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Moreno Galleni - One of the best experts on this subject based on the ideXlab platform.

  • Clonal diversity and Metallo-Beta-Lactamase production in clinical isolates of Stenotrophomonas maltophilia.
    Microbial drug resistance (Larchmont N.Y.), 2002
    Co-Authors: Paola Sandra Mercuri, Nicola Franceschini, Gian Maria Rossolini, Jeanmarie Frere, Yoshikazu Ishii, Francesco Luzzaro, G. Amicosante, Moreno Galleni
    Abstract:

    Stenotrophomonas maltophilia is a nosocomial pathogen with an intrinsic broad-spectrum resistance to Beta-lactam compounds and other antibacterial agents. It produces two chromosomal Beta-Lactamases: a clavulanic acid-sensitive class A (L2) and a tetrameric carbapenemase (L1 or BlaS). We screened 40 S. maltophilia multidrug-resistant clinical isolates recovered between 1995 and 1998 in the Varese Hospital (Italy) for the presence of the Metallo-Beta-Lactamase. The isolates were investigated by phenotypic profiling (enzymatic activity and antibiotic resistance pattern) and molecular methods such as PCR and pulsed-field gel electrophoresis (PFGE) to reveal intraspecies diversity. For the tested S. maltophilia strains, we showed that the Beta-Lactamase production could be induced by the presence of imipenem (50 microg/ml) in the culture media. Addition of 1 mM dipicolinic acid completely inhibited the hydrolysis of imipenem but decreased that nitrocefin in a strain-dependent manner. Full activity of crude extract towards imipenem could be restored by addition of 1 mM ZnCl2. Finally, the gene encoding the carbapenem-hydrolyzing Beta-Lactamase from S. maltophilia ULA-511 and 39/95, a clinical strain, were isolated and sequenced. These two strains have a different profile of multidrug resistance. The two Metallo-Beta-Lactamases were found to be isologous. The difference of sensitivity of these two strains was associated to the level of production of the Metallo-Beta-Lactamase.

  • CAU-1, a subclass B3 Metallo-Beta-Lactamase of low substrate affinity encoded by an ortholog present in the Caulobacter crescentus chromosome
    Antimicrobial agents and chemotherapy, 2002
    Co-Authors: Jean Denis Docquier, Gianfranco Amicosante, Jeanmarie Frere, Moreno Galleni, Fabrizio Pantanella, Francesco Giuliani, Maria Cristina Thaller, Karen Bush, Gian Maria Rossolini
    Abstract:

    The sequenced chromosome of Caulobacter crescentus CB15 encodes a hypothetical protein that exhibits significant similarity (30 to 35% identical residues) to Metallo-Beta-Lactamases of subclass B3. An allelic variant of this gene (divergent by 3% of its nucleotides) was cloned in Escherichia coli from C. crescentus type strain DSM4727. Expression studies confirmed the Metallo-Beta-Lactamase activity of its product, CAU-1. The enzyme produced in E. coli was purified by two ion-exchange chromatography steps. CAU-1 contains a 29-kDa polypeptide with an alkaline isoelectric pH (> 9), and unlike the L1 enzyme of Stenotrophomonas maltophilia, the native form is monomeric. Kinetic analysis revealed a preferential activity toward penicillins, carbapenems, and narrow-spectrum cephalosporins, while oxyimino cephalosporins were poorly or not hydrolyzed. Affinities for the various Beta-lactams were poor overall (K(m) values were always > 100 microM and often > 400 microM). The interaction with divalent ion chelators appeared to occur by a mechanism similar to that prevailing in other members of subclass B3. In C. crescentus, the CAU-1 enzyme is produced independently of Beta-lactam exposure and, interestingly, the bla(CAU) determinant is bracketed by three other genes, including two genes encoding enzymes involved in methionine biosynthesis and a gene encoding a putative transcriptional regulator, in an operon-like structure. The CAU-1 enzyme is the first example of a Metallo-Beta-Lactamase in a member of the alpha subdivision of the class Proteobacteria:

  • biochemical characterization of the pseudomonas aeruginosa 101 1477 Metallo Beta Lactamase imp 1 produced by escherichia coli
    Antimicrobial Agents and Chemotherapy, 1999
    Co-Authors: Nezha Laraki, Gianfranco Amicosante, Nicola Franceschini, Gian Maria Rossolini, Pasqualino Santucci, Cecile Meunier, Edwin De Pauw, Jeanmarie Frere, Moreno Galleni
    Abstract:

    The blaIMP gene coding for the IMP-1 Metallo-Beta-Lactamase produced by a Pseudomonas aeruginosa clinical isolate (isolate 101/1477) was overexpressed via a T7 expression system in Escherichia coli BL21 (DE3), and its product was purified to homogeneity with a final yield of 35 mg/liter of culture. The structural and functional properties of the enzyme purified from E. coli were identical to those of the enzyme produced by P. aeruginosa. The IMP-1 Metallo-Beta-Lactamase exhibits a broad-spectrum activity profile that includes activity against penicillins, cephalosporins, cephamycins, oxacephamycins, and carbapenems. Only monobactams escape its action. The enzyme activity was inhibited by metal chelators, of which 1,10-o-phenanthroline and dipicolinic acid were the most efficient. Two zinc-binding sites were found. The zinc content of the P. aeruginosa 101/1477 Metallo-Beta-Lactamase was not pH dependent.

  • Biochemical characterization of the Pseudomonas aeruginosa 101/1477 Metallo-Beta-Lactamase IMP-1 produced by Escherichia coli.
    Antimicrobial agents and chemotherapy, 1999
    Co-Authors: Nezha Laraki, Gianfranco Amicosante, Nicola Franceschini, Gian Maria Rossolini, Pasqualino Santucci, Cecile Meunier, Edwin De Pauw, Jeanmarie Frere, Moreno Galleni
    Abstract:

    The blaIMP gene coding for the IMP-1 Metallo-Beta-Lactamase produced by a Pseudomonas aeruginosa clinical isolate (isolate 101/1477) was overexpressed via a T7 expression system in Escherichia coli BL21 (DE3), and its product was purified to homogeneity with a final yield of 35 mg/liter of culture. The structural and functional properties of the enzyme purified from E. coli were identical to those of the enzyme produced by P. aeruginosa. The IMP-1 Metallo-Beta-Lactamase exhibits a broad-spectrum activity profile that includes activity against penicillins, cephalosporins, cephamycins, oxacephamycins, and carbapenems. Only monobactams escape its action. The enzyme activity was inhibited by metal chelators, of which 1,10-o-phenanthroline and dipicolinic acid were the most efficient. Two zinc-binding sites were found. The zinc content of the P. aeruginosa 101/1477 Metallo-Beta-Lactamase was not pH dependent.

Michio Ohta - One of the best experts on this subject based on the ideXlab platform.

  • multifocal outbreaks of Metallo Beta Lactamase producing pseudomonas aeruginosa resistant to broad spectrum Beta lactams including carbapenems
    Antimicrobial Agents and Chemotherapy, 1996
    Co-Authors: Kazuyoshi Senda, Yoshichika Arakawa, Hideo Ito, Nobuo Kato, Kazumitsu Nakashima, Satoshi Ichiyama, Kaoru Shimokata, Michio Ohta
    Abstract:

    A total of 3,700 Pseudomonas aeruginosa isolates were collected from 17 general hospitals in Japan from 1992 to 1994. Of these isolates, 132 carbapenem-resistant strains were subjected to DNA hybridization analysis with the Metallo-Beta-Lactamase gene (blaIMP)-specific probe. Fifteen strains carrying the Metallo-Beta-Lactamase gene were identified in five hospitals in different geographical areas. Three strains of P. aeruginosa demonstrated high-level imipenem resistance (MIC, > or = 128 micrograms/ml), two strains exhibited low-level imipenem resistance (MIC, < or = 4 micrograms/ml), and the rest of the strains were in between. These results revealed that the acquisition of a Metallo-Beta-Lactamase gene alone does not necessarily confer elevated resistance to carbapenems. In several strains, the Metallo-Beta-Lactamase gene was carried by large plasmids, and carbapenem resistance was transferred from P. aeruginosa to Escherichia coli by electroporation in association with the acquisition of the large plasmid. Southern hybridization analysis and genomic DNA fingerprinting profiles revealed different genetic backgrounds for these 15 isolates, although considerable similarity was observed for the strains isolated from the same hospital. These findings suggest that the Metallo-Beta-Lactamase-producing P. aeruginosa strains are not confined to a unique clonal lineage but proliferated multifocally by plasmid-mediated dissemination of the Metallo-Beta-Lactamase gene in strains of different genetic backgrounds. Thus, further proliferation of Metallo-Beta-Lactamase-producing strains with resistance to various Beta-lactams may well be inevitable in the future, which emphasizes the need for early recognition of Metallo-Beta-Lactamase-producing strains, rigorous infection control, and restricted clinical use of broad-spectrum Beta-lactams including carbapenems.

  • a novel integron like element carrying the Metallo Beta Lactamase gene blaimp
    Antimicrobial Agents and Chemotherapy, 1995
    Co-Authors: Yoshichika Arakawa, M Murakami, K Suzuki, Hideo Ito, R Wacharotayankun, Shinji Ohsuka, Nobuo Kato, Michio Ohta
    Abstract:

    A plasmid-mediated Metallo-Beta-Lactamase gene was cloned from a carbapenem-resistant Serratia marcescens strain, AK9373. The Metallo-Beta-Lactamase gene was identical to the blaIMP, and it was located in the space between an integrase-like gene and an aac(6')-Ib-like gene. The deduced amino acid sequence for the putative integrase gene showed considerable identity (60.9%) to that of the Escherichia coli integrase reported. Sequences similar to the GTTRRRY and an atypical 59-base element containing a 67-bp inverted repeat sequence, which were peculiar to the integrase-dependent recombination, were also conserved in the flanking regions of the blaIMP gene. These findings imply that the Metallo-Beta-Lactamase gene in S. marcescens AK9373 is carried by a novel integron-like element that is mediated by a transferable large plasmid.

  • plasmid mediated dissemination of the Metallo Beta Lactamase gene blaimp among clinically isolated strains of serratia marcescens
    Antimicrobial Agents and Chemotherapy, 1995
    Co-Authors: Yoshichika Arakawa, R Wacharotayankun, Shinji Ohsuka, Nobuo Kato, Michio Ohta
    Abstract:

    The distribution of strains producing Metallo-Beta-Lactamase among 105 strains of Serratia marcescens was investigated. All of these strains were isolated in seven general hospitals located in Aichi Prefecture, Japan, from April to May 1993. Southern hybridization analysis suggested that four S. marcescens strains, AK9373, AK9374, AK9385, and AK9391, had a Metallo-Beta-Lactamase genes similar to the blaIMP gene found by our laboratory (E. Osano, Y. Arakawa, R. Wacharotayankun, M. Ohta, T. Horii, H. Ito, F. Yoshimura, and N. Kato, Antimicrob. Agents Chemother. 38:71-78, 1994), and these four strains showed resistance to carbapenems as well as to the other broad-spectrum Beta-lactams. In particular, strains AK9373, AK9374, and AK9391 showed an extraordinarily high-level resistance to imipenem (MICs, > or = 64 micrograms/ml), whereas strain AK9385 demonstrated moderate imipenem resistance (MIC, 8 micrograms/ml). The imipenem resistance of AK9373 was transferred to Escherichia coli CSH2 by conjugation with a frequency of 10(-5). The DNA probe of the blaIMP gene hybridized to a large plasmid (approximately 120 kb) transferred into the E. coli transconjugant as well as to the large plasmids harbored by AK9373. On the other hand, although we failed in the conjugational transfer of imipenem resistance from strains AK9374, AK9385, and AK9391 to E. coli CSH2, imipenem resistance was transferred from these strains to E. coli HB101 by transformation. A plasmid (approximately 25 kb) was observed in each transformant which acquired imipenem resistance. The amino acid sequence at the N terminus of the enzyme purified from strain AK9373 was identical to that of the Metallo-Beta-Lactamase IMP-1. In contrast, strains ES9348, AK9386, and AK93101, which were moderately resistant to imipenem (MICs, > or = 4 to or = 2 micrograms/ml), and strains which showed high-level imipenem resistance because of acquisition of a plasmid-mediated blaIMP-like Metallo-Beta-Lactamase gene had already proliferated as nosocomial infections, at least in a general hospital.

  • plasmid mediated dissemination of the Metallo Beta Lactamase gene blaimp among clinically isolated strains of serratia marcescens
    Antimicrobial Agents and Chemotherapy, 1995
    Co-Authors: Hideo Ito, Yoshichika Arakawa, R Wacharotayankun, Shinji Ohsuka, Nobuo Kato, Michio Ohta
    Abstract:

    The distribution of strains producing Metallo-Beta-Lactamase among 105 strains of Serratia marcescens was investigated. All of these strains were isolated in seven general hospitals located in Aichi Prefecture, Japan, from April to May 1993. Southern hybridization analysis suggested that four S. marcescens strains, AK9373, AK9374, AK9385, and AK9391, had a Metallo-Beta-Lactamase genes similar to the blaIMP gene found by our laboratory (E. Osano, Y. Arakawa, R. Wacharotayankun, M. Ohta, T. Horii, H. Ito, F. Yoshimura, and N. Kato, Antimicrob. Agents Chemother. 38:71-78, 1994), and these four strains showed resistance to carbapenems as well as to the other broad-spectrum Beta-lactams. In particular, strains AK9373, AK9374, and AK9391 showed an extraordinarily high-level resistance to imipenem (MICs, > or = 64 micrograms/ml), whereas strain AK9385 demonstrated moderate imipenem resistance (MIC, 8 micrograms/ml). The imipenem resistance of AK9373 was transferred to Escherichia coli CSH2 by conjugation with a frequency of 10(-5). The DNA probe of the blaIMP gene hybridized to a large plasmid (approximately 120 kb) transferred into the E. coli transconjugant as well as to the large plasmids harbored by AK9373. On the other hand, although we failed in the conjugational transfer of imipenem resistance from strains AK9374, AK9385, and AK9391 to E. coli CSH2, imipenem resistance was transferred from these strains to E. coli HB101 by transformation. A plasmid (approximately 25 kb) was observed in each transformant which acquired imipenem resistance. The amino acid sequence at the N terminus of the enzyme purified from strain AK9373 was identical to that of the Metallo-Beta-Lactamase IMP-1. In contrast, strains ES9348, AK9386, and AK93101, which were moderately resistant to imipenem (MICs, > or = 4 to or = 2 micrograms/ml), and strains which showed high-level imipenem resistance because of acquisition of a plasmid-mediated blaIMP-like Metallo-Beta-Lactamase gene had already proliferated as nosocomial infections, at least in a general hospital.

  • molecular characterization of an enterobacterial Metallo Beta Lactamase found in a clinical isolate of serratia marcescens that shows imipenem resistance
    Antimicrobial Agents and Chemotherapy, 1994
    Co-Authors: E Osano, Yoshichika Arakawa, R Wacharotayankun, Michio Ohta, Toshinobu Horii, F Yoshimura, Nobuo Kato
    Abstract:

    Abstract A clinical isolate of Serratia marcescens (TN9106) produced a Metallo Beta-Lactamase (IMP-1) which conferred resistance to imipenem and broad-spectrum Beta-lactams. The blaIMP gene providing imipenem resistance was cloned and expressed in Escherichia coli HB101. The IMP-1 was purified from E. coli HB101 that harbors pSMBNU24 carrying blaIMP, and its apparent molecular mass was calculated to be about 30 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Kinetic studies of IMP-1 against various Beta-lactams revealed that this enzyme hydrolyzes not only various broad-spectrum Beta-lactams but also carbapenems. However, aztreonam was relatively stable against IMP-1. Although clavulanate or cloxacillin failed to inhibit IMP-1, Hg2+, Fe2+, or Cu2+ blocked the enzyme's activity. Moreover, the presence of EDTA in the reaction buffer resulted in a decrease in the enzyme's activity. Carbapenem resistance was not transferred from S. marcescens TN9106 to E. coli CSH2 by conjugation. A hybridization study confirmed that blaIMP was encoded on the chromosome of S. marcescens TN9106. By nucleotide sequencing analysis, blaIMP was found to encode a protein of 246 amino acid residues and was shown to have considerable homology to the Metallo Beta-Lactamase genes of Bacillus cereus, Bacteroides fragilis, and Aeromonas hydrophila. The G+C content of blaIMP was 39.4%. Four consensus amino acid residues, His-95, His-97, Cys-176, and His-215, which form putative zinc ligands, were conserved in the deduced amino acid sequence of IMP-1. By determination of the amino acid sequence at the N terminus of purified mature IMP-1, 18 amino acid residues were found to be processed from the N terminus of the premature enzyme as a signal peptide. These results clearly show that IMP-1 is an enterobacterial Metallo Beta-Lactamase, of which the primary structure has been completely determined, that confers resistance to carbapenems and other broad-spectrum Beta-lactams.

Nobuo Kato - One of the best experts on this subject based on the ideXlab platform.

  • multifocal outbreaks of Metallo Beta Lactamase producing pseudomonas aeruginosa resistant to broad spectrum Beta lactams including carbapenems
    Antimicrobial Agents and Chemotherapy, 1996
    Co-Authors: Kazuyoshi Senda, Yoshichika Arakawa, Hideo Ito, Nobuo Kato, Kazumitsu Nakashima, Satoshi Ichiyama, Kaoru Shimokata, Michio Ohta
    Abstract:

    A total of 3,700 Pseudomonas aeruginosa isolates were collected from 17 general hospitals in Japan from 1992 to 1994. Of these isolates, 132 carbapenem-resistant strains were subjected to DNA hybridization analysis with the Metallo-Beta-Lactamase gene (blaIMP)-specific probe. Fifteen strains carrying the Metallo-Beta-Lactamase gene were identified in five hospitals in different geographical areas. Three strains of P. aeruginosa demonstrated high-level imipenem resistance (MIC, > or = 128 micrograms/ml), two strains exhibited low-level imipenem resistance (MIC, < or = 4 micrograms/ml), and the rest of the strains were in between. These results revealed that the acquisition of a Metallo-Beta-Lactamase gene alone does not necessarily confer elevated resistance to carbapenems. In several strains, the Metallo-Beta-Lactamase gene was carried by large plasmids, and carbapenem resistance was transferred from P. aeruginosa to Escherichia coli by electroporation in association with the acquisition of the large plasmid. Southern hybridization analysis and genomic DNA fingerprinting profiles revealed different genetic backgrounds for these 15 isolates, although considerable similarity was observed for the strains isolated from the same hospital. These findings suggest that the Metallo-Beta-Lactamase-producing P. aeruginosa strains are not confined to a unique clonal lineage but proliferated multifocally by plasmid-mediated dissemination of the Metallo-Beta-Lactamase gene in strains of different genetic backgrounds. Thus, further proliferation of Metallo-Beta-Lactamase-producing strains with resistance to various Beta-lactams may well be inevitable in the future, which emphasizes the need for early recognition of Metallo-Beta-Lactamase-producing strains, rigorous infection control, and restricted clinical use of broad-spectrum Beta-lactams including carbapenems.

  • a novel integron like element carrying the Metallo Beta Lactamase gene blaimp
    Antimicrobial Agents and Chemotherapy, 1995
    Co-Authors: Yoshichika Arakawa, M Murakami, K Suzuki, Hideo Ito, R Wacharotayankun, Shinji Ohsuka, Nobuo Kato, Michio Ohta
    Abstract:

    A plasmid-mediated Metallo-Beta-Lactamase gene was cloned from a carbapenem-resistant Serratia marcescens strain, AK9373. The Metallo-Beta-Lactamase gene was identical to the blaIMP, and it was located in the space between an integrase-like gene and an aac(6')-Ib-like gene. The deduced amino acid sequence for the putative integrase gene showed considerable identity (60.9%) to that of the Escherichia coli integrase reported. Sequences similar to the GTTRRRY and an atypical 59-base element containing a 67-bp inverted repeat sequence, which were peculiar to the integrase-dependent recombination, were also conserved in the flanking regions of the blaIMP gene. These findings imply that the Metallo-Beta-Lactamase gene in S. marcescens AK9373 is carried by a novel integron-like element that is mediated by a transferable large plasmid.

  • plasmid mediated dissemination of the Metallo Beta Lactamase gene blaimp among clinically isolated strains of serratia marcescens
    Antimicrobial Agents and Chemotherapy, 1995
    Co-Authors: Yoshichika Arakawa, R Wacharotayankun, Shinji Ohsuka, Nobuo Kato, Michio Ohta
    Abstract:

    The distribution of strains producing Metallo-Beta-Lactamase among 105 strains of Serratia marcescens was investigated. All of these strains were isolated in seven general hospitals located in Aichi Prefecture, Japan, from April to May 1993. Southern hybridization analysis suggested that four S. marcescens strains, AK9373, AK9374, AK9385, and AK9391, had a Metallo-Beta-Lactamase genes similar to the blaIMP gene found by our laboratory (E. Osano, Y. Arakawa, R. Wacharotayankun, M. Ohta, T. Horii, H. Ito, F. Yoshimura, and N. Kato, Antimicrob. Agents Chemother. 38:71-78, 1994), and these four strains showed resistance to carbapenems as well as to the other broad-spectrum Beta-lactams. In particular, strains AK9373, AK9374, and AK9391 showed an extraordinarily high-level resistance to imipenem (MICs, > or = 64 micrograms/ml), whereas strain AK9385 demonstrated moderate imipenem resistance (MIC, 8 micrograms/ml). The imipenem resistance of AK9373 was transferred to Escherichia coli CSH2 by conjugation with a frequency of 10(-5). The DNA probe of the blaIMP gene hybridized to a large plasmid (approximately 120 kb) transferred into the E. coli transconjugant as well as to the large plasmids harbored by AK9373. On the other hand, although we failed in the conjugational transfer of imipenem resistance from strains AK9374, AK9385, and AK9391 to E. coli CSH2, imipenem resistance was transferred from these strains to E. coli HB101 by transformation. A plasmid (approximately 25 kb) was observed in each transformant which acquired imipenem resistance. The amino acid sequence at the N terminus of the enzyme purified from strain AK9373 was identical to that of the Metallo-Beta-Lactamase IMP-1. In contrast, strains ES9348, AK9386, and AK93101, which were moderately resistant to imipenem (MICs, > or = 4 to or = 2 micrograms/ml), and strains which showed high-level imipenem resistance because of acquisition of a plasmid-mediated blaIMP-like Metallo-Beta-Lactamase gene had already proliferated as nosocomial infections, at least in a general hospital.

  • plasmid mediated dissemination of the Metallo Beta Lactamase gene blaimp among clinically isolated strains of serratia marcescens
    Antimicrobial Agents and Chemotherapy, 1995
    Co-Authors: Hideo Ito, Yoshichika Arakawa, R Wacharotayankun, Shinji Ohsuka, Nobuo Kato, Michio Ohta
    Abstract:

    The distribution of strains producing Metallo-Beta-Lactamase among 105 strains of Serratia marcescens was investigated. All of these strains were isolated in seven general hospitals located in Aichi Prefecture, Japan, from April to May 1993. Southern hybridization analysis suggested that four S. marcescens strains, AK9373, AK9374, AK9385, and AK9391, had a Metallo-Beta-Lactamase genes similar to the blaIMP gene found by our laboratory (E. Osano, Y. Arakawa, R. Wacharotayankun, M. Ohta, T. Horii, H. Ito, F. Yoshimura, and N. Kato, Antimicrob. Agents Chemother. 38:71-78, 1994), and these four strains showed resistance to carbapenems as well as to the other broad-spectrum Beta-lactams. In particular, strains AK9373, AK9374, and AK9391 showed an extraordinarily high-level resistance to imipenem (MICs, > or = 64 micrograms/ml), whereas strain AK9385 demonstrated moderate imipenem resistance (MIC, 8 micrograms/ml). The imipenem resistance of AK9373 was transferred to Escherichia coli CSH2 by conjugation with a frequency of 10(-5). The DNA probe of the blaIMP gene hybridized to a large plasmid (approximately 120 kb) transferred into the E. coli transconjugant as well as to the large plasmids harbored by AK9373. On the other hand, although we failed in the conjugational transfer of imipenem resistance from strains AK9374, AK9385, and AK9391 to E. coli CSH2, imipenem resistance was transferred from these strains to E. coli HB101 by transformation. A plasmid (approximately 25 kb) was observed in each transformant which acquired imipenem resistance. The amino acid sequence at the N terminus of the enzyme purified from strain AK9373 was identical to that of the Metallo-Beta-Lactamase IMP-1. In contrast, strains ES9348, AK9386, and AK93101, which were moderately resistant to imipenem (MICs, > or = 4 to or = 2 micrograms/ml), and strains which showed high-level imipenem resistance because of acquisition of a plasmid-mediated blaIMP-like Metallo-Beta-Lactamase gene had already proliferated as nosocomial infections, at least in a general hospital.

  • molecular characterization of an enterobacterial Metallo Beta Lactamase found in a clinical isolate of serratia marcescens that shows imipenem resistance
    Antimicrobial Agents and Chemotherapy, 1994
    Co-Authors: E Osano, Yoshichika Arakawa, R Wacharotayankun, Michio Ohta, Toshinobu Horii, F Yoshimura, Nobuo Kato
    Abstract:

    Abstract A clinical isolate of Serratia marcescens (TN9106) produced a Metallo Beta-Lactamase (IMP-1) which conferred resistance to imipenem and broad-spectrum Beta-lactams. The blaIMP gene providing imipenem resistance was cloned and expressed in Escherichia coli HB101. The IMP-1 was purified from E. coli HB101 that harbors pSMBNU24 carrying blaIMP, and its apparent molecular mass was calculated to be about 30 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Kinetic studies of IMP-1 against various Beta-lactams revealed that this enzyme hydrolyzes not only various broad-spectrum Beta-lactams but also carbapenems. However, aztreonam was relatively stable against IMP-1. Although clavulanate or cloxacillin failed to inhibit IMP-1, Hg2+, Fe2+, or Cu2+ blocked the enzyme's activity. Moreover, the presence of EDTA in the reaction buffer resulted in a decrease in the enzyme's activity. Carbapenem resistance was not transferred from S. marcescens TN9106 to E. coli CSH2 by conjugation. A hybridization study confirmed that blaIMP was encoded on the chromosome of S. marcescens TN9106. By nucleotide sequencing analysis, blaIMP was found to encode a protein of 246 amino acid residues and was shown to have considerable homology to the Metallo Beta-Lactamase genes of Bacillus cereus, Bacteroides fragilis, and Aeromonas hydrophila. The G+C content of blaIMP was 39.4%. Four consensus amino acid residues, His-95, His-97, Cys-176, and His-215, which form putative zinc ligands, were conserved in the deduced amino acid sequence of IMP-1. By determination of the amino acid sequence at the N terminus of purified mature IMP-1, 18 amino acid residues were found to be processed from the N terminus of the premature enzyme as a signal peptide. These results clearly show that IMP-1 is an enterobacterial Metallo Beta-Lactamase, of which the primary structure has been completely determined, that confers resistance to carbapenems and other broad-spectrum Beta-lactams.

Gianfranco Amicosante - One of the best experts on this subject based on the ideXlab platform.

  • CAU-1, a subclass B3 Metallo-Beta-Lactamase of low substrate affinity encoded by an ortholog present in the Caulobacter crescentus chromosome
    Antimicrobial agents and chemotherapy, 2002
    Co-Authors: Jean Denis Docquier, Gianfranco Amicosante, Jeanmarie Frere, Moreno Galleni, Fabrizio Pantanella, Francesco Giuliani, Maria Cristina Thaller, Karen Bush, Gian Maria Rossolini
    Abstract:

    The sequenced chromosome of Caulobacter crescentus CB15 encodes a hypothetical protein that exhibits significant similarity (30 to 35% identical residues) to Metallo-Beta-Lactamases of subclass B3. An allelic variant of this gene (divergent by 3% of its nucleotides) was cloned in Escherichia coli from C. crescentus type strain DSM4727. Expression studies confirmed the Metallo-Beta-Lactamase activity of its product, CAU-1. The enzyme produced in E. coli was purified by two ion-exchange chromatography steps. CAU-1 contains a 29-kDa polypeptide with an alkaline isoelectric pH (> 9), and unlike the L1 enzyme of Stenotrophomonas maltophilia, the native form is monomeric. Kinetic analysis revealed a preferential activity toward penicillins, carbapenems, and narrow-spectrum cephalosporins, while oxyimino cephalosporins were poorly or not hydrolyzed. Affinities for the various Beta-lactams were poor overall (K(m) values were always > 100 microM and often > 400 microM). The interaction with divalent ion chelators appeared to occur by a mechanism similar to that prevailing in other members of subclass B3. In C. crescentus, the CAU-1 enzyme is produced independently of Beta-lactam exposure and, interestingly, the bla(CAU) determinant is bracketed by three other genes, including two genes encoding enzymes involved in methionine biosynthesis and a gene encoding a putative transcriptional regulator, in an operon-like structure. The CAU-1 enzyme is the first example of a Metallo-Beta-Lactamase in a member of the alpha subdivision of the class Proteobacteria:

  • Biochemical characterization of the Pseudomonas aeruginosa 101/1477 Metallo-Beta-Lactamase IMP-1 produced by Escherichia coli.
    Antimicrobial agents and chemotherapy, 1999
    Co-Authors: Nezha Laraki, Gianfranco Amicosante, Nicola Franceschini, Gian Maria Rossolini, Pasqualino Santucci, Cecile Meunier, Edwin De Pauw, Jeanmarie Frere, Moreno Galleni
    Abstract:

    The blaIMP gene coding for the IMP-1 Metallo-Beta-Lactamase produced by a Pseudomonas aeruginosa clinical isolate (isolate 101/1477) was overexpressed via a T7 expression system in Escherichia coli BL21 (DE3), and its product was purified to homogeneity with a final yield of 35 mg/liter of culture. The structural and functional properties of the enzyme purified from E. coli were identical to those of the enzyme produced by P. aeruginosa. The IMP-1 Metallo-Beta-Lactamase exhibits a broad-spectrum activity profile that includes activity against penicillins, cephalosporins, cephamycins, oxacephamycins, and carbapenems. Only monobactams escape its action. The enzyme activity was inhibited by metal chelators, of which 1,10-o-phenanthroline and dipicolinic acid were the most efficient. Two zinc-binding sites were found. The zinc content of the P. aeruginosa 101/1477 Metallo-Beta-Lactamase was not pH dependent.

  • biochemical characterization of the pseudomonas aeruginosa 101 1477 Metallo Beta Lactamase imp 1 produced by escherichia coli
    Antimicrobial Agents and Chemotherapy, 1999
    Co-Authors: Nezha Laraki, Gianfranco Amicosante, Nicola Franceschini, Gian Maria Rossolini, Pasqualino Santucci, Cecile Meunier, Edwin De Pauw, Jeanmarie Frere, Moreno Galleni
    Abstract:

    The blaIMP gene coding for the IMP-1 Metallo-Beta-Lactamase produced by a Pseudomonas aeruginosa clinical isolate (isolate 101/1477) was overexpressed via a T7 expression system in Escherichia coli BL21 (DE3), and its product was purified to homogeneity with a final yield of 35 mg/liter of culture. The structural and functional properties of the enzyme purified from E. coli were identical to those of the enzyme produced by P. aeruginosa. The IMP-1 Metallo-Beta-Lactamase exhibits a broad-spectrum activity profile that includes activity against penicillins, cephalosporins, cephamycins, oxacephamycins, and carbapenems. Only monobactams escape its action. The enzyme activity was inhibited by metal chelators, of which 1,10-o-phenanthroline and dipicolinic acid were the most efficient. Two zinc-binding sites were found. The zinc content of the P. aeruginosa 101/1477 Metallo-Beta-Lactamase was not pH dependent.

  • The renal membrane dipeptidase (dehydropeptidase I) inhibitor, cilastatin, inhibits the bacterial Metallo-Beta-Lactamase enzyme CphA.
    Antimicrobial agents and chemotherapy, 1995
    Co-Authors: Shoshana Keynan, Nigel M. Hooper, Antonio Felici, Gianfranco Amicosante, Andanthony J. Turner
    Abstract:

    The Aeromonas hydrophila AE036 chromosome contains a cphA gene encoding a Metallo-Beta-Lactamase which is highly active against carbapenem antibiotics such as imipenem. Here we show that the cphA gene product shares inhibitory similarities with a mammalian zinc peptidase, membrane dipeptidase (MDP; dehydropeptidase I). Both enzymes are able to hydrolyze imipenem and are inhibited by cilastatin. The active site similarities of these enzymes are not reflected in any significant primary sequence similarity.

  • High specificity of cphA-encoded Metallo-Beta-Lactamase from Aeromonas hydrophila AE036 for carbapenems and its contribution to Beta-lactam resistance.
    Antimicrobial agents and chemotherapy, 1993
    Co-Authors: Bernardetta Segatore, Orietta Massidda, Giuseppe Satta, Domenico Setacci, Gianfranco Amicosante
    Abstract:

    The Aeromonas hydrophila AE036 chromosome contains a cphA gene encoding a Metallo-Beta-Lactamase highly active against carbapenem antibiotics. This enzyme was induced in strain AE036 to the same extent by both benzylpenicillin and imipenem. When the cphA gene was inserted into plasmid pACYC184, used to transform Escherichia coli DH5 alpha, the MICs of imipenem, meropenem, and penem HRE664 for recombinant clone DH5 alpha(pAA20R), expressing the Aeromonas Metallo-Beta-Lactamase, were significantly increased, but those of penicillins and cephalosporins were not. When the Metallo-Beta-Lactamase purified from E. coli DH5 alpha(pAA20R) was assayed with several Beta-lactam substrates, it hydrolyzed carbapenems but not penicillins or cephalosporins efficiently. These results demonstrate that this Metallo-Beta-Lactamase possesses an unusual spectrum of activity compared with all the other class B enzymes identified so far, being active on penems and carbapenems only. This enzyme may thus contribute to the development of resistance to penems and carbapenems but not other Beta-lactams.

Yoshichika Arakawa - One of the best experts on this subject based on the ideXlab platform.

  • multifocal outbreaks of Metallo Beta Lactamase producing pseudomonas aeruginosa resistant to broad spectrum Beta lactams including carbapenems
    Antimicrobial Agents and Chemotherapy, 1996
    Co-Authors: Kazuyoshi Senda, Yoshichika Arakawa, Hideo Ito, Nobuo Kato, Kazumitsu Nakashima, Satoshi Ichiyama, Kaoru Shimokata, Michio Ohta
    Abstract:

    A total of 3,700 Pseudomonas aeruginosa isolates were collected from 17 general hospitals in Japan from 1992 to 1994. Of these isolates, 132 carbapenem-resistant strains were subjected to DNA hybridization analysis with the Metallo-Beta-Lactamase gene (blaIMP)-specific probe. Fifteen strains carrying the Metallo-Beta-Lactamase gene were identified in five hospitals in different geographical areas. Three strains of P. aeruginosa demonstrated high-level imipenem resistance (MIC, > or = 128 micrograms/ml), two strains exhibited low-level imipenem resistance (MIC, < or = 4 micrograms/ml), and the rest of the strains were in between. These results revealed that the acquisition of a Metallo-Beta-Lactamase gene alone does not necessarily confer elevated resistance to carbapenems. In several strains, the Metallo-Beta-Lactamase gene was carried by large plasmids, and carbapenem resistance was transferred from P. aeruginosa to Escherichia coli by electroporation in association with the acquisition of the large plasmid. Southern hybridization analysis and genomic DNA fingerprinting profiles revealed different genetic backgrounds for these 15 isolates, although considerable similarity was observed for the strains isolated from the same hospital. These findings suggest that the Metallo-Beta-Lactamase-producing P. aeruginosa strains are not confined to a unique clonal lineage but proliferated multifocally by plasmid-mediated dissemination of the Metallo-Beta-Lactamase gene in strains of different genetic backgrounds. Thus, further proliferation of Metallo-Beta-Lactamase-producing strains with resistance to various Beta-lactams may well be inevitable in the future, which emphasizes the need for early recognition of Metallo-Beta-Lactamase-producing strains, rigorous infection control, and restricted clinical use of broad-spectrum Beta-lactams including carbapenems.

  • a novel integron like element carrying the Metallo Beta Lactamase gene blaimp
    Antimicrobial Agents and Chemotherapy, 1995
    Co-Authors: Yoshichika Arakawa, M Murakami, K Suzuki, Hideo Ito, R Wacharotayankun, Shinji Ohsuka, Nobuo Kato, Michio Ohta
    Abstract:

    A plasmid-mediated Metallo-Beta-Lactamase gene was cloned from a carbapenem-resistant Serratia marcescens strain, AK9373. The Metallo-Beta-Lactamase gene was identical to the blaIMP, and it was located in the space between an integrase-like gene and an aac(6')-Ib-like gene. The deduced amino acid sequence for the putative integrase gene showed considerable identity (60.9%) to that of the Escherichia coli integrase reported. Sequences similar to the GTTRRRY and an atypical 59-base element containing a 67-bp inverted repeat sequence, which were peculiar to the integrase-dependent recombination, were also conserved in the flanking regions of the blaIMP gene. These findings imply that the Metallo-Beta-Lactamase gene in S. marcescens AK9373 is carried by a novel integron-like element that is mediated by a transferable large plasmid.

  • plasmid mediated dissemination of the Metallo Beta Lactamase gene blaimp among clinically isolated strains of serratia marcescens
    Antimicrobial Agents and Chemotherapy, 1995
    Co-Authors: Yoshichika Arakawa, R Wacharotayankun, Shinji Ohsuka, Nobuo Kato, Michio Ohta
    Abstract:

    The distribution of strains producing Metallo-Beta-Lactamase among 105 strains of Serratia marcescens was investigated. All of these strains were isolated in seven general hospitals located in Aichi Prefecture, Japan, from April to May 1993. Southern hybridization analysis suggested that four S. marcescens strains, AK9373, AK9374, AK9385, and AK9391, had a Metallo-Beta-Lactamase genes similar to the blaIMP gene found by our laboratory (E. Osano, Y. Arakawa, R. Wacharotayankun, M. Ohta, T. Horii, H. Ito, F. Yoshimura, and N. Kato, Antimicrob. Agents Chemother. 38:71-78, 1994), and these four strains showed resistance to carbapenems as well as to the other broad-spectrum Beta-lactams. In particular, strains AK9373, AK9374, and AK9391 showed an extraordinarily high-level resistance to imipenem (MICs, > or = 64 micrograms/ml), whereas strain AK9385 demonstrated moderate imipenem resistance (MIC, 8 micrograms/ml). The imipenem resistance of AK9373 was transferred to Escherichia coli CSH2 by conjugation with a frequency of 10(-5). The DNA probe of the blaIMP gene hybridized to a large plasmid (approximately 120 kb) transferred into the E. coli transconjugant as well as to the large plasmids harbored by AK9373. On the other hand, although we failed in the conjugational transfer of imipenem resistance from strains AK9374, AK9385, and AK9391 to E. coli CSH2, imipenem resistance was transferred from these strains to E. coli HB101 by transformation. A plasmid (approximately 25 kb) was observed in each transformant which acquired imipenem resistance. The amino acid sequence at the N terminus of the enzyme purified from strain AK9373 was identical to that of the Metallo-Beta-Lactamase IMP-1. In contrast, strains ES9348, AK9386, and AK93101, which were moderately resistant to imipenem (MICs, > or = 4 to or = 2 micrograms/ml), and strains which showed high-level imipenem resistance because of acquisition of a plasmid-mediated blaIMP-like Metallo-Beta-Lactamase gene had already proliferated as nosocomial infections, at least in a general hospital.

  • plasmid mediated dissemination of the Metallo Beta Lactamase gene blaimp among clinically isolated strains of serratia marcescens
    Antimicrobial Agents and Chemotherapy, 1995
    Co-Authors: Hideo Ito, Yoshichika Arakawa, R Wacharotayankun, Shinji Ohsuka, Nobuo Kato, Michio Ohta
    Abstract:

    The distribution of strains producing Metallo-Beta-Lactamase among 105 strains of Serratia marcescens was investigated. All of these strains were isolated in seven general hospitals located in Aichi Prefecture, Japan, from April to May 1993. Southern hybridization analysis suggested that four S. marcescens strains, AK9373, AK9374, AK9385, and AK9391, had a Metallo-Beta-Lactamase genes similar to the blaIMP gene found by our laboratory (E. Osano, Y. Arakawa, R. Wacharotayankun, M. Ohta, T. Horii, H. Ito, F. Yoshimura, and N. Kato, Antimicrob. Agents Chemother. 38:71-78, 1994), and these four strains showed resistance to carbapenems as well as to the other broad-spectrum Beta-lactams. In particular, strains AK9373, AK9374, and AK9391 showed an extraordinarily high-level resistance to imipenem (MICs, > or = 64 micrograms/ml), whereas strain AK9385 demonstrated moderate imipenem resistance (MIC, 8 micrograms/ml). The imipenem resistance of AK9373 was transferred to Escherichia coli CSH2 by conjugation with a frequency of 10(-5). The DNA probe of the blaIMP gene hybridized to a large plasmid (approximately 120 kb) transferred into the E. coli transconjugant as well as to the large plasmids harbored by AK9373. On the other hand, although we failed in the conjugational transfer of imipenem resistance from strains AK9374, AK9385, and AK9391 to E. coli CSH2, imipenem resistance was transferred from these strains to E. coli HB101 by transformation. A plasmid (approximately 25 kb) was observed in each transformant which acquired imipenem resistance. The amino acid sequence at the N terminus of the enzyme purified from strain AK9373 was identical to that of the Metallo-Beta-Lactamase IMP-1. In contrast, strains ES9348, AK9386, and AK93101, which were moderately resistant to imipenem (MICs, > or = 4 to or = 2 micrograms/ml), and strains which showed high-level imipenem resistance because of acquisition of a plasmid-mediated blaIMP-like Metallo-Beta-Lactamase gene had already proliferated as nosocomial infections, at least in a general hospital.

  • molecular characterization of an enterobacterial Metallo Beta Lactamase found in a clinical isolate of serratia marcescens that shows imipenem resistance
    Antimicrobial Agents and Chemotherapy, 1994
    Co-Authors: E Osano, Yoshichika Arakawa, R Wacharotayankun, Michio Ohta, Toshinobu Horii, F Yoshimura, Nobuo Kato
    Abstract:

    Abstract A clinical isolate of Serratia marcescens (TN9106) produced a Metallo Beta-Lactamase (IMP-1) which conferred resistance to imipenem and broad-spectrum Beta-lactams. The blaIMP gene providing imipenem resistance was cloned and expressed in Escherichia coli HB101. The IMP-1 was purified from E. coli HB101 that harbors pSMBNU24 carrying blaIMP, and its apparent molecular mass was calculated to be about 30 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Kinetic studies of IMP-1 against various Beta-lactams revealed that this enzyme hydrolyzes not only various broad-spectrum Beta-lactams but also carbapenems. However, aztreonam was relatively stable against IMP-1. Although clavulanate or cloxacillin failed to inhibit IMP-1, Hg2+, Fe2+, or Cu2+ blocked the enzyme's activity. Moreover, the presence of EDTA in the reaction buffer resulted in a decrease in the enzyme's activity. Carbapenem resistance was not transferred from S. marcescens TN9106 to E. coli CSH2 by conjugation. A hybridization study confirmed that blaIMP was encoded on the chromosome of S. marcescens TN9106. By nucleotide sequencing analysis, blaIMP was found to encode a protein of 246 amino acid residues and was shown to have considerable homology to the Metallo Beta-Lactamase genes of Bacillus cereus, Bacteroides fragilis, and Aeromonas hydrophila. The G+C content of blaIMP was 39.4%. Four consensus amino acid residues, His-95, His-97, Cys-176, and His-215, which form putative zinc ligands, were conserved in the deduced amino acid sequence of IMP-1. By determination of the amino acid sequence at the N terminus of purified mature IMP-1, 18 amino acid residues were found to be processed from the N terminus of the premature enzyme as a signal peptide. These results clearly show that IMP-1 is an enterobacterial Metallo Beta-Lactamase, of which the primary structure has been completely determined, that confers resistance to carbapenems and other broad-spectrum Beta-lactams.