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1.  Plasmid-mediated dissemination of the metallo-beta-lactamase gene blaIMP among clinically isolated strains of Serratia marcescens. 
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 = 8 micrograms/ml), had no detectable blaIMP gene. As a conclusion, 19% of clinically isolated S. marcescens strains in Aichi Prefecture, Japan, in 1993 were resistant to imipenem (MICs, > 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.
PMCID: PMC162636  PMID: 7785978
2.  Molecular characterization of an enterobacterial metallo beta-lactamase found in a clinical isolate of Serratia marcescens that shows imipenem resistance. 
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.
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PMCID: PMC284399  PMID: 8141584
3.  Carbapenemases: the Versatile β-Lactamases 
Clinical Microbiology Reviews  2007;20(3):440-458.
Carbapenemases are β-lactamases with versatile hydrolytic capacities. They have the ability to hydrolyze penicillins, cephalosporins, monobactams, and carbapenems. Bacteria producing these β-lactamases may cause serious infections in which the carbapenemase activity renders many β-lactams ineffective. Carbapenemases are members of the molecular class A, B, and D β-lactamases. Class A and D enzymes have a serine-based hydrolytic mechanism, while class B enzymes are metallo-β-lactamases that contain zinc in the active site. The class A carbapenemase group includes members of the SME, IMI, NMC, GES, and KPC families. Of these, the KPC carbapenemases are the most prevalent, found mostly on plasmids in Klebsiella pneumoniae. The class D carbapenemases consist of OXA-type β-lactamases frequently detected in Acinetobacter baumannii. The metallo-β-lactamases belong to the IMP, VIM, SPM, GIM, and SIM families and have been detected primarily in Pseudomonas aeruginosa; however, there are increasing numbers of reports worldwide of this group of β-lactamases in the Enterobacteriaceae. This review updates the characteristics, epidemiology, and detection of the carbapenemases found in pathogenic bacteria.
doi:10.1128/CMR.00001-07
PMCID: PMC1932750  PMID: 17630334
4.  Laboratory Surveillance for Prospective Plasmid-Mediated AmpC β-Lactamases in the Kinki Region of Japan▿  
Journal of Clinical Microbiology  2010;48(9):3267-3273.
Extended-spectrum β-lactamases, plasmid-mediated AmpC β-lactamases (PABLs), and plasmid-mediated metallo-β-lactamases confer resistance to many β-lactams. In Japan, although several reports exist on the prevalence of extended-spectrum β-lactamases and metallo-β-lactamases, the prevalence and characteristics of PABLs remain unknown. To investigate the production of PABLs, a total of 22,869 strains of 4 enterobacterial species, Escherichia coli, Klebsiella pneumoniae, Klebsiella oxytoca, and Proteus mirabilis, were collected during six 6-month periods from 17 clinical laboratories in the Kinki region of Japan. PABLs were detected in 29 (0.13%) of 22,869 isolates by the 3-dimensional test, PCR analysis, and DNA sequencing analysis. PABL-positive isolates were detected among isolates from 13 laboratories. Seventeen of 13,995 (0.12%) E. coli isolates, 8 of 5,970 (0.13%) K. pneumoniae isolates, 3 of 1,722 (0.17%) K. oxytoca isolates, and 1 of 1,182 (0.08%) P. mirabilis isolates were positive for PABLs. Of these 29 PABL-positive strains, 20 (69.0%), 6 (20.7%), 2 (6.9%), and 1 (3.4%) carried the genes for CMY-2, DHA-1, CMY-8, and MOX-1 PABLs, respectively. Pattern analysis of randomly amplified polymorphic DNA and pulsed-field gel electrophoretic analysis revealed that the prevalence of CMY-2-producing E. coli strains was not due to epidemic strains and that 3 DHA-1-producing K. pneumoniae strains were identical, suggesting their clonal relatedness. In conclusion, the DHA-1 PABLs were predominantly present in K. pneumoniae strains, but CMY-2 PABLs were predominantly present in E. coli strains. The present findings will provide significant information to assist in preventing the emergence and further spread of PABL-producing bacteria.
doi:10.1128/JCM.02111-09
PMCID: PMC2937711  PMID: 20610688
5.  PCR detection of metallo-beta-lactamase gene (blaIMP) in gram-negative rods resistant to broad-spectrum beta-lactams. 
Journal of Clinical Microbiology  1996;34(12):2909-2913.
We applied PCR to the rapid detection of the metallo-beta-lactamase gene, blaIMP, in clinically isolated gram-negative rods. A total of 54 high-level ceftazidime-resistant strains (MICs, > 128 micrograms/ml) were subjected to PCR analyses with the blaIMP-specific primers, since the blaIMP-bearing clinical isolates tested in our previous study always demonstrated high-level resistance to ceftazidime. Twenty-two blaIMP-positive strains including 9 Pseudomonas aeruginosa, 9 Serratia marcescens, 2 Alcaligenes xylosoxidans, 1 Pseudomonas putida, and 1 Klebsiella pneumoniae strains were newly identified from 18 different hospitals in Japan. These strains were mostly isolated from urine samples and showed high-level resistance to almost every cephem, while their levels of resistance to carbapenems were diverse. The PCR analyses with novel integrase gene-specific (intI3) and acc(6')-Ib gene-specific primers suggested that the integron structure found in a large plasmid harbored by S. marcescens AK9373 was also well conserved among blaIMP-positive strains. These results imply that the blaIMP gene cassettes have been dispersing into various gram-negative rods with the help of the newly identified integron element. Thus, the PCR-aided rapid detection will be helpful for the early recognition of emerging blaIMP-positive clinical isolates which demonstrate consistent resistance to beta-lactams.
PMCID: PMC229432  PMID: 8940421
6.  Plasmid-Encoded Metallo-β-Lactamase (IMP-6) Conferring Resistance to Carbapenems, Especially Meropenem 
In 1996, Serratia marcescens KU3838 was isolated from the urine of a patient with a urinary tract infection at a hospital in northern Japan and was found to contain the plasmid pKU501. Previously, we determined that pKU501 carries blaIMP and the genes for TEM-1-type β-lactamases as well as producing both types of β-lactamases (H. Yano, A. Kuga, K. Irinoda, R. Okamoto, T. Kobayashi, and M. Inoue, J. Antibiot. 52:1135–1139, 1999). pKU502 is a recombinant plasmid that contains a 1.5-kb DNA fragment, including the metallo-β-lactamase gene, and is obtained by PCR amplification of pKU501. The sequence of the metallo-β-lactamase gene in pKU502 was determined and revealed that this metallo-β-lactamase gene differed from the gene encoding IMP-1 by one point mutation, leading to one amino acid substitution: 640-A in the base sequence of the IMP-1 gene was replaced by G, and Ser-196 was replaced by Gly in the mature enzyme. This enzyme was designated IMP-6. The strains that produced IMP-6 were resistant to carbapenems. The MICs of panipenem and especially meropenem were higher than the MIC of imipenem for these strains. The kcat/Km value of IMP-6 was about sevenfold higher against meropenem than against imipenem, although the MIC of meropenem for KU1917, which produced IMP-1, was lower than that of imipenem, and the MIC of panipenem was equal to that of imipenem. These results support the hypothesis that IMP-6 has extended substrate profiles against carbapenems. However, the activity of IMP-6 was very low against penicillin G and piperacillin. These results suggest that IMP-6 acquired high activity against carbapenems, especially meropenem, via the point mutation but in the process lost activity against penicillins. Although IMP-6 has reduced activity against penicillins due to this point mutation, pKU501 confers resistance to a variety of antimicrobial agents because it also produces TEM-1-type enzyme.
doi:10.1128/AAC.45.5.1343-1348.2001
PMCID: PMC90471  PMID: 11302793
7.  Multifocal outbreaks of metallo-beta-lactamase-producing Pseudomonas aeruginosa resistant to broad-spectrum beta-lactams, including carbapenems. 
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.
PMCID: PMC163114  PMID: 8834878
8.  New Delhi metallo-β-lactamase-1: local acquisition in Ontario, Canada, and challenges in detection 
New Delhi metallo-β-lactamase-1 (NDM-1) is a recently identified metallo-β-lactamase that confers resistance to carbapenems and all other β-lactam antibiotics, with the exception of aztreonam. NDM-1 is also associated with resistance to many other classes of antibiotics. The enzyme was first identified in organisms isolated from a patient in Sweden who had previously received medical treatment in India, but it is now recognized as endemic throughout India and Pakistan and has spread worldwide. The gene encoding NDM-1 has been found predominantly in Escherichia coli and Klebsiella pneumoniae. We describe the isolation NDM-1–producing organisms from two patients in Toronto, Ontario. To the best of our knowledge, this is the first report of an organism producing NDM-1 that was locally acquired in Canada. We also discuss the evidence that NDM-1 can affect bacterial species other than E. coli and K. pneumoniae, the limited options for treatment and the difficulty laboratories face in detecting organisms that produce NDM-1.
doi:10.1503/cmaj.110477
PMCID: PMC3153514  PMID: 21624908
9.  A novel integron-like element carrying the metallo-beta-lactamase gene blaIMP. 
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.
PMCID: PMC162793  PMID: 7492116
10.  Use of Microdilution Panels with and without β-Lactamase Inhibitors as a Phenotypic Test for β-Lactamase Production among Escherichia coli, Klebsiella spp., Enterobacter spp., Citrobacter freundii, and Serratia marcescens 
Over the past decade, a number of new β-lactamases have appeared in clinical isolates of Enterobacteriaceae that, unlike their predecessors, do not confer β-lactam resistance that is readily detected in routine antibiotic susceptibility tests. Because optimal methodologies are needed to detect these important new β-lactamases, a study was designed to evaluate the ability of a panel of various β-lactam antibiotics tested alone and in combination with β-lactamase inhibitors to discriminate between the production of extended-spectrum β-lactamases, AmpC β-lactamases, high levels of K1 β-lactamase, and other β-lactamases in 141 isolates of Escherichia coli, Klebsiella pneumoniae, Klebsiella oxytoca, Enterobacter cloacae, Enterobacter aerogenes, Citrobacter freundii, and Serratia marcescens possessing well-characterized β-lactamases. The microdilution panels studied contained aztreonam, cefpodoxime, ceftazidime, cefotaxime, and ceftriaxone, with and without 1, 2, and 4 μg of clavulanate per ml or 8 μg of sulbactam per ml and cefoxitin and cefotetan with and without 8 μg of sulbactam per ml. The results indicated that a minimum panel of five tests would provide maximum separation of extended-spectrum β-lactamase high AmpC, high K1, and other β-lactamase production in Enterobacteriaceae. These included cefpodoxime, cefpodoxime plus 4 μg of clavulanate per ml, ceftazidime, ceftriaxone, and ceftriaxone plus 8 μg of sulbactam per ml. Ceftriaxone plus 2 μg of clavulanate per ml could be substituted for cefpodoxime plus 4 μg of clavulanate per ml without altering the accuracy of the tests. This study indicated that tests with key β-lactam drugs, alone and in combination with β-lactamase inhibitors, could provide a convenient approach to the detection of a variety of β-lactamases in members of the family Enterobacteriaceae.
PMCID: PMC89285  PMID: 10348759
11.  Structure of In31, a blaIMP-Containing Pseudomonas aeruginosa Integron Phyletically Related to In5, Which Carries an Unusual Array of Gene Cassettes 
The location and environment of the acquired blaIMP gene, which encodes the IMP-1 metallo-β-lactamase, were investigated in a Japanese Pseudomonas aeruginosa clinical isolate (isolate 101/1477) that produced the enzyme. In this isolate, blaIMP was carried on a 36-kb plasmid, and similar to the identical alleles found in Serratia marcescens and Klebsiella pneumoniae clinical isolates, it was located on a mobile gene cassette inserted into an integron. The entire structure of this integron, named In31, was determined. In31 is a class 1 element belonging to the same group of defective transposon derivatives that originated from Tn402-like ancestors such as In0, In2, and In5. The general structure of In31 appeared to be most closely related to that of In5 from pSCH884, suggesting a recent common phylogeny for these two elements. In In31, the blaIMP cassette is the first of an array of five gene cassettes that also includes an aacA4 cassette and three original cassettes that have never been described in other integrons. The novel cassettes carry, respectively, (i) a new chloramphenicol acetyltransferase-encoding allele of the catB family, (ii) a qac allele encoding a new member of the small multidrug resistance family of proteins, and (iii) an open reading frame encoding a protein of unknown function. All the resistance genes carried on cassettes inserted in In31 were found to be functional in decreasing the in vitro susceptibilities of host strains to the corresponding antimicrobial agents.
PMCID: PMC89222  PMID: 10103196
12.  Convenient Test Using a Combination of Chelating Agents for Detection of Metallo-β-Lactamases in the Clinical Laboratory▿  
Journal of Clinical Microbiology  2007;45(9):2798-2801.
Although transmissible metallo-β-lactamases (MBLs) are a serious threat to β-lactam antibiotic therapy, the CLSI currently does not recommend testing methods for the detection of MBLs. The aim of this study was to evaluate the capability of double-disk tests (DDTs) by using disks containing a combination of the chelators 2-mercaptopropionic acid (MPA) and Tris-EDTA (TE) to detect MBLs. Sixteen isolates (4 Acinetobacter baumannii isolates, 6 Pseudomonas aeruginosa isolates, 1 Serratia marcescens isolate, 1 Aeromonas hydrophila isolate, 1 Aeromonas veronii isolate, 2 Chryseobacterium meningosepticum isolates, and 1 Stenotrophomonas maltophilia isolate) producing IMP-1, IMP-1-like, IMP-18, GIM-1, SPM-1, VIM-2, VIM-2-like, and chromosomal MBLs and 20 isolates (7 Klebsiella pneumoniae isolates, 3 Escherichia coli isolates, 5 Enterobacter cloacae isolates, 2 S. marcescens isolates, 1 Proteus mirabilis isolate, and 2 A. baumannii isolates) producing non-MBL carbapenemases, AmpC β-lactamases, and extended-spectrum β-lactamases were tested. The DDT method was evaluated by using four types of chelator disks (TE, high-strength TE, MPA, and TE plus 20 μl of MPA [at various concentrations]) and the β-lactams imipenem (IPM), meropenem (MEM), ertapenem (ERT), and ceftazidime (CAZ). DDTs with IPM and a TE disk supplemented with 1:320 MPA detected all MBLs and yielded no false-positive results. Some, but not all, MBL producers were detected in IPM-based tests involving the single chelator TE or MPA alone or by ERT- or CAZ-based tests. IPM-based tests with MPA concentrations other than 1:320 and all MEM-based tests had suboptimal sensitivities or specificities. DDT with IPM and a TE disk supplemented with 20 μl of 1:320 MPA appears to be convenient for the detection of MBLs in the clinical laboratory.
doi:10.1128/JCM.02486-06
PMCID: PMC2045267  PMID: 17596358
13.  Biochemical properties of inducible beta-lactamases produced from Xanthomonas maltophilia. 
Four different beta-lactamases have been found in several strains of Xanthomonas maltophilia isolated from blood cultures during 1984 to 1991 at the Edinburgh Royal Infirmary. One was a metallo-beta-lactamase with predominantly penicillinase activity and an isoelectric point of 6.8. Its molecular size as determined by gel filtration was 96 kDa but was only 26 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), suggesting a tetramer of four equal subunits. The enzyme hydrolyzed all classes of beta-lactams except the monobactam aztreonam. This enzyme was not inhibited by potassium clavulanate or BRL 42715 but was inhibited by p-chloromercuribenzoate, mercuric chloride, and EDTA. The beta-lactamase was unstable in 50 mM sodium phosphate buffer (pH 8.0) but stable in 50 mM Tris HCl (pH 8.0). The other beta-lactamases focused as a series of different isoelectric points, ranging from pI 5.2 to 6.6. Together, these enzymes exhibited a broad spectrum of activity, hydrolyzing most classes of beta-lactams but not imipenem or aztreonam. Their molecular size was 48 kDa by Sephadex gel filtration and 24 kDa by SDS-PAGE, indicating that they were enzymes consisting of two equal subunits. They were inhibited by p-chloromercuribenzoate, mercuric chloride, potassium clavulanate, and BRL 42715 but not EDTA. This study demonstrated that X. maltophilia produces more than just the L1 and L2 beta-lactamases.
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PMCID: PMC284698  PMID: 7811033
14.  Biochemical Characterization of the Pseudomonas aeruginosa 101/1477 Metallo-β-Lactamase IMP-1 Produced by Escherichia coli 
The blaIMP gene coding for the IMP-1 metallo-β-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-β-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-β-lactamase was not pH dependent.
PMCID: PMC89223  PMID: 10103197
15.  Isolation of the first IMP-4 metallo-β-lactamase producing Klebsiella pneumoniae in Tianjin, China 
Brazilian Journal of Microbiology  2012;43(3):917-922.
This study shows for the first time the mechanism of carbapenem resistance of a Klebsiella pneumoniae clinical isolate TJ8 recovered from Tianjin Medical University General Hospital ,China. The modified Hodge test and EDTA synergy test were performed for the screening of carbapenemases and metallo-β-lactamases (MBLs), respectively. Polymerase chain reactions and DNA sequencing confirmed that the strain carried IMP-4 metallo-β-lactamase (MBL) , SHV-11 and TEM-1 β-lactamase.
Class I integron was positive and gave a 3.0-kb PCR amplicon .IMP-4 was located in Class I integron 5’CS. The gene determinants were organized in the order of blaIMP-4-orfII-orfIII.In all, the results show that IMP-4 MBL production caused the TJ8 resistance to carbapenems.
doi:10.1590/S1517-838220120003000010
PMCID: PMC3768900  PMID: 24031907
Klebsiella pneumoniae; metallo-β-lactamases; integron
16.  Contribution of enzymatic properties, cell permeability, and enzyme expression to microbiological activities of beta-lactams in three Bacteroides fragilis isolates that harbor a metallo-beta-lactamase gene. 
The metallo-beta-lactamase gene, ccrA, has been cloned from three clinical isolates of Bacteroides fragilis, TAL3636, QMCN3, and QMCN4. Although all three isolates harbored a gene encoding a potent beta-lactamase, the MICs of benzylpenicillin, piperacillin, cefotaxime, ceftazidime, imipenem, and biapenem for the three isolates varied from 4- to > 128-fold. QMCN4 was the most susceptible of the three isolates, followed by QMCN3. TAL3636 was resistant to all of the beta-lactams. Previous DNA sequence analysis of the three ccrA genes revealed that the enzymes differed at 5 amino acid residues (B. A. Rasmussen, Y. Gluzman, and F. P. Tally, Mol. Microbiol. 5:1211-1219, 1991). Biochemical characterization of the three enzymes revealed only small differences in kcat and Km values for the majority of beta-lactams tested. Thus, the 5 amino acid substitutions affected the hydrolyzing activity of the enzymes only modestly. Crypticity differences between the three isolates showed that QMCN4 was the least permeable of the isolates to cephaloridine, followed by TAL3636, and that QMCN3 was highly permeable to cephaloridine. Therefore, neither catalytic activity nor permeability was a major contributor to the dramatic differences in the MICs. Instead, microbiological susceptibility was closely related to the level of metallo-beta-lactamase present in each isolate. Both biochemical and physical studies indicated that TAL3636 produced 5- to 10-fold and 50- to 100-fold more metallo-beta-lactamase than QMCN3 and QMCN4, respectively. Therefore, the level of CcrA enzyme production is the dominant contributing factor to high-level resistance among strains harboring a ccrA gene.
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PMCID: PMC284694  PMID: 7811029
17.  Outbreak of Infection with Multidrug-Resistant Klebsiella pneumoniae Carrying blaIMP-8 in a University Medical Center in Taiwan 
Journal of Clinical Microbiology  2001;39(12):4433-4439.
Klebsiella pneumoniae strains with the transferable carbapenem-hydrolyzing metallo-β-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 blaIMP-8, a metallo-β-lactamase gene recently identified from a K. pneumoniae strain in Taiwan. Carriage of blaVIM or other blaIMP genes was detected in none of the remaining isolates. Of the 17 nonrepetitive blaIMP-8-positive isolates, 15 isolates (88.2%) appeared susceptible to imipenem (MICs, ≤4 μg/ml) and meropenem (MICs, ≤1 μg/ml), indicating the difficulty in detecting blaIMP-8 in K. pneumoniae by routine susceptibility tests; 14 isolates (82.4%) produced SHV-12 as well; and 14 isolates (82.4%) were also resistant to fluoroquinolones. The organisms caused wound infections in eight patients and bloodstream infections in three patients. They were not directly associated with the death of nine patients. Before the recovery of the blaIMP-8-positive isolates, all 16 patients had undergone various surgical procedures, and 15 patients had been admitted to the surgical intensive care unit, suggesting a nosocomial outbreak. Two major patterns were observed by pulsed-field gel electrophoresis for 14 of the 17 nonrepetitive isolates, indicating that the clonal spread was mainly responsible for the outbreak.
doi:10.1128/JCM.39.12.4433-4439.2001
PMCID: PMC88561  PMID: 11724857
18.  Global Spread of Carbapenemase-producing Enterobacteriaceae 
Emerging Infectious Diseases  2011;17(10):1791-1798.
These resistance traits have been identified among nosocomial and community-acquired infections.
Carbapenemases increasingly have been reported in Enterobacteriaceae in the past 10 years. Klebsiella pneumoniae carbapenemases have been reported in the United States and then worldwide, with a marked endemicity at least in the United States and Greece. Metallo-enzymes (Verona integron–encoded metallo-β-lactamase, IMP) also have been reported worldwide, with a higher prevalence in southern Europe and Asia. Carbapenemases of the oxacillinase-48 type have been identified mostly in Mediterranean and European countries and in India. Recent identification of New Delhi metallo-β-lactamase-1 producers, originally in the United Kingdom, India, and Pakistan and now worldwide, is worrisome. Detection of infected patients and carriers with carbapenemase producers is necessary for prevention of their spread. Identification of the carbapenemase genes relies mostly on molecular techniques, whereas detection of carriers is possible by using screening culture media. This strategy may help prevent development of nosocomial outbreaks caused by carbapenemase producers, particularly K. pneumoniae.
doi:10.3201/eid1710.110655
PMCID: PMC3310682  PMID: 22000347
antimicrobial resistance; bacteria; carbapenems; carbapenemase; Enterobacteriaceae; metallo-β-lactamase; oxacillinase-48; New Delhi metallo-β-lactamase-1; Verona integron–encoded metallo-β-lactamase; IMP; Klebsiella pneumonia carbapenemase; perspective
19.  Extended-Spectrum β-Lactamase-Producing Klebsiella pneumoniae Strains Causing Nosocomial Outbreaks of Infection in the United Kingdom 
Journal of Clinical Microbiology  1998;36(10):3105-3110.
Representative isolates from 10 distinct extended-spectrum β-lactamase-producing strains of Klebsiella pneumoniae that caused hospital outbreaks in the United Kingdom from 1991 to 1994 were examined for relationships between their enzymes and plasmids. The β-lactamases were identified by a combination of isoelectric focusing and gene sequencing. SHV-2 β-lactamase was produced by isolates from four outbreaks, SHV-5 was involved in three, and SHV-4, TEM-15, and TEM-26 were involved in one outbreak each. All of the extended-spectrum β-lactamases were encoded by self-transmissible plasmids, with sizes ranging from about 70 to 160 kb. No similarities between the restriction digest patterns of the extended-spectrum β-lactamase-encoding plasmids were detected, except to some extent between those that produced TEM-15 and TEM-26. Thus, outbreaks of hospital infection with these organisms in the United Kingdom from 1991 to 1994 involved distinct organisms and resistance plasmids and appeared to be unrelated.
PMCID: PMC105128  PMID: 9738084
20.  Bench-to-bedside review: The role of β-lactamases in antibiotic-resistant Gram-negative infections 
Critical Care  2010;14(3):224.
Multidrug resistance has been increasing among Gram-negative bacteria and is strongly associated with the production of both chromosomal- and plasmid-encoded β-lactamases, whose number now exceeds 890. Many of the newer enzymes exhibit broad-spectrum hydrolytic activity against most classes of β-lactams. The most important plasmid-encoded β-lactamases include (a) AmpC cephalosporinases produced in high quantities, (b) the expanding families of extended-spectrum β-lactamases such as the CTX-M enzymes that can hydrolyze the advanced-spectrum cephalosporins and monobactams, and (c) carbapenemases from multiple molecular classes that are responsible for resistance to almost all β-lactams, including the carbapenems. Important plasmid-encoded carbapenemases include (a) the KPC β-lactamases originating in Klebsiella pneumoniae isolates and now appearing worldwide in pan-resistant Gram-negative pathogens and (b) metallo-β-lactamases that are produced in organisms with other deleterious β-lactamases, causing resistance to all β-lactams except aztreonam. β-Lactamase genes encoding these enzymes are often carried on plasmids that bear additional resistance determinants for other antibiotic classes. As a result, some infections caused by Gram-negative pathogens can now be treated with only a limited number, if any, antibiotics. Because multidrug resistance in Gram-negative bacteria is observed in both nosocomial and community isolates, eradication of these resistant strains is becoming more difficult.
doi:10.1186/cc8892
PMCID: PMC2911681  PMID: 20594363
21.  Analysis of the Functional Contributions of Asn233 in Metallo-β-Lactamase IMP-1 ▿ 
Antimicrobial Agents and Chemotherapy  2011;55(12):5696-5702.
Metallo-β-lactamases, such as IMP-1, are a major global health threat, as they provide for bacterial resistance to a wide range of β-lactam antibiotics, including carbapenems. Understanding the molecular details of the enzymatic process and the sequence requirements for function are essential aids in overcoming β-lactamase-mediated resistance. An asparagine residue is conserved at position 233 in approximately 67% of all metallo-β-lactamases. Despite its conservation, the molecular basis of Asn233 function is poorly understood and remains controversial. It has previously been shown that mutations at this site exhibit context-dependent sequence requirements in that the importance of a given amino acid depends on the antibiotic being tested. To provide a more thorough examination as to the function and sequence requirements at this position, a collection of IMP-1 mutants encoding each of the 19 possible amino acid substitutions was generated. The resistance levels toward four β-lactam antibiotics were measured for Escherichia coli containing each of these mutants. The sequence requirements at position 233 for wild-type levels of resistance toward two cephalosporins were the most relaxed, while there were more stringent sequence requirements for resistance to ampicillin or imipenem. Enzyme kinetic analysis and determinations of steady-state protein levels indicated that the effects of the substitutions on resistance are due to changes in the kinetic parameters of the enzyme. Taken together, the results indicate that substitutions at position 233 significantly alter the kinetic parameters of the enzyme, but most substituted enzymes are able to provide for a high level of resistance to a broad range of β-lactams.
doi:10.1128/AAC.00340-11
PMCID: PMC3232802  PMID: 21896903
22.  Chromosomal beta-lactamase genes of Klebsiella oxytoca are divided into two main groups, blaOXY-1 and blaOXY-2. 
The chromosomally encoded beta-lactamase gene (blaOXY-2) of the wild-type Klebsiella oxytoca SL911 was cloned and sequenced. Its nucleotide sequence similarity with the previously sequenced K. oxytoca beta-lactamase gene (blaOXY-1) (Y. Arakawa, M. Ohta, N. Kido, M. Mori, H. Ito, T. Komatsu, Y. Fujii, and N. Kato, Antimicrob. Agents Chemother. 33:63-70, 1989) is 87.3%, and its amino acid similarity is 89.7%. This group of K. oxytoca beta-lactamases is related to chromosomal beta-lactamases of Citrobacter diversus, Proteus vulgaris, and Yersinia enterocolitica and to the plasmid-mediated extended-spectrum beta-lactamases MEN-1 and Toho-1. By colony hybridization with 86 strains susceptible and resistant to aztreonam, isolated in six countries, K. oxytoca beta-lactamase genes hybridized with either a specific blaOXY-1 DNA probe (668 bp) or a blaOXY-2 DNA probe (723 bp). Thus, beta-lactamase genes could be divided into two groups: blaOXY-1 (47% of the strains) and blaOXY-2 (53% of the strains). A study of isoelectric points confirmed the great variability reported in the literature. However, the two beta-lactamase groups were each represented by four different pIs: for OXY-2, 5.2, 5.7, 6.4, and 6.8, with the 5.2 form representing 59% of all OXY-2 enzymes, and for OXY-1, 7.1, 7.5, 8.2, and 8.8, with the 7.5 form representing 88% of all OXY-1 enzymes.
PMCID: PMC163133  PMID: 8834897
23.  Distribution of the cfiA Gene among Bacteroides fragilis Strains in Japan and Relatedness of cfiA to Imipenem Resistance 
Antimicrobial Agents and Chemotherapy  1999;43(11):2808-2810.
The cfiA gene, encoding an imipenem-hydrolyzing metallo-β-lactamase produced by Bacteroides fragilis, and insertion-like elements were detected by PCR amplification with B. fragilis strains isolated in Japan. The cfiA gene was found in 1.9 and 4.1% of the imipenem-susceptible B. fragilis isolates collected from 1987 to 1988 and from 1992 to 1994, respectively. Insertion-like elements adjacent to the cfiA gene were found in all nine metallo-β-lactamase-producing imipenem-resistant strains tested but not in nine cfiA-positive strains with no detectable metallo-β-lactamase activity.
PMCID: PMC89567  PMID: 10543771
24.  Nosocomial Infections Caused by Multidrug-Resistant Isolates of Pseudomonas putida Producing VIM-1 Metallo-β-Lactamase 
Journal of Clinical Microbiology  2002;40(11):4051-4055.
Successful carbapenem-based chemotherapy for the treatment of Pseudomonas infections has been seriously hindered by the recent appearance of IMP- and VIM-type metallo-β-lactamases, which confer high-level resistance to carbapenems and most other β-lactams. Recently, multidrug-resistant Pseudomonas putida isolates for which carbapenem MICs were ≥32 μg/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-β-lactamase activity, while molecular analysis detected in each isolate a blaVIM-1 determinant carried by an apparently identical medium-sized plasmid. Conjugation experiments were unsuccessful in transferring the β-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 blaVIM-1 determinant (named In110) showed that the blaVIM-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 blaVIM-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.
doi:10.1128/JCM.40.11.4051-4055.2002
PMCID: PMC139695  PMID: 12409373
25.  Metallo-β-Lactamases: the Quiet before the Storm? 
Clinical Microbiology Reviews  2005;18(2):306-325.
The ascendancy of metallo-β-lactamases within the clinical sector, while not ubiquitous, has nonetheless been dramatic; some reports indicate that nearly 30% of imipenem-resistant Pseudomonas aeruginosa strains possess a metallo-β-lactamase. Acquisition of a metallo-β-lactamase gene will invariably mediate broad-spectrum β-lactam resistance in P. aeruginosa, but the level of in vitro resistance in Acinetobacter spp. and Enterobacteriaceae is less dependable. Their clinical significance is further embellished by their ability to hydrolyze all β-lactams and by the fact that there is currently no clinical inhibitor, nor is there likely to be for the foreseeable future. The genes encoding metallo-β-lactamases are often procured by class 1 (sometimes class 3) integrons, which, in turn, are embedded in transposons, resulting in a highly transmissible genetic apparatus. Moreover, other gene cassettes within the integrons often confer resistance to aminoglycosides, precluding their use as an alternative treatment. Thus far, the metallo-β-lactamases encoded on transferable genes include IMP, VIM, SPM, and GIM and have been reported from 28 countries. Their rapid dissemination is worrisome and necessitates the implementation of not just surveillance studies but also metallo-β-lactamase inhibitor studies securing the longevity of important anti-infectives.
doi:10.1128/CMR.18.2.306-325.2005
PMCID: PMC1082798  PMID: 15831827

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