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.     

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.     

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).     

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.     

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.     

Nosocomial spread of Pseudomonas aeruginosa isolates expressing the metallo-beta-lactamase VIM-2 in a hematology unit of a French hospital

Dissemination of metallo-beta-lactamases (carbapenemases) was investigated retrospectively among ceftazidimeand imipenem-resistant Pseudomonas aeruginosa isolates in a hematology unit in Marseilles, France, from September, 1995, to March, 1999. Sixteen clinical isolates and 23 environmental strains were identified, with a same bla (VIM-2) gene that encoded a carbapenemase identified in Southern Europe and South Korea. Five different genotypes were identified among clinical and environmental P. aeruginosa isolates all harboring an approximately 45-kb plasmid with bla (VIM-2)-positive class 1 integrons varying in structures. This study identified a hidden reservoir of carbapenemase producers.     

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.     

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.     

Outbreak caused by a multidrug-resistant Klebsiella pneumoniae clone carrying blaVIM-12 in a university hospital

From November 2006 to April 2007, nine nonrepetitive isolates of Klebsiella pneumoniae with reduced susceptibility or resistance to carbapenems were recovered from clinical specimens from separate patients hospitalized in a tertiary care hospital. The imipenem-EDTA synergy test was positive for all isolates. PCR, sequencing, and transferability experiments revealed the novel bla(VIM-12) metallo-beta-lactamase gene, which was plasmid mediated and located in a class 1 integron. Pulsed-field gel electrophoresis demonstrated a single macrorestriction pattern, indicating the clonal spread of VIM-12-producing K. pneumoniae.     

Diversity of β-lactamases in Pseudomonas aeruginosa isolates producing metallo-β-lactamase in two Tunisian hospitals

This study was conducted to identify the β-lactamase content of 30 metallo-β-lactamase-producing Pseudomonas aeruginosa isolated in 2007 from two Tunisian hospitals and to investigate their genetic relatedness. All these isolates produced VIM-2. bla(PER-1), bla(PSE-1), bla(OXA-2), and bla(OXA-10) were identified in 17, 5, 21, and 1 isolates, respectively. These enzymes were often associated in the same isolate: 26 isolates had at least two β-lactamases. The predominant serotype was O12. Pulsed-field gel electrophoresis revealed genetic diversity among the metallo-β-lactamase-producing P. aeruginosa isolates. This is the first report on the existence of bla(PER-1), bla(PSE-1), bla(OXA-2), and bla(OXA-10) in Tunisia.     

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.     

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.     

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.     

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.     

Fecal carriage of carbapenemase-producing Enterobacteriaceae: a hidden reservoir in hospitalized and nonhospitalized patients

Fecal carriage of carbapenemase-producing Enterobacteriaceae (CPE) has not been extensively investigated, except in the cases of selected patients at risk, mostly during outbreaks. A total of 1,100 fecal samples randomly collected in our institution in two different periods in 2006 (n = 600) and 2009-2010 (n = 500) from hospitalized (26.8%) and nonhospitalized (73.2%) patients were screened for CPE. The first period coincided with an outbreak of VIM-1-producing Enterobacteriaceae, and the second one coincided with the emergence of KPC enzymes in our hospital. Diluted samples in saline were cultured in Luria-Bertani broth with 1 μg/ml imipenem and subcultured in MacConkey agar plates with 4 μg/ml ceftazidime. Growing colonies were screened for CPE (modified Hodge test and EDTA and boronic acid synergy tests). Carbapenemase genes, plasmids in which they are located, and clonal relatedness were determined. Individuals who exhibited fecal carriage of CPE (11/1,043, 1.1%; 95% confidence interval [CI], 0.53 to 1.88) included 8 hospitalized (carriage rate, 2.9%; 95% CI, 1.24 to 5.55) and 3 nonhospitalized patients (carriage rate, 0.4%; 95% CI, 0.08 to 1.14), the latter being identified in 2009. Eighty-two percent of colonized patients were not infected with CPE. Isolates harboring bla(VIM-1) with or without bla(SHV-12) were identified as Klebsiella pneumoniae (n = 8; ST39, ST688, ST253, and ST163), Enterobacter cloacae (n = 3; two pulsed-field gel electrophoresis [PFGE] types), Escherichia coli (n = 2; ST155 and ST2441), and Citrobacter freundii (n = 1). Some of these lineages had previously been detected in our institution. The bla(VIM-1) gene was a member of the class 1 integrons In110 (bla(VIM-1)-aacA4-aadA1) and In113 (bla(VIM-1)-aacA4-dhfrII) located on plasmids IncN (n = 11; 30 to 50 kb) and IncHI2 (n = 3; 300 kb), respectively. Dissemination of bla(VIM-1) class-1 integrons within highly transferable plasmids in a polyclonal population has potentially contributed to the maintenance and spread of CPE.     

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.