. They yielded amplicons with bla(VIM)-specific primers, and sequencing of DNA from a representative isolate revealed bla(VIM-8), a novel allele with three polymorphisms compared with the sequence of bla(VIM-2). Two of these nucleotide changes were silent, but the third determined a Thr139Ala substitution. Only 4 of 13 resistant isolates (2 clinical isolates and 2 environmental isolates) assigned to other PFGE types carried bla(VIM) alleles, whereas the others were less multiresistant and mostly had lower levels of imipenem resistance (MICs, < or =32 microg/ml) which was not significantly reduced by EDTA. No bla(IMP) alleles were detected. During 2003, when the environmental study was undertaken, serotype O12 isolates with bla(VIM) were recovered from sinks and stethoscopes in the most-affected units, although not from the hands of staff; the problem declined once these reservoirs were disinfected and hygienic precautions were reinforced.     

Molecular evolution of metallo-beta-lactamase-producing Pseudomonas aeruginosa in a nosocomial setting of high-level endemicity

An outbreak of multidrug-resistant Pseudomonas aeruginosa strains producing VIM-type metallo-beta-lactamases (MBLs) has occurred in an Italian hospital since 2000 (C. Lagatolla, E. A. Tonin, C. Monti-Bragadin, L. Dolzani, F. Gombac, C. Bearzi, E. Edalucci, F. Gionechetti, and G. M. Rossolini, Emerg. Infect. Dis. 10:535-538, 2004). In this work, using molecular methods, we characterized 128 carbapenem-resistant isolates (including 98 VIM-positive isolates) collected from that hospital from 2000 to 2002 to investigate the dynamics of the dissemination of MBL producers in the clinical setting. Genotyping by random amplification of polymorphic DNA and pulsed-field gel electrophoresis showed that most VIM-positive isolates belonged to two different clonal lineages, producing either a VIM-1- or a VIM-2-like MBL, whose ancestors were detected for the first time in the hospital in 1999, suggesting that clonal expansion played a predominant role in the dissemination of these isolates. The 86 clonally related isolates carrying a blaVIM-1-like gene on an In70-like integron were clearly related to a VIM-1-positive P. aeruginosa clone circulating in various Italian hospitals since the late 1990s. VIM-negative P. aeruginosa strains related to the VIM-1-positive clone were detected during the same period, suggesting that the latter strain was derived from a clonal lineage already circulating in the hospital. In the VIM-2-like positive clone, the MBL gene was carried by an unusual class 1 integron, named In71, lacking the 3' conserved sequence region typical of sul1-associated integrons. A different class 1 integron with an original structure carrying a blaVIM-2 determinant, named In74, was detected in a sporadic isolate. A retrospective investigation did not reveal the presence of strains related to any of the VIM-producing isolates earlier than 1997.     

Local dissemination of multidrug-resistant Acinetobacter baumannii clones in a Thai hospital

A total of 83 Acinetobacter baumannii isolates from patients attending a tertiary care university hospital in Thailand were investigated for their clonal relatedness, antimicrobial susceptibility profiles, and integron carriage. Susceptibility profiles showed that 56 (67%) of these isolates exhibited multiple drug resistance (MDR). Pulsed-field gel electrophoresis (PFGE) showed that 73% of these resistant isolates were clustered into three predominant PFGE types: 6, 7, and 36. This suggested that the high number of isolates exhibiting MDR phenotypes observed in the hospital is, to some extent, due to the spread of these three resistant clones. Class 1 integrase genes were detected in all MDR isolates belonging to PFGE type 6, most MDR isolates belonging to PFGE type 7 and none of the isolates belonging to PFGE type 36. Five different class 1 gene cassette arrays, dfrA1-orfC, bla(IMP-14)-aac6', aacA4- catB8-aadA1, aacC1-orfX-orfX'-aadA1a, and aacC1-orfX-orfX-orfX'-aadA1a, were identified, of which the bla(IMP-14)-aac6' array has only been found in Thai isolates. Two isolates identified in this study carried a class 2 integrase gene with a 2.2 kb cassette array containing aadA1-sat-dfrA1.     

Detection and characterization of metallo beta lactamases producing Pseudomonas aeruginosa

This study was undertaken to evaluate phenotypic and genotypic methods for detection of Metallo-Beta-Lactamases (MBLs) among nosocomial Pseudomonas aeruginosa. Sixty one among 176 P. aeruginosa isolates, collected as part of a multicentric study (2005-2007), were evaluated for carbapenem resistance (CARB-R; resistant to either imipenem/meropenem) and screened for MBL by Combination Disk Diffusion Test (CDDT) using imipenem (IMP), meropenem (MER) and ceftazidime (CAZ) with EDTA. MBL positives were further confirmed by IMP + EDTA Etest. Twenty strains (42.6%) were found to be MBL producers among the 61 P. aeruginosa. PCR for IMP and VIM MBL was performed on 48 of the 61, 15 were positive for VIM MBL type. CDDT using IMP + EDTA had the highest sensitivity and specificity of 87.8% and 84.4% when compared to Etest, which was higher than the values obtained for CAZ + EDTA and MER + EDTA. CDDT using IMP + EDTA also compared very well with the PCR (specificity = 90.9%, sensitivity = 93.3%). CARB-R among P. aeruginosa is mediated predominantly via MBL production. Clinical P. aeruginosa isolates can be screened routinely using the less expensive IMP + EDTA CDDT in clinical microbiology laboratories.     

汕头地区耐亚胺培南铜绿假单胞菌耐药分析

目的：了解汕头地区对亚胺培南耐药的铜绿假单胞菌（PA）的耐药情况及耐药机制。方法：收集临床分离耐亚胺培南的PA共141株，双纸片协同实验检测金属酶表型，PCR法检测外膜孔蛋白OprD2和金属β内酰胺酶（IMP、VIM、SPM）基因。结果：耐亚胺培南的铜绿假单胞菌均为多重耐药茵，对头孢哌酮／舒巴坦的耐药率较低，未发现产金属酶菌株，仅22株菌株扩增出OprD2基因。结论：头孢哌酮／舒巴坦可作为本地区临床治疗耐亚胺培南铜绿假单胞茵所致感染的首选经验用药，OprDa表达减少或不表达可能是临床分离铜绿假单胞茵对亚胺培南耐药的主要机制。