Hydrolysis Of Imipenem Research Articles

Carbapenemase-producing Enterobacteriaceae in Finland: the first years (2008-11)

Carbapenemase-producing Enterobacteriaceae (CPE) are becoming a global problem; they are often resistant to nearly all available antibiotics. Here we report details on all Finnish CPE isolates found until the end of 2011: carbapenemase genes, travel history and multilocus sequence typing (MLST) data. Enterobacteriaceae sent to the Antimicrobial Resistance Unit of the National Institute for Health and Welfare were tested for susceptibility to carbapenems, screened for carbapenemases by PCR and isolates with decreased susceptibility to carbapenems were tested for hydrolysis of imipenem. Carbapenemase-producing Escherichia coli and Klebsiella pneumoniae isolates were typed by MLST. In all, 26 CPE strains were found from 25 patients: 10 with OXA-48-like enzymes, 5 with KPC, 4 with VIM, 3 with NDM, 3 with IMI/NMC-A and 1 with GES-14. The species were K. pneumoniae (n = 16), E. coli (n = 6), Enterobacter cloacae (n = 3) and Raoultella planticola (n = 1). Of the 25 patients, 18 had a known travel history/hospital transfer from abroad. Local spread/transmission was suspected in 2011, but there were no hospital outbreaks. The K. pneumoniae multilocus sequence types ST258, ST182, ST147, ST244, ST14, ST13, ST383, ST101 and ST15, and the E. coli sequence types ST38 and ST90 were found. Many of these are global epidemic clones. CPE strains are increasingly found in Finland, but still at a very low prevalence.

Abstract 2659: Cilastatin attenuates cisplatin-induced proximal tubular cell damage without compromising antitumoral activity

Abstract Introduction: Cisplatin (CDDP) is a very effective and common treatment in solid malignancies. One of the most important side effects of CDDP is the nephrotoxicity. Nephrotoxicity is a limitating side effect on treatment with CDDP, preventing patients with limit kidney function of receiving the drug and stopping treatment once kidney function has worsened. Cilastatin (Cls) is a specific inhibitor of renal dedydrodipeptidase I (DHP-I) which prevents hydrolysis of imipenem and its accumulation in the proximal tubule. In this work we hypothesized that Cls acts as a nephroprotector against CDDP-induced damage without compromising antitumor activity. Methods: Primary cultures of proximal tubular cells (PTCs) and cell lines of different malignancies (colon, breast, ovarian, bladder) were cultured with different concentrations of CDDP (1, 10 and 30 μM) in the presence or absence of Cls. Cell viability was assessed with MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide) assay. Raft staining was measured with toxine B choleric and FasL by confocal microscopy. Results: Cls interfered with CDDP-induced FasL signalling at raft level on PTCs brush border. Concomitant treatment with Cls reduced CDDP-induced changes. Several tumoral cell lines were tested with CDDP and Cls together. Cls did not increase or decreased tumor growth alone or in combination with CDDP. CDDP sensitivity was not affected by Cls. Conclusion: By binding a DHP-I, Cls blocks CDDP-induced PTCs apoptosis but it does not affect the CDDP antitumoral activity. Our findings suggest that the affinity of Cls for renal DHP-I make this effect specific for proximal tubular cells and may be related to a reduction in intracellular drug accumulation. Cls administration might represent a novel strategy in the prevention of CDDP-induced acute renal injury. Cls treatment could potentially increase the number of patients undergoing CDDP treatment or maintaining treatment. Further clinical trials for renal function preservation in cancer patients are warranted. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2659. doi:1538-7445.AM2012-2659

Cilastatin Attenuates Cisplatin-Induced Proximal Tubular Cell Damage

A major area in cancer therapy is the search for protective strategies against cisplatin-induced nephrotoxicity. We investigated the protective effect of cilastatin on cisplatin-induced injury to renal proximal tubular cells. Cilastatin is a specific inhibitor of renal dehydrodipeptidase I (DHP-I), which prevents hydrolysis of imipenem and its accumulation in the proximal tubule. Primary cultures of proximal cells were treated with cisplatin (1-30 microM) in the presence or absence of cilastatin (200 microg/ml). Apoptosis and mitochondrial injury were assessed by different techniques. Cisplatin uptake and DNA binding were measured by inductively coupled plasma spectrometry. HeLa cells were used to control the effect of cilastatin on the tumoricidal activity of cisplatin. Cisplatin increased cell death, apoptotic-like morphology, caspase activation, and mitochondrial injury in proximal tubular cells in a dose- and time-dependent way. Concomitant treatment with cilastatin reduced cisplatin-induced changes. Cilastatin also reduced the DNA-bound platinum but did not modify cisplatin-dependent up-regulation of death receptors (Fas) or ligands (tumor necrosis factor alpha, Fas ligand). In contrast, cilastatin did not show any effects on cisplatin-treated HeLa cells. Renal DHP-I was virtually absent in HeLa cells. Cilastatin attenuates cisplatin-induced cell death in proximal tubular cells without reducing the cytotoxic activity of cisplatin in tumor cells. Our findings suggest that the affinity of cilastatin for renal dipeptidase makes this effect specific for proximal tubular cells and may be related to a reduction in intracellular drug accumulation. Therefore, cilastatin administration might represent a novel strategy in the prevention of cisplatin-induced acute renal injury.

First Report of Molecular Characterization of Carbapenem-Resistant Acinetobacter baumannii in Different Intensive Care Units in University Hospital Split, Croatia

Acinetobacter baumannii is an important cause of nosocomial infections such as pneumonia, septicaemia, urinary tract infections, and wound infections 1. It may become resistant to a wide range of antibiotics, thus complicating the treatment of nosocomial infections 1. The emergence of carbapenem resistance in Acinetobacter baumannii has become a global concern since these β -lactams are often the only effective treatment left against many multiresistant strains . According to a recent report, the overall resistance rate to imipenem in A. baumannii isolated from 17 representative laboratories in Croatia was 1 % (range 0%-8%) . However, molecular basis of carbapenem resistance was not investigated yet. We would like to report the molecular epidemiology of carbapenem resistant strains obtained from clinical samples of several patients hospitalised on different intensive care units inside University Hospital Split, Croatia. Eleven isolates of A. baumannii with an unusual resistance profile were isolated during 2003 and 2004, from patients hospitalised in three different intensive care units (two adults and one children ICU) on different locations inside UHS. Routine susceptibility testing was done by standard disc diffusion method according to the National Committee for Clinical Laboratory Standards guidelines. All collected isolates of A. baumannii displayed intermediate (MICs>8 mg/L) or resistant (MICs>16 mg/L) profile to imipenem and/or meropenem. Minimum inhibitory concentrations were also determined for ceftazidime, cefepime, ceftriaxone, amikacin, gentamicin, ciprofloxacin and piperacillin-tazobactam by broth microdilution method according to CLSI recommendation.. All isolates were multidrug-resistant exhibiting highly resistance to ceftazidime, cefepime, piperacillin/tazobactam, amikacin, gentamicin, netilmicin and ciprofloxacin. No resistance to ampicillin/sulbactam was observed which is not in use in our hospital. The isolates were genetically characterized with pulsed-field gel electrophoresis (PFGE). The preparation of genomic DNA of A. baumannii isolates was performed as previously described. Oxacillinases were detected by using a standard PCR technique with primers specific for blaOXA-23, blaOXA-40, blaOXA-58, and blaOXA-69 genes. Production of metallo-β -lactamases was screened by E test-MBL and PCR with primers specific for VIM and IMP β -lactamases. Β -lactamases were prepared from overnight broth cultures, released by sonication and cell debris was removed by centrifugation. PCRs were positive for the OXA-69 like enzyme in ten Acinetobacter strains belonging to unique PFGE profile characterised to pulsotype A, while strain characterised to pusotype B was the only strain isolated from children intensive care unit and carbapenemase gene was not found with tested primers. No significant hydrolysis of imipenem was detected. Acccording to the bibliographical data OXA-69 is a naturally occurring oxacillinase in A. baumannii and it does not confer on the producing isolates significant resistance to carbapenems, thus it can be concluded that resistance to imipenem and meropenem in our isolates is due to other mechanisms such as porin loss or efflux. Spread of the pulsotype A with OXA-69-type oxacillinases was due to clonal dissemination of multi-drug resistant A. baumannii between two different adult Intensive Care Units, probably by hospital staff during medical procedures. The infection control team of the hospital implemented restriction of carbapenem usage and strict antiseptic techniques, which included the rigorous use of alcohol-clorhexidine solutions before and between patient and equipment contact and before leaving the unit. To our knowledge, this is the first report on the molecular characterization carbapenem resistance in A. baumannii in Croatia. A similar finding underscores the progressive emergence of these determinants in different geographic areas. Consequently, incidence and spread of multidrug-resistant A. baumannii nosocomial infections suggest the necessity of a surveillance program and enforcing adequate control measures in different hospital settings

Biochemical and Molecular Characterization of Three New Variants of AmpC β-Lactamases from Morganella morganii

Morganella morganii produces an inducible, chromosomally encoded AmpC beta-lactamase. We describe in this study three new variants of AmpC within this species with apparent pIs of 6.6 (M19 from M. morganii strain PP19), 7.4 (M29 from M. morganii strain PP29), and 7.8 (M37 from M. morganii strain PP37). After gene sequencing, deduced amino acid sequences displayed one to six substitutions when compared to the available Morganella AmpC sequences. An AmpR-encoding gene was also found upstream of ampC, including the LysR regulators' helix-turn-helix DNA-binding domain and the putative T-N11-A-protected region in the ampR-ampC intercistronic sequence. All three AmpC variants were purified from in vitro-generated derepressed mutants and showed overall similar kinetic parameters. None of the observed amino acid changes, occurring at the surface of the protein, appear to have a major influence in their catalytic properties. Morganella AmpCs exhibit the highest catalytic efficiencies (k(cat)/K(m)) on classical penicillins, cefoxitin, narrow-spectrum cephalosporins, and cefotaxime. Cefotaxime was more effectively hydrolyzed than other oxyimino-cephalosporins, whereas cefepime was 3 log-fold less efficiently hydrolyzed than other cephalosporins such as cephalothin. Several differences with other AmpC beta-lactamases were found. Ampicillin was more efficiently hydrolyzed than benzylpenicillin. High k(cat)/K(m) values were observed for oxacillin and piperacillin, which are usually poor substrates for AmpC. A fairly efficient hydrolysis of imipenem was detected as well. Aztreonam, carbenicillin, and tazobactam were effective transient inactivators of these variants.

Use of Etest MBL strips for the detection of carbapenemases in Acinetobacter baumannii

Sir, In2002,wesawtheemergenceofcarbapenemresistanceinclinical isolates of Acinetobacter baumannii isolated from patients at two local hospitals, Groote Schuur Hospital (GSH) and Tygerberg Hospital (TH), Western Cape, South Africa. The high levels of imipenem (16–256 mg/L) and meropenem (>32 mg/L) resistance observed in these A. baumannii isolates suggested the presence of a metallo-b-lactamase (MBL) or an oxacillinase, since these carbapenemases are considered the major mechanism of carbapenem resistance in these organisms. 1 To screen for the presence of MBLs, Etest strips (AB Biodisk, Solna, Sweden) were used according to the manufacturer’s instructions. Etest MBL strips contain increasing concentrations of imipenem (IP) on one end and imipenem overlaid with EDTA (IPI) on the other. The EDTA chelates the zinc ions required by MBLs to catalyse hydrolysis of imipenem and meropenem, thereby inhibitingMBLactivity.AreductionintheimipenemMICinthepresence of EDTA of greater than or equal to eight-fold (IP/IPI ‡ 8) is interpreted as indicating MBL activity. A control Pseudomonas aeruginosa strain expressing a metallo-b-lactamase VIM-2 (gift from P. Nordmann, France) had an IP/IPI ratio of >64. Of the 49 A. baumannii isolates included in this study (GSH, n = 13; TH, n = 36), all had an IP/IPI ratio of 8–64, suggesting the presence of an MBL in each of these strains. To elucidate which enzyme was responsible for the observed MBL activity, PCR assays were carried out using primers for amplification of MBL-encoding genes blaIMP and blaVIM. Although PCR products of the expected size were obtained fromP. aeruginosastrainscarryingblaIMP-1(giftfromY.Hirakata, Japan) and blaVIM-2, no PCR products were obtained from any of the 49 carbapenem-resistant A. baumannii isolates screened. This suggested that the strains tested do not contain IMP- or VIMtype MBLs. Notwithstanding the Etest MBL result, PCR assays were carried out to screen for the presence of genes encoding oxacillinases. Amplicons of the expected size were obtained from a control A. baumannii strain containing OXA-23 (gift from L. Poirel, France) and from all 49 test strains. PCR products from a representative strain from each hospital were purified and sequenced. Analysis of the sequencing data obtained showed that the blaOXA genes were identical to blaOXA-23. It is likely, therefore, that the production of OXA-23 is the major mechanism of carbapenem resistance in the GSH and TH isolates, since this enzyme confers high levels of imipenem and meropenem resistance (MIC > 32 mg/L) on an A. baumannii host. 2 One explanation for the anomalous results obtained with the

Probing the Role of Asp-120(81) of Metallo-β-lactamase (IMP-1) by Site-directed Mutagenesis, Kinetic Studies, and X-ray Crystallography

Metallo-beta-lactamase IMP-1 is a di-Zn(II) metalloenzyme that efficiently hydrolyzes beta-lactam antibiotics. Wild-type (WT) IMP-1 has a conserved Asp-120(81) in the active site, which plays an important role in catalysis. To probe the catalytic role of Asp-120(81) in IMP-1, the IMP-1 mutants, D120(81)A and D120(81)E, were prepared by site-directed mutagenesis, and various kinetics studies were conducted. The IMP-1 mutants exhibited 10(2)-10(4)-fold drops in k(cat) values compared with WT despite the fact that they contained two Zn(II) ions in the active site. To evaluate the acid-base characteristics of Asp-120(81), the pH dependence for hydrolysis was examined by stopped-flow studies. No observable pK(a) values between pH 5 and 9 were found for WT and D120(81)A. The rapid mixing of equimolar amounts of nitrocefin and all enzymes failed to result in the detection of an anion intermediate of nitrocefin at 650 nm. These results suggest that Asp-120(81) of IMP-1 is not a factor in decreasing the pK(a) for the water bridging two Zn(II) ions and is not a proton donor to the anionic intermediate. In the case of D120(81)E, the nitrocefin hydrolysis product, which shows a maximum absorption at 460 nm, was bound to D120(81)E in the protonated form. The three-dimensional structures of D120(81)A and D120(81)E were also determined at 2.0 and 3.0 A resolutions, respectively. In the case of D120(81)E, the Zn-Zn distance was increased by 0.3 A compared with WT, due to the change in the coordination mode of Glu-120(81)OE1 and the positional shift in the conserved His-263(197) at the active site.

Analysis of the Context Dependent Sequence Requirements of Active Site Residues in the Metallo-β-lactamase IMP-1

The metallo-beta-lactamase IMP-1 catalyzes the hydrolysis of a broad range of beta-lactam antibiotics to provide bacterial resistance to these compounds. In this study, 29 amino acid residue positions in and near the active-site pocket of the IMP-1 enzyme were randomized individually by site-directed mutagenesis of the corresponding codons in the bla(IMP-1) gene. The 29 random libraries were used to identify positions that are critical for the catalytic and substrate-specific properties of the IMP-1 enzyme. Mutants from each of the random libraries were selected for the ability to confer to Escherichia coli resistance to ampicillin, cefotaxime, imipenem or cephaloridine. The DNA sequence of several functional mutants was determined for each of the substrates. Comparison of the sequences of mutants obtained from the different antibiotic selections indicates the sequence requirements for each position in the context of each substrate. The zinc-chelating residues in the active site were found to be essential for hydrolysis of all antibiotics tested. Several positions, however, displayed context-dependent sequence requirements, in that they were essential for one substrate(s) but not others. The most striking examples included Lys69, Asp84, Lys224, Pro225, Gly232, Asn233, Asp236 and Ser262. In addition, comparison of the results for all 29 positions indicates that hydrolysis of imipenem, cephaloridine and ampicillin has stringent sequence requirements, while the requirements for hydrolysis of cefotaxime are more relaxed. This suggests that more information is required to specify active-site pockets that carry out imipenem, cephaloridine or ampicillin hydrolysis than one that catalyzes cefotaxime hydrolysis.

Dependence of hydrolysis of beta-lactams with a zinc(II)-beta-lactamase produced from Serratia marcescens (IMP-1) on pH and concentration of zinc(II) ion: dissociation of Zn(II) from IMP-1 in acidic medium.

The pH dependence for the hydrolysis of beta-lactam antibiotics by a metallo-beta-lactamase (IMP-1) produced from Serratia marcescens was investigated varying the concentration of Zn(II). The activity of IMP-1 for imipenem was decreased at pH less than pH 5.3 without external addition of Zn(II) ions but was recovered with addition of Zn(II). Varying the concentration of external Zn(II), the molar activity of the enzyme, k(obs), that was defined by the velocity of hydrolysis of imipenem/concentration of IMP-1 was expressed by k(obs)=v(init)/[E](T)=k(max)[Zn]/(K(d)+[Zn]) in which K(d) stands for the dissociation constant between Zn(II) and IMP-1. The dissociation constants, K(d), vary with pH; K(d)=840 x 10(-6) M at pH 4.3 and K(d)=0.19 x 10(-6) M at pH 6.0. The plot of -log K(d) against pH showed a straight line having a slope of 4.0 below pH 5.0, showing the existence of four functional groups which may be protonated upon dissociation of Zn(II) ion(s). The k(cat), K(m), and k(cat)/K(m) of hydrolysis of imipenem and cephalothin in the presence of sufficient concentration of Zn(NO(3))(2) for saturation of IMP-1 with Zn(II) showed similar dependency to each other on pH between pH 6.0 and 9.0.

Clonal diversity and metallo-beta-lactamase production in clinical isolates of Stenotrophomonas maltophilia.

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.

Plasmid location and molecular heterogeneity of the L1 and L2 beta-lactamase genes of Stenotrophomonas maltophilia.

An approximately 200-kb plasmid has been purified from clinical isolates of Stenotrophomonas maltophilia. This plasmid was found in all of the 10 isolates examined and contains both the L1 and the L2 beta-lactamase genes. The location of L1 and L2 on a plasmid makes it more likely that they could spread to other gram-negative bacteria, potentially causing clinical problems. Sequence analysis of the 10 L1 genes revealed three novel genes, L1c, L1d, and L1e, with 8, 12, and 20% divergence from the published strain IID 1275 L1 (L1a), respectively. The most unusual L1 enzyme (L1e) displayed markedly different kinetic properties, with respect to hydrolysis of nitrocefin and imipenem, compared to those of L1a (250- and 100-fold lower k(cat)/K(m) ratios respectively). L1c and L1d, in contrast, displayed levels of hydrolysis very similar to that of L1a. Several nonconservative amino acid differences with respect to L1a, L1b, L1c, and L1d were observed in the substrate binding-catalytic regions of L1e, and this could explain the kinetic differences. Three novel L2 genes (L2b, L2c, and L2d) were sequenced from the same isolates, and their sequences diverge from the published sequence of strain IID 1275 L2 (L2a) by 4, 9, and 25%, respectively. Differences in L1 and L2 gene sequences were not accompanied by similar divergences in 16S rRNA gene sequences, for which differences of <1% were found. It is therefore apparent that the L1 and L2 genes have evolved relatively quickly, perhaps because of their presence on a plasmid.

Amino acid substitutions in a variant of IMP-1 metallo-beta-lactamase.

In the course of surveying for the carbapenem-hydrolyzing metallo-beta-lactamase gene bla(IMP) in pathogenic bacteria by the PCR method, we detected a gene encoding a variant metallo-beta-lactamase, designated IMP-3, which differed from IMP-1 by having low hydrolyzing activity for penicillins and carbapenems. PCR product direct sequencing of a 2.2-kb segment revealed that the gene bla(IMP-3) was located on a cassette inserted within a class I integron in the pMS390 plasmid. The 741-bp nucleotide sequence of bla(IMP-3) was identical to that of bla(IMP-1), except for seven base substitutions. Among these were two, at nucleotide positions 314 and 640, which caused amino acid alterations. Hybrid bla genes were constructed from bla(IMP-3) and bla(IMP-1) by recombinant DNA techniques, and beta-lactamases encoded by these genes were compared with those of the parents IMP-3 and IMP-1 under the same experimental conditions. The kinetic parameters indicated that the inefficient hydrolysis of benzylpenicillin, ampicillin, imipenem, and ceftazidime by IMP-3 was due to the substitution of glycine for serine at amino acid residue 196 in the mature enzyme. This alteration corresponded to the presence of guanine instead of an adenine at nucleotide position 640 of the bla(IMP-3) gene. This indicated that extension of the substrate profile in the metallo-beta-lactamase IMP-1 compared to IMP-3 is the result of a one-step single-base mutation, suggesting that the gene bla(IMP-3) is an ancestor of bla(IMP-1).

Interaction between class B β-lactamases and suicide substrates of active-site serine β-lactamases

The most widely used inactivators of active-site serine β-lactamases behave as substrates of four class B metallo-β-lactamases, but the efficiency of the catalytic process can vary by several orders of magnitude. A comparison of the kinetic parameters for the α and β isomers of 6-iodopenicillanic acid shows that there is no general preference for the α isomer and that the efficient hydrolysis of imipenem by these enzymes must rest on other factors.

Transferable antibiotic resistance in nosocomial Stenotrophomonas maltophilia strain

Stenotrophomonas maltophilia ( 298 85 ) was isolated from the extensively inflamed conjunctiva of a neonate in a regional hospital in Ostrava, Czech Republic. It was resistant to all available antibiotics except cefepime and trimethoprim. The donor S. maltophilia strain 298 85 transferred carbenicillin and cephaloridine resistance determinants to recipient strains of Escherichia coli K-12 3110 rif+ and Proteus mirabilis P-38 rif+. All transconjugant colonies were coresistant also to kanamycin, cefotaxime, and aztreonam. Active hydrolysis of imipenem in the original strain was inhibited by ethylene diamine tetra-acetic acid, and hydrolysis of cefotaxime and aztreonam in the original strain and in the E. coli K-12 transconjugant was inhibited by clavulanate. In contrast, ceftazidime was hydrolyzed by the original strain and was not inhibited by clavulanate, indicating a different character of the resistance to cefotaxime or aztreonam and ceftazidime.

Cloning and expression of a cloxacillin-hydrolyzing enzyme and a cephalosporinase from Aeromonas sobria AER 14M in Escherichia coli: requirement for an E. coli chromosomal mutation for efficient expression of the class D enzyme

Two beta-lactamase genes, asbA1 and asbB1, encoding AsbA1 and AsbB1, respectively, have been cloned from Aeromonas sobria AER 14M into Escherichia coli. AsbA1 was expressed at low but detectable levels in all E. coli laboratory cloning strains tested. AsbB1 was expressed well in the E. coli cloning strain DH5 alpha. However, no enzyme activity could be detected from the same clone when placed in E. coli MC1061. Ampicillin-resistant mutants of E. coli MC1061 were obtained that expressed high levels of enzymatically active AsbB1. Four independent mutants were examined. All four mutations mapped to one locus, designated blpA (beta-lactamase permissive). The blpA locus was distinct from other known loci that play a role in beta-lactamase expression, i.e., the two loci that affect expression of the Bacteroides fragilis metallo-beta-lactamase and the ampC regulatory genes, ampD, ampE, and ampG. Sequence analysis of asbA1 and asbB1 revealed that AsbA1 was a class C beta-lactamase most closely related to the Pseudomonas aeruginosa chromosomal cephalosporinase and probably represents the common A. sobria cephalosporinase. AsbB1 was a class D enzyme most closely related to the oxacillin-hydrolyzing enzyme OXA-1, with 34% amino acid sequence identity. Purified AsbA1 was a typical cephalosporinase with a substrate profile that reflected high rates of hydrolysis of cephaloridine compared with benzylpenicillin. Purified AsbB1 showed strong penicillinase activity, with hydrolysis rates for carbenicillin and cloxacillin 2 to 2.5 times that for benzylpenicillin. Hydrolysis of imipenem was < or = 1% of that for benzylpenicillin. Both clavulanic acid and tazobactam strongly inhibited AsbB1, while sulbactam inhibited the AsbB1 enzyme less effectively. None of the inhibitors worked well against the AsbA1 enzyme. The chelators EDTA and 1,10-o-phenanthroline did not affect the activity of either enzyme. A. sobria AER 14M was found to produce both a group 1 cephalosporinase and a novel group 2d cloxacillin-hydrolyzing beta-lactamase that has been designated here OXA-12.

Substrate-induced inactivation of the OXA2 β-lactamase

The hydrolysis time courses of 22 beta-lactam antibiotics by the class D OXA2 beta-lactamase were studied. Among these, only three appeared to correspond to the integrated Henri-Michaelis equation. 'Burst' kinetics, implying branched pathways, were observed with most penicillins, cephalosporins and with flomoxef and imipenem. Kinetic parameters characteristic of the different phases of the hydrolysis were determined for some substrates. Mechanisms generally accepted to explain such reversible partial inactivations involving branches at either the free enzyme or the acyl-enzyme were inadequate to explain the enzyme behaviour. The hydrolysis of imipenem was characterized by the occurrence of two 'bursts', and that of nitrocefin by a partial substrate-induced inactivation complicated by a competitive inhibition by the hydrolysis product.

Catalysis of hydrolysis and aminolysis of non-classical beta-lactam antibiotics by metal ions and metal chelates.

The Zn(2+)-tris (hydroxymethyl)aminomethane (Tris) system has a great catalytic effect on the hydrolysis and aminolysis of some beta-lactam antibiotics. In order to ascertain the mechanism of this catalysis we have analysed the effects of the beta-lactam antibiotic structure. First we studied the kinetics of the decomposition of imipenem, SCH 29482, aztreonam and nocardicin A in aqueous solution of Tris at 35.0 degrees C, 0.5 mol.dm-3 ionic strength and in the presence of metal ions (Zn2+, Cd2+, Co2+, Cu2+, Ni2+ and Mn2+). From these studies, we conclude that Tris and metal ions (in separate solutions) exert a great catalytic effect on the hydrolysis of imipenem and SCH 29482. We suggest that in metal ion solutions a 1:1 complex is formed between the metal ion and beta-lactam antibiotic, which is attacked by hydroxide ions. Studies of the degradation of the antibiotics studied in solutions of Tris and metal ions together indicate that the systems Cd(2+)-Tris and Zn(2+)-Tris have a great catalytic effect on the hydrolysis and aminolysis of imipenem and SCH 29482. We suggest that this catalysis takes place via a ternary complex in which the metal ion plays a double role by (a) placing the antibiotic and the Tris in the right position for the reaction and (b) lowering the pKa of the hydroxide group of Tris, which is coordinated with the metal ion, generating a strong nucleophile.