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1.  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
2.  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
3.  Interactions of β-Lactamases with Sanfetrinem (GV 104326) Compared to Those with Imipenem and with Oral β-Lactams 
Sanfetrinem is a trinem β-lactam which can be administered orally as a hexatil ester. We examined whether its β-lactamase interactions resembled those of the available carbapenems, i.e., stable to AmpC and extended-spectrum β-lactamases but labile to class B and functional group 2f enzymes. The comparator drugs were imipenem, oral cephalosporins, and amoxicillin. MICs were determined for β-lactamase expression variants, and hydrolysis was examined directly with representative enzymes. Sanfetrinem was a weak inducer of AmpC β-lactamases below the MIC and had slight lability, with a kcat of 0.00033 s−1 for the Enterobacter cloacae enzyme. Its MICs for AmpC-derepressed E. cloacae and Citrobacter freundii were 4 to 8 μg/ml, compared with MICs of 0.12 to 2 μg/ml for AmpC-inducible and -basal strains; MICs for AmpC-derepressed Serratia marcescens and Morganella morganii were not raised. Cefixime and cefpodoxime were more labile than sanfetrinem to the E. cloacae AmpC enzyme, and AmpC-derepressed mutants showed much greater resistance; imipenem was more stable and retained full activity against derepressed mutants. Like imipenem, sanfetrinem was stable to TEM-1 and TEM-10 enzymes and retained full activity against isolates and transconjugants with various extended-spectrum TEM and SHV enzymes, whereas these organisms were resistant to cefixime and cefpodoxime. Sanfetrinem, like imipenem and cefixime but unlike cefpodoxime, also retained activity against Proteus vulgaris and Klebsiella oxytoca strains that hyperproduced potent chromosomal class A β-lactamases. Functional group 2f enzymes, including Sme-1, NMC-A, and an unnamed enzyme from Acinetobacter spp., increased the sanfetrinem MICs by up to 64-fold. These enzymes also compromised the activities of imipenem and amoxicillin but not those of the cephalosporins. The hydrolysis of sanfetrinem was examined with a purified Sme-1 enzyme, and biphasic kinetics were found. Finally, zinc β-lactamases, including IMP-1 and the L1 enzyme of Stenotrophomonas maltophilia, conferred resistance to sanfetrinem and all other β-lactams tested, and hydrolysis was confirmed with the IMP-1 enzyme. We conclude that sanfetrinem has β-lactamase interactions similar to those of the available carbapenems except that it is a weaker inducer of AmpC types, with some tendency to select derepressed mutants, unlike imipenem and meropenem.
PMCID: PMC105767  PMID: 9593145
4.  The Structure of the Dizinc Subclass B2 Metallo-β-Lactamase CphA Reveals that the Second Inhibitory Zinc Ion Binds in the Histidine Site▿  
Antimicrobial Agents and Chemotherapy  2009;53(10):4464-4471.
Bacteria can defend themselves against β-lactam antibiotics through the expression of class B β-lactamases, which cleave the β-lactam amide bond and render the molecule harmless. There are three subclasses of class B β-lactamases (B1, B2, and B3), all of which require Zn2+ for activity and can bind either one or two zinc ions. Whereas the B1 and B3 metallo-β-lactamases are most active as dizinc enzymes, subclass B2 enzymes, such as Aeromonas hydrophila CphA, are inhibited by the binding of a second zinc ion. We crystallized A. hydrophila CphA in order to determine the binding site of the inhibitory zinc ion. X-ray data from zinc-saturated crystals allowed us to solve the crystal structures of the dizinc forms of the wild-type enzyme and N220G mutant. The first zinc ion binds in the cysteine site, as previously determined for the monozinc form of the enzyme. The second zinc ion occupies a slightly modified histidine site, where the conserved His118 and His196 residues act as metal ligands. This atypical coordination sphere probably explains the rather high dissociation constant for the second zinc ion compared to those observed with enzymes of subclasses B1 and B3. Inhibition by the second zinc ion results from immobilization of the catalytically important His118 and His196 residues, as well as the folding of the Gly232-Asn233 loop into a position that covers the active site.
doi:10.1128/AAC.00288-09
PMCID: PMC2764157  PMID: 19651913
5.  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
6.  Characterization of VIM-2, a Carbapenem-Hydrolyzing Metallo-β-Lactamase and Its Plasmid- and Integron-Borne Gene from a Pseudomonas aeruginosa Clinical Isolate in France 
Pseudomonas aeruginosa COL-1 was identified in a blood culture of a 39-year-old-woman treated with imipenem in Marseilles, France, in 1996. This strain was resistant to β-lactams, including ureidopenicillins, ticarcillin-clavulanic acid, cefepime, ceftazidime, imipenem, and meropenem, but remained susceptible to the monobactam aztreonam. The carbapenem-hydrolyzing β-lactamase gene of P. aeruginosa COL-1 was cloned, sequenced, and expressed in Escherichia coli DH10B. The deduced 266-amino-acid protein was an Ambler class B β-lactamase, with amino acid identities of 32% with B-II from Bacillus cereus; 31% with IMP-1 from several gram-negative rods in Japan, including P. aeruginosa; 27% with CcrA from Bacteroides fragilis; 24% with BlaB from Chryseobacterium meningosepticum; 24% with IND-1 from Chryseobacterium indologenes; 21% with CphA-1 from Aeromonas hydrophila; and 11% with L-1 from Stenotrophomonas maltophilia. It was most closely related to VIM-1 β-lactamase recently reported from Italian P. aeruginosa clinical isolates (90% amino acid identity). Purified VIM-2 β-lactamase had a pI of 5.6, a relative molecular mass of 29.7 kDa, and a broad substrate hydrolysis range, including penicillins, cephalosporins, cephamycins, oxacephamycins, and carbapenems, but not monobactams. As a metallo-β-lactamase, its activity was zinc dependent and inhibited by EDTA (50% inhibitory concentration, 50 μM). VIM-2 conferred a resistance pattern to β-lactams in E. coli DH10B that paralleled its in vitro hydrolytic properties, except for susceptibility to ureidopenicillins, carbapenems, and cefepime. blaVIM-2 was located on a ca. 45-kb plasmid that in addition conferred resistance to sulfamides and that was not self-transmissible either from P. aeruginosa to E. coli or from E. coli to E. coli. blaVIM-2 was the only gene cassette located within the variable region of a novel class 1 integron, In56, that was weakly related to the blaVIM-1-containing integron. VIM-2 is the second carbapenem-hydrolyzing metalloenzyme characterized from a P. aeruginosa isolate outside Japan.
PMCID: PMC89788  PMID: 10722487
7.  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
8.  One origin for metallo-β-lactamase activity, or two? An investigation assessing a diverse set of reconstructed ancestral sequences based on a sample of phylogenetic trees 
Journal of Molecular Evolution  2014;79(3-4):117-129.
Bacteria use metallo-β-lactamase enzymes to hydrolyse lactam rings found in many antibiotics, rendering them ineffective. Metallo-β-lactamase activity is thought to be polyphyletic, having arisen on more than one occasion within a single functionally diverse homologous superfamily. Since discovery of multiple origins of enzymatic activity conferring antibiotic resistance has broad implications for the continued clinical use of antibiotics, we test the hypothesis of polyphyly further; if lactamase function has arisen twice independently, the most recent common ancestor (MRCA) is not expected to possess lactam-hydrolysing activity. Two major problems present themselves. Firstly, even with a perfectly known phylogeny, ancestral sequence reconstruction is error prone. Secondly, the phylogeny is not known, and in fact reconstructing a single, unambiguous phylogeny for the superfamily has proven impossible. To obtain a more statistical view of the strength of evidence for or against MRCA lactamase function, we reconstructed a sample of 98 MRCAs of the metallo-β-lactamases, each based on a different tree in a bootstrap sample of reconstructed phylogenies. InterPro sequence signatures and homology modelling were then used to assess our sample of MRCAs for lactamase functionality. Only 5 % of these models conform to our criteria for metallo-β-lactamase functionality, suggesting that the ancestor was unlikely to have been a metallo-β-lactamase. On the other hand, given that ancestral proteins may have had metallo-β-lactamase functionality with variation in sequence and structural properties compared with extant enzymes, our criteria are conservative, estimating a lower bound of evidence for metallo-β-lactamase functionality but not an upper bound.
Electronic supplementary material
The online version of this article (doi:10.1007/s00239-014-9639-7) contains supplementary material, which is available to authorized users.
doi:10.1007/s00239-014-9639-7
PMCID: PMC4185109  PMID: 25185655
Metallo-β-lactamase; Antibiotic resistance; Phylogenetics; Most recent common ancestor; Ancestral sequence reconstruction; Exaptation
9.  Mutagenesis of Zinc Ligand Residue Cys221 Reveals Plasticity in the IMP-1 Metallo-β-Lactamase Active Site 
Antimicrobial Agents and Chemotherapy  2012;56(11):5667-5677.
Metallo-β-lactamases catalyze the hydrolysis of a broad range of β-lactam antibiotics and are a concern for the spread of drug resistance. To analyze the determinants of enzyme structure and function, the sequence requirements for the subclass B1 IMP-1 β-lactamase zinc binding residue Cys221 were tested by saturation mutagenesis and evaluated for protein expression, as well as hydrolysis of β-lactam substrates. The results indicated that most substitutions at position 221 destabilized the enzyme. Only the enzymes containing C221D and C221G substitutions were expressed well in Escherichia coli and exhibited catalytic activity toward β-lactam antibiotics. Despite the lack of a metal-chelating group at position 221, the C221G enzyme exhibited high levels of catalytic activity in the presence of exogenous zinc. Molecular modeling suggests the glycine substitution is unique among substitutions in that the complete removal of the cysteine side chain allows space for a water molecule to replace the thiol and coordinate zinc at the Zn2 zinc binding site to restore function. Multiple methods were used to estimate the C221G Zn2 binding constant to be 17 to 43 μM. Studies of enzyme function in vivo in E. coli grown on minimal medium showed that both IMP-1 and the C221G mutant exhibited compromised activity when zinc availability was low. Finally, substitutions at residue 121, which is the IMP-1 equivalent of the subclass B3 zinc-chelating position, failed to rescue C221G function, suggesting the coordination schemes of subclasses B1 and B3 are not interchangeable.
doi:10.1128/AAC.01276-12
PMCID: PMC3486559  PMID: 22908171
10.  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
11.  The Legionella (Fluoribacter) gormanii Metallo-β-Lactamase: a New Member of the Highly Divergent Lineage of Molecular-Subclass B3 β-Lactamases 
A metallo-β-lactamase determinant was cloned from a genomic library of Legionella (Fluoribacter) gormanii ATCC 33297T constructed in the plasmid vector pACYC184 and transformed into Escherichia coli DH5α, by screening for clones showing a reduced susceptibility to imipenem. The product of the cloned determinant, named FEZ-1, contains a 30-kDa polypeptide and exhibits an isoelectric pH of 7.6. Sequencing revealed that FEZ-1 is a molecular-class B β-lactamase which shares the closest structural similarity (29.7% of identical residues) with the L1 enzyme of Stenotrophomonas maltophilia, being a new member of the highly divergent subclass B3 lineage. All the residues that in L1 are known to be directly or indirectly involved in coordination of the zinc ions were found to be conserved also in FEZ-1, suggesting that the geometry of zinc coordination in the active site of the latter enzyme is identical to that of L1. Unlike L1, however, FEZ-1 appeared to be monomeric in gel permeation chromatography experiments and exhibited a distinctive substrate specificity with a marked preference for cephalosporins and meropenem. The properties of FEZ-1 overall resembled those of a β-lactamase previously purified from the same strain of L. gormanii (T. Fujii, K. Sato, K. Miyata, M. Inoue, and S. Mitsuhashi, Antimicrob. Agents Chemother. 29:925–926, 1986) and are as yet unique among class B enzymes, reinforcing the notion that considerable functional heterogeneity can be encountered among members of this class. A system for overexpression of the blaFEZ-1 gene in E. coli, based on the T7 phage promoter, was also developed.
PMCID: PMC89909  PMID: 10817705
12.  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
13.  Rapid identification of metallo- and serine beta-lactamases. 
Simple methods to detect, identify, and differentiate metallo- and serine beta-lactamases were developed and used to differentiate enzymes produced by 17 clinical isolates of Xanthomonas maltophilia. All isolates exhibited beta-lactamase activity, and in 16 strains this was induced by imipenem. All but one isolate hydrolyzed imipenem (and meropenem), and in all cases this activity was inhibited by 1 mM EDTA. The metallo- and serine beta-lactamases in the cell extracts were distinguished on isoelectric focusing (IEF) gels by using the following procedures. (i) Cell lysates were preincubated with 83 mM EDTA prior to IEF and subsequent visualization with nitrocefin, and (ii) after IEF, the gels were overlaid with either 1 mM zinc sulfate or 100 microM BRL 42715 before staining with nitrocefin. Bands of beta-lactamase activity which were removed by BRL 42715 but unaffected by EDTA or zinc sulfate were categorized as serine beta-lactamases. Bands which were unaffected by BRL 42715 but inhibited by EDTA or enhanced by zinc sulfate were classified as metallo-beta-lactamases. By using this approach, seven metallo-beta-lactamases were differentiated with pI values of 4.8 (two strains), 5.5 (four strains), 5.7 (one strain), 6.0 (one strain), 6.4 (four strains), 6.6 (one strain), and 6.8 (three strains). The metallo-beta-lactamase band with a pI of 6.4 aligned with the recently characterized metallo-beta-lactamase from X. maltophilia 511. Heterogeneity was also observed for the serine beta-lactamases: 14 isolates elaborated serine beta-lactamase activity which focused with major bands with at least eight different pIs. The remaining three strains produced serine beta-lactamases which focused with five distinct bands with pIs of 6.4, 6.2, 5.7, 5.5, and 5.2. We conclude that X. maltophilia produces many types of metallo- and serine beta-lactamases distinguishable by these new methods and that the previously reported L-1 and L-2 enzymes are not solely representative of the beta-lactamases produced by this species.
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PMCID: PMC188139  PMID: 8067782
14.  Biochemical Characterization of the FEZ-1 Metallo-β-Lactamase of Legionella gormanii ATCC 33297T Produced in Escherichia coli 
The blaFEZ-1 gene coding for the metallo-β-lactamase of Legionella (Fluoribacter) gormanii ATCC 33297T was overexpressed via a T7 expression system in Escherichia coli BL21(DE3)(pLysS). The product was purified to homogeneity in two steps with a yield of 53%. The FEZ-1 metallo-β-lactamase exhibited a broad-spectrum activity profile, with a preference for cephalosporins such as cephalothin, cefuroxime, and cefotaxime. Monobactams were not hydrolyzed. The β-lactamase was inhibited by metal chelators. FEZ-1 is a monomeric enzyme with a molecular mass of 29,440 Da which possesses two zinc-binding sites. Its zinc content did not vary in the pH range of 5 to 9, but the presence of zinc ions modified the catalytic efficiency of the enzyme. A model of the FEZ-1 three-dimensional structure was built.
doi:10.1128/AAC.45.4.1254-1262.2001
PMCID: PMC90452  PMID: 11257043
15.  Updated Functional Classification of β-Lactamases▿  
Two classification schemes for β-lactamases are currently in use. The molecular classification is based on the amino acid sequence and divides β-lactamases into class A, C, and D enzymes which utilize serine for β-lactam hydrolysis and class B metalloenzymes which require divalent zinc ions for substrate hydrolysis. The functional classification scheme updated herein is based on the 1995 proposal by Bush et al. (K. Bush, G. A. Jacoby, and A. A. Medeiros, Antimicrob. Agents Chemother. 39:1211-1233, 1995). It takes into account substrate and inhibitor profiles in an attempt to group the enzymes in ways that can be correlated with their phenotype in clinical isolates. Major groupings generally correlate with the more broadly based molecular classification. The updated system includes group 1 (class C) cephalosporinases; group 2 (classes A and D) broad-spectrum, inhibitor-resistant, and extended-spectrum β-lactamases and serine carbapenemases; and group 3 metallo-β-lactamases. Several new subgroups of each of the major groups are described, based on specific attributes of individual enzymes. A list of attributes is also suggested for the description of a new β-lactamase, including the requisite microbiological properties, substrate and inhibitor profiles, and molecular sequence data that provide an adequate characterization for a new β-lactam-hydrolyzing enzyme.
doi:10.1128/AAC.01009-09
PMCID: PMC2825993  PMID: 19995920
16.  Purification and Biochemical Characterization of the VIM-1 Metallo-β-Lactamase 
Antimicrobial Agents and Chemotherapy  2000;44(11):3003-3007.
VIM-1 is a new group 3 metallo-β-lactamase recently detected in carbapenem-resistant nosocomial isolates of Pseudomonas aeruginosa from the Mediterranean area. In this work, VIM-1 was purified from an Escherichia coli strain carrying the cloned blaVIM-1 gene by means of an anion-exchange chromatography step followed by a gel permeation chromatography step. The purified enzyme exhibited a molecular mass of 26 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and an acidic pI of 5.1 in analytical isoelectric focusing. Amino-terminal sequencing showed that mature VIM-1 results from the removal of a 26-amino-acid signal peptide from the precursor. VIM-1 hydrolyzes a broad array of β-lactam compounds, including penicillins, narrow- to expanded-spectrum cephalosporins, carbapenems, and mechanism-based serine-β-lactamase inactivators. Only monobactams escape hydrolysis. The highest catalytic constant/Km ratios (>106 M−1 · s−1) were observed with carbenicillin, azlocillin, some cephalosporins (cephaloridine, cephalothin, cefuroxime, cefepime, and cefpirome), imipenem, and biapenem. Kinetic parameters showed remarkable variability with different β-lactams and also within the various penam, cephem, and carbapenem compounds, resulting in no clear preference of the enzyme for any of these β-lactam subfamilies. Significant differences were observed with some substrates between the kinetic parameters of VIM-1 and those of other metallo-β-lactamases. Inactivation assays carried out with various chelating agents (EDTA, 1,10-o-phenanthroline, and pyridine-2,6-dicarboxylic acid) indicated that formation of a ternary enzyme-metal-chelator complex precedes metal removal from the zinc center of the protein and revealed notable differences in the inactivation parameters of VIM-1 with different agents.
PMCID: PMC101593  PMID: 11036013
17.  SMB-1, a Novel Subclass B3 Metallo-β-Lactamase, Associated with ISCR1 and a Class 1 Integron, from a Carbapenem-Resistant Serratia marcescens Clinical Isolate▿ 
Antimicrobial Agents and Chemotherapy  2011;55(11):5143-5149.
A carbapenem-resistant Serratia marcescens strain, 10mdr148, was identified in a Japanese hospital in 2010. The carbapenem resistance of this strain was attributed to the production of a novel metallo-β-lactamase (MBL), named SMB-1 (Serratia metallo-β-lactamase). SMB-1 possessed a zinc binding motif, H(Q)XHXDH (residues 116 to 121), H196, and H263 and was categorized as a member of subclass B3 MBL. SMB-1 has 75% amino acid identity with the most closely related MBL, AMO1, of uncultured bacterium, recently identified through the metagenomic analysis of apple orchard soil. The introduction of blaSMB-1 into Escherichia coli conferred resistance to a variety of β-lactam antibiotics, penicillins, cephalosporins, and carbapenems, but not aztreonam, a resistance pattern consistent with those of other MBLs. SMB-1 demonstrated high kcat values of >500 s−1 for carbapenems, resulting in the highest hydrolyzing efficiency (kcat/Km) among the agents tested. The hydrolyzing activity of SMB-1 was well inhibited by chelating agents. The blaSMB-1 gene was located on the chromosome of S. marcescens strain 10mdr148 and at the 3′ end of the ISCR1 element in complex with a typical class 1 integron carrying aac(6′)-Ib and catB3 gene cassettes. Downstream of blaSMB-1, the second copy of the 3′conserved segment and ISCR1 were found. To our knowledge, this is the first subclass B3 MBL gene associated with an ISCR1 element identified in an Enterobacteriaceae clinical isolate. A variety of antibiotic resistance genes embedded with ISCR1 have been widely spread among Enterobacteriaceae clinical isolates, thus the further dissemination of blaSMB-1 mediated by ISCR1 transposition activity may become a future concern.
doi:10.1128/AAC.05045-11
PMCID: PMC3195065  PMID: 21876060
18.  Biochemical Characterization of the Acquired Metallo-β-Lactamase SPM-1 from Pseudomonas aeruginosa 
SPM-1 is a new metallo-β-lactamase recently identified in Pseudomonas aeruginosa strain 48-1997A, isolated in Sao Paulo, Brazil. Kinetic analysis demonstrated that SPM-1 has a broad hydrolytic profile across a wide range of β-lactam antibiotics. Considerable variation was observed within the penicillin, cephalosporin, and carbapenem subfamilies; however, on the whole, SPM-1 appears to preferentially hydrolyze cephalosporins. The highest kcat/Km ratios (in micromolar per second) overall were observed for this subgroup. The hydrolytic profile of SPM-1 bears the most similarity to that of the metallo-β-lactamase IMP-1, yet for the most part, SPM-1 has kcat/Km values higher than those of IMP-1. Zinc chelator studies established that progressive inhibition of SPM-1 by EDTA, dipicolinic acid, and 1-10-o-phenanthroline demonstrated a biexponential pattern in which none of the chelators completely inhibited SPM-1. A homology model of SPM-1 was developed on the basis of the IMP-1 crystal structure, which showed the protein folding and active-site structure characteristic of metallo-β-lactamases and which provides an explanation for the kinetic profiles observed.
doi:10.1128/AAC.47.2.582-587.2003
PMCID: PMC151762  PMID: 12543663
19.  Probing the role of Met221 in the unusual metallo-β-lactamase GOB-18 
Inorganic chemistry  2012;51(22):12419-12425.
Metallo-β-lactamases (MβLs) are the main mechanism of bacterial resistance against last generation β-lactam antibiotics such as carbapenems. Most MβLs display unusual structural features in their active sites, such as binuclear zinc centers without carboxylate bridging ligands, and/or a Cys ligand in a catalytic zinc site. Cys221 is an essential residue for catalysis conserved in B1 and B2 lactamases, while most B3 enzymes present a Ser in this position. GOB lactamases stand as an exception within this picture, with a Met residue in position 221. Then, we obtained a series of GOB-18 point mutants in order to analyze the role of this unusual Met221 residue. We found that Met221 is essential for the protein stability, most likely due to its involvement in a hydrophobic core. In contrast to other known MβLs, residue 221 is not involved in metal binding or in catalysis in GOB enzymes, according to spectroscopic and kinetic studies. Our findings show that the essential catalytic features are maintained despite the structural heterogeneity among MβLs, and suggest that a strategy to design general inhibitors should be undertaken based on mechanistic rather than on structural information.
doi:10.1021/ic301801h
PMCID: PMC3593996  PMID: 23113650
20.  In Vitro and In Vivo Properties of BAL30376, a β-Lactam and Dual β-Lactamase Inhibitor Combination with Enhanced Activity against Gram-Negative Bacilli That Express Multiple β-Lactamases ▿  
BAL30376 is a triple combination comprising a siderophore monobactam, BAL19764; a novel bridged monobactam, BAL29880, which specifically inhibits class C β-lactamases; and clavulanic acid, which inhibits many class A and some class D β-lactamases. The MIC90 was ≤4 μg/ml (expressed as the concentration of BAL19764) for most species of the Enterobacteriaceae family, including strains that produced metallo-β-lactamases and were resistant to all of the other β-lactams tested. The MIC90 for Stenotrophomonas maltophilia was 2 μg/ml, for multidrug-resistant (MDR) Pseudomonas aeruginosa it was 8 μg/ml, and for MDR Acinetobacter and Burkholderia spp. it was 16 μg/ml. The presence of the class C β-lactamase inhibitor BAL29880 contributed significantly to the activity of BAL30376 against strains of Citrobacter freundii, Enterobacter species, Serratia marcescens, and P. aeruginosa. The presence of clavulanic acid contributed significantly to the activity against many strains of Escherichia coli and Klebsiella pneumoniae that produced class A extended-spectrum β-lactamases. The activity of BAL30376 against strains with metallo-β-lactamases was largely attributable to the intrinsic stability of the monobactam BAL19764 toward these enzymes. Considering its three components, BAL30376 was unexpectedly refractory toward the development of stable resistance.
doi:10.1128/AAC.01370-10
PMCID: PMC3067176  PMID: 21245441
21.  Cloning of a Chryseobacterium (Flavobacterium) meningosepticum Chromosomal Gene (blaACME) Encoding an Extended-Spectrum Class A β-Lactamase Related to the Bacteroides Cephalosporinases and the VEB-1 and PER β-Lactamases 
In addition to the BlaB metallo-β-lactamase, Chryseobacterium (Flavobacterium) meningosepticum CCUG 4310 (NCTC 10585) constitutively produces a 31-kDa active-site serine β-lactamase, named CME-1, with an alkaline isoelectric pH. The blaACME gene that encodes the latter enzyme was isolated from a genomic library constructed in the Escherichia coli plasmid vector pACYC184 by screening for cefuroxime-resistant clones. Sequence analysis revealed that the CME-1 enzyme is a new class A β-lactamase structurally divergent from the other members of this class, being most closely related to the VEB-1 (also named CEF-1) and PER β-lactamases and the Bacteroides chromosomal cephalosporinases. The blaACME determinant is located on the chromosome and exhibits features typical of those of C. meningosepticum resident genes. The CME-1 protein was purified from an E. coli strain that overexpresses the cloned gene via a T7-based expression system by means of an anion-exchange chromatography step followed by a gel permeation chromatography step. Kinetic parameters for several substrates were determined. CME-1 is a clavulanic acid-susceptible extended-spectrum β-lactamase that hydrolyzes most cephalosporins, penicillins, and monobactams but that does not hydrolyze cephamycins and carbapenems. The enzyme exhibits strikingly different kinetic parameters for different classes of β-lactams, with both Km and kcat values much higher for cephalosporins than for penicillins and monobactams. However, the variability of both kinetic parameters resulted in overall similar acylation rates (kcat/Km ratios) for all types of β-lactam substrates.
PMCID: PMC89445  PMID: 10471563
22.  Complete 1H, 15N and 13C resonance assignments of Bacillus cereus metallo-β-lactamase and its complex with the inhibitor R-thiomandelic acid 
Biomolecular Nmr Assignments  2013;8(2):313-318.
β-Lactamases inactivate β-lactam antibiotics by hydrolysis of their endocyclic β-lactam bond and are a major cause of antibiotic resistance in pathogenic bacteria. The zinc dependent metallo-β-lactamase enzymes are of particular concern since they are located on highly transmissible plasmids and have a broad spectrum of activity against almost all β-lactam antibiotics. We present here essentially complete (>96 %) backbone and sidechain sequence-specific NMR resonance assignments for the Bacillus cereus subclass B1 metallo-β-lactamase, BcII, and for its complex with R-thiomandelic acid, a broad spectrum inhibitor of metallo-β-lactamases. These assignments have been used as the basis for determination of the solution structures of the enzyme and its inhibitor complex and can also be used in a rapid screen for other metallo-β-lactamase inhibitors.
doi:10.1007/s12104-013-9507-1
PMCID: PMC4145196  PMID: 23838816
Metallo-β-lactamase; R-thiomandelic acid; BcII; BcII–inhibitor complex
23.  Cloning and sequence analysis of the gene for a carbapenem-hydrolyzing class A beta-lactamase, Sme-1, from Serratia marcescens S6. 
Serratia marcescens S6 produces a pI 9.7 carbapenem-hydrolyzing beta-lactamase that is probably encoded by the chromosome (Y. Yang, P. Wu, and D. M. Livermore, Antimicrob. Agents Chemother. 34:755-758, 1990). A total of 11.3 kb of genomic DNA from this strain was cloned into plasmid pACYC184 in Escherichia coli. After further subclonings, the carbapenem-hydrolyzing beta-lactamase gene (blaSme-1) was sequenced (EMBL accession number Z28968). The gene corresponded to an 882-bp open reading frame which encoded a 294-amino-acid polypeptide. This open reading frame was preceded by a -10 and a -35 region consistent with a putative promoter sequence of members of the family Enterobacteriaceae. This promoter was active in E. coli and S. marcescens, as demonstrated by primer extension analysis. N-terminal sequencing showed that the Sme-1 enzyme had a 27-amino-acid leader peptide and enabled calculation of the molecular mass of the mature protein (29.3 kDa). Sequence alignment revealed that Sme-1 is a class A serine beta-lactamase and not a class B metalloenzyme. The earlier view that the enzyme was zinc dependent was discounted. Among class A beta-lactamases, Sme-1 had the greatest amino acid identity (70%) with the pI 6.9 carbapenem-hydrolyzing beta-lactamase, NMC-A, from Enterobacter cloacae NOR-1. Comparison of these two protein sequences suggested a role for specific residues in carbapenem hydrolysis. The relatedness of Sme-1 to other class A beta-lactamases such as the TEM and SHV types was remote. This work details the sequence of the second carbapenem-hydrolyzing class A beta-lactamase from an enterobacterial species and the first in the genus Serratia.
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PMCID: PMC188196  PMID: 8092824
24.  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
25.  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

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