Metallo-beta-lactamase VIM-2 in clinical isolates of Pseudomonas aeruginosa from Portugal

Resistance to carbapenems is emerging, and it is a great problem to therapeutics. Three isolates of Pseudomonas aeruginosa from a Portuguese hospital identified in urine and sputum, in 1995, presented a high-level resistance to imipenem (> 32 mg/L). Afterward, one isolate of P. aeruginosa recovered from urine of an ambulatory patient in 1998 showed high resistance to imipenem and meropenem. The resistance to carbapenems in these strains was associated with the production of a class B beta-lactamase, as was demonstrated by imipenem hydrolysis and inhibition by EDTA. Using primers described for bla(IMP) and bla(VIM), the amplification of the latter was observed in all isolates and a VIM-2 metallo-enzyme was identified. The pulsed-field gel electrophoresis (PFGE) patterns of these isolates were indistinguishable, suggesting dissemination to the community of this VIM-2 producer.     

Evaluation of imipenem/imipenem+EDTA disk method for detection of metallo-beta-lactamase-producing Klebsiella pneumoniae isolated from blood cultures

The objective of this study was to evaluate the imipenem (IMP) and IMP+EDTA (IMP/IMP+EDTA) disk method for the detection of metallo-beta-lactamases (MBLs) in clinical isolates of Klebsiella pneumoniae with various MIC levels to IMP. Forty-one blood isolates of K. pneumoniae with MIC to IMP ranging from < or =0.5 to > or =16 microg/ml were examined. The MICs were determined by VITEK-2 (bioMerieux Vitek two, France). Disks of 10 microg IMP with and without the addition of 0.5 M EDTA were used for the IMP/IMP+EDTA disk method. The E-test (AB Biodisk, Solna, Sweden) for MBL detection was also used. All isolates were examined for the bla (VIM-1) gene by PCR and for clonality of VIM-1-producing isolates by pulsed-field gel electrophoresis (PFGE). All isolates with MIC values of IMP < or =0.5 microg/ml exhibited no differences in inhibition zone diameters (IZD) produced by IMP and IMP+EDTA disks, whereas the isolates with MICs > or =1 microg/ml showed an increase in IZD, ranging from 8 to 26 mm. All isolates with MIC values of > or =1 microg/ml were found positive for the bla (VIM-1) gene by PCR and for MBL production by the E-test, whereas none of isolates with MICs <0.5 microg/ml was found positive by any of the tests. DNA restriction fragments generated by PFGE of VIM-1-producing isolates were classified in four main types. The IMP/IMP+EDTA disk method is simple to perform, sensitive, and specific for detection of MBL-producing K. pneumoniae clinical isolates. K. pneumoniae isolates with MICs of IMP > or =1 microg/ml and/or IZD produced by IMP disk <19 mm should be tested for MBL production.     

Outbreak of infections caused by Pseudomonas aeruginosa producing VIM-1 carbapenemase in Greece

Resistance to imipenem and meropenem was observed in 211 (16.5%) isolates of Pseudomonas aeruginosa recovered in a Greek university hospital during 1996 to 1998. In six isolates selected from throughout this period, high-level resistance to both carbapenems (MICs >/= 128 microg/ml) was associated with production of the class B beta-lactamase VIM-1. bla(VIM)-bearing isolates belonged to serotype O:12 and were indistinguishable by pulsed-field gel electrophoresis.     

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.     

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.     

Molecular typing indicates an important role for two international clonal complexes in dissemination of VIM-producing Pseudomonas aeruginosa clinical isolates in Hungary

VIM metallo-beta-lactamase-producing serotype O11 or O12 Pseudomonas aeruginosa isolates infecting or colonising 19 patients from seven hospitals were reported in Hungary between 2003 and 2005. In this study we characterised VIM-producing Pseudomonas spp. clinical isolates from two novel locations in Hungary; we identified three new bla(VIM) carrying integron types and the presence of the bla(VIM-2) allele in Hungary. By applying various typing techniques, including multilocus sequence typing, we revealed an important role of two international clonal complexes, CC4 and CC11, in the dissemination of bla(VIM)-positive P. aeruginosa in hospitals in Hungary. Isolate P12-Q, a representative strain from France of the major European multiresistant P12 clone, displayed ST111 which, according to eBURST analysis, is the presently calculated founder sequence type of CC4. This is in accordance with the wide geographic distribution of the P12 clone. Our data indicate that, although the CC4 clonal complex includes serotype O1 and O6 isolates as well, it also contains the P12 clone. We characterised a P. aeruginosa nosocomial clone with a singleton sequence type (ST313), that may have acquired bla(VIM-2) and bla(VIM-4) gene cassettes from a yet unidentified local gene pool in Hungary.     

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).     

Molecular epidemiology of clinical Pseudomonas aeruginosa isolates carrying IMP-1 metallo-beta-lactamase gene in a University Hospital in Turkey

Pseudomonas aeruginosa isolates carrying IMP- or VIM-type metallo-beta-lactamase (MBL) have been increasingly reported in hospitals worldwide. One hundred P. aeruginosa clinical isolates from unrelated inpatients hospitalized at a Turkish university hospital were screened for the presence of bla(IMP) and bla(VIM) genes by polymerase chain reaction (PCR). One (1%) isolate was found to carry a VIM-type MBL gene, whereas nine (9%) carried an IMP-1 MBL gene carried on a cassette inserted into a class 1 integron. Only four of the IMP producers were detected as MBL producers according to E-test MBL. Minimum inhibitory concentrations (MICs) of imipenem for the IMP-1 and VIM-type MBL-producers were highly variable (MIC values, 8-128 mug/ml). Imipenem resistance was not plasmid-mediated according to the transformation assays. Piperacillin/tazobactam was the only effective drug in antimicrobial susceptibility testing. No aztreonam-resistant IMP and VIM producers were detected to produce an extended-spectrum beta-lactamase (ESBL). Three class 1 integrons of approximately 2,300 bp, 1,800 bp, and 1,500 bp in size were detected in each of the nine IMP-positive isolates. Sequencing revealed three novel gene cassette arrays, aac(3)-1c-cmlA5, bla(IMP-1)-aadA7-like, and aacA7-smr-2-orfD. Enterobacterial repetitive intergenic consensus PCR (ERIC-PCR) indicated that a clonal spread of IMP-1-producers had occurred in this hospital.     

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.     

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   

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.     

VIM-1 Metallo-beta-lactamase-producing Klebsiella pneumoniae strains in Greek hospitals

Seventeen Klebsiella pneumoniae clinical isolates carrying the bla(VIM-1) metallo-beta-lactamase gene were collected in the intensive care units of three hospitals in Athens, Greece, in 2002. They exhibited various carbapenem resistance levels (Etest MICs of imipenem ranged from 4 to 32 microg/ml). All isolates gave positive results by the imipenem-EDTA synergy Etest. The isolates were classified into four main types by pulsed-field gel electrophoresis; the majority of the isolates (5 and 10 isolates) belonged to two types. The bla(VIM-1) gene cassette was part of the variable region of a class 1 integron that also included aac6, dhfrI, and aadA. This structure was carried by transferable plasmids.     

First outbreak of multidrug-resistant Klebsiella pneumoniae producing both SHV-12-type extended-spectrum beta-lactamase and DHA-1-type AmpC beta-lactamase at a Korean hospital

PURPOSE: Coexistence of different classes of beta-lactamases in a single bacterial isolate may pose diagnostic and therapeutic challenges. We investigated a spread of Klebsiella pneumoniae isolates co-producing an AmpC beta-lactamase and an extended-spectrum beta-lactamase (ESBL) in a university hospital. MATERIALS AND METHODS: Over a three-month period, a total of 11 K. pneumoniae isolates, which exhibited resistance to cefotaxime, aztreonam, and cefoxitin, were isolated. These isolates showed positive to ESBLs by double disk tests. Minimal inhibitory concentrations (MICs) were determined by broth microdilution testing. All isolates were examined by isoelectric focusing, PCR and sequence analysis to identify bla(SHV) and bla(DHA), and molecular typing by pulsed-field gel electrophoresis (PFGE). RESULTS: All 11 isolates were highly resistant (MIC, > or = 128microg/ml) to ceftazidime, aztreonam, and cefoxitin, while they were susceptible (MIC, < or = 2microg/ml) to imipenem. The bla(SHV-12) and bla(DHA-1) genes were detected by PCR and sequence analysis. PFGE revealed a similar pattern in 10 of the 11 strains tested. CONCLUSION: This is the first outbreak report of K. pneumoniae in Korea which co-produced SHV-12 and DHA-1 beta-lactamase, and we suggest a clonal spread of multidrug-resistant K. pneumoniae at a hospital.     

Detection of carbapenemase-producing Acinetobacter baumannii in a hospital

Acinetobacter baumannii strains resistant to both imipenem (IPM) and ceftazidime (CAZ) were isolated from 1994 through 1996 at Gunma University Hospital. Nine isolates from different inpatients were examined for carbapenem-hydrolyzing activity and for the carbapemase gene bla(IMP) by the PCR method. All nine isolates were carbapenemase-producing strains that hydrolyzed IPM and that harbored bla(IMP). The bla(IMP) gene was transmissible by conjugation to an IPM-susceptible recipient strain of A. baumannii and conferred resistance to IPM, CAZ, cefotaxime (CTX), ampicillin (AMP), and piperacillin (PIP). Either intermediate or high-level resistance to amikacin (AMK) was transferred from two and five strains, respectively, concomitantly with bla(IMP), and gentamicin (GEN) resistance was also transferred in one instance of high-level AMK resistance. Comparative examination of clinical isolates for resistance patterns to nine drugs, IPM, CAZ, CTX, aztreonam, AMP, PIP, AMK, GEN, and norfloxacin, in addition to pulsed-field gel electrophoresis patterns with NotI-digested genomic DNA, confirmed nosocomial transmission of infections involving carbapenemase-producing A. baumannii strains.     

Molecular epidemiology of a clonal outbreak of multidrug-resistant Acinetobacter baumannii in a university hospital in Italy

This study investigated the molecular epidemiology of a clonal outbreak of multidrug-resistant Acinetobacter baumannii that occurred between June 2003 and June 2004 in a tertiary-care hospital in Naples, Italy. A. baumannii was isolated from 74 patients, of whom 38 were infected and 36 were colonised. Thirty-three patients had ventilator-associated pneumonia, three had hospital-acquired pneumonia, and two had sepsis. Genotypic analysis of 45 available A. baumannii isolates revealed two distinct pulsed-field gel electrophoresis (PFGE) patterns. Of these, PFGE pattern 1 was represented by isolates from 44 patients and was identical to that of an epidemic A. baumannii clone isolated in another hospital of Naples during 2002. All A. baumannii isolates of PFGE type 1 showed identical multiresistant antibiotypes, characterised by resistance to all antimicrobial agents tested, including carbapenems, with the exception of colistin. In these isolates, inhibition of OXA enzymes by 200 mM NaCl reduced the imipenem MIC by up to four-fold. Molecular analysis of antimicrobial resistance genes showed that all A. baumannii isolates of PFGE type 1 harboured a class 1 integron containing the aacA4, orfX and bla(OXA-20) gene cassettes, an ampC gene and a bla(OXA-51)-like allele. Moreover, a bla(OXA-58)-like gene surrounded by the regulatory elements ISAba2 and ISAba3 was identified in a 30-kb plasmid from A. baumannii isolates of PFGE type 1, but not PFGE type 2. Thus, selection of a single A. baumannii clone producing an OXA-58-type carbapenem-hydrolysing oxacillinase was responsible for the increase in the number of A. baumannii infections that occurred in this hospital.     

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.     

Presence of Pseudomonas putida strains harboring plasmids bearing the metallo-beta-lactamase gene bla(IMP) in a hospital in Japan

To determine the persistence and spread of antibiotic-resistant strains in Gunma University Hospital, 83 Pseudomonas putida strains (each from a different patient) were isolated from January 1997 through December 2001. Of the 83 strains isolated, 27 were resistant to carbapenems. All 27 produced metallo-beta-lactamase and were found to be PCR positive for the bla(IMP) gene. Most (22 strains) were primarily isolated from the wards (W7 [9 strains] and W4 [8 strains]). Another five bla(IMP)-positive P. putida strains from wards W7 and W4 were obtained by swabbing around the water pipes. A total of 32 bla(IMP)-positive P. putida strains were assessed by pulsed-field gel electrophoresis (PFGE) and testing of drug susceptibility to 10 chemotherapeutic agents. Both PFGE and MIC patterns revealed that there were long-term resident strains among inpatients and hospital environments. The bla(IMP) genes of 22 of 32 strains were all transferable to a recipient strain of Pseudomonas aeruginosa by conjugation or transformation and conferred resistance to carbapenems and cephems. The bla(IMP) plasmids were conjugally transmissible among P. aeruginosa strains and mediated resistance to amikacin as well as beta-lactams. Ten of the 22 plasmids mediated additional resistance to gentamicin and tobramycin. Plasmids with identical DNA and drug resistance patterns were found in P. putida strains with identical PFGE patterns and with different PFGE patterns. We presumed that P. putida was one of the resident species in inpatients and especially in hospital environments, spreading drug resistance genes via plasmids among P. putida strains and supplying them to more pathogenically important species, such as P. aeruginosa.     

Simple microdilution test for detection of metallo-beta-lactamase production in Pseudomonas aeruginosa

A microdilution test measuring imipenem MICs in the presence or absence of a mixture of EDTA plus 1,10-phenanthroline was developed and tested on 190 Pseudomonas aeruginosa isolates, including 18 VIM- and 4 IMP-type metallo-beta-lactamase (MBL) producers. The chelator mixture reduced by fourfold or more the imipenem MICs for MBL producers, while a lower effect or no effect was usually observed with MBL nonproducers.