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D M Livermore - One of the best experts on this subject based on the ideXlab platform.
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Detection of Beta-Lactamase-mediated resistance.
The Journal of antimicrobial chemotherapy, 2001Co-Authors: D M Livermore, D F BrownAbstract:beta-Lactams are the most widely used antibiotics, and Beta-Lactamases are the greatest source of resistance to them. An understanding of Beta-Lactamase detection and identification is therefore valuable. Colorimetric, acidimetric and iodometric tests of Beta-Lactamase production are good, rapid indicators of penicillin and ampicillin resistance in Haemophilus, Moraxella and Neisseria spp. These methods can also be applied to Gram-negative aerobic bacilli but are less useful, since the usual question is not whether a Beta-Lactamase is produced by these organisms, but which Beta-Lactamase? Accurate identification of the Beta-Lactamases of Enterobacteriaceae demands gene or protein sequencing, but the broad type of enzyme produced by an isolate can often be inferred from antibiotic susceptibility data. Resistance to ceftazidime or cefpodoxime implies extended-spectrum Beta-Lactamase (ESBL) production in Escherichia coli and Klebsiella spp., especially if susceptibility to cefoxitin is retained. ESBL production can be confirmed with double disc tests or with various commercial kits. Derepression of AmpC Beta-Lactamases in Enterobacter spp. and Citrobacter freundii is another important mechanism and can be inferred from cross-resistance to Beta-Lactamase inhibitor combinations and to all cephalosporins except fourth-generation agents. Antagonism between cefoxitin and cefotaxime can be used to infer the presence of inducible AmpC enzymes in these species, indicating the risk of segregation of derepressed mutants, but in general this risk is better predicted from accurate speciation.
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Evolution of Beta-Lactamase inhibitors.
Intensive care medicine, 1994Co-Authors: D M LivermoreAbstract:Beta-Lactamases present the greatest single challenge to beta-lactam antibiotics, including piperacillin. Beta-Lactamase-mediated resistance to supposedly Beta-Lactamase stable agents such as second- and third-generation cephalosporins is now emerging and inhibitor combinations provide an alternative strategy to overcome this problem. The success of this strategy depends on 1) how efficiently the inhibitor inhibits important Beta-Lactamases, 2) on how much Beta-Lactamase the bacteria produce, 3) on the drug that is to be protected, 4) on the permeability and intrinsic susceptibility of the organisms and 5) on the conditions, notably the pH. Tazobactam inhibits most of the clinically important Beta-Lactamases that give piperacillin resistance, except for the Class I types. Piperacillin itself is a relatively easy drug to protect, particularly against the TEM-type enzymes. The result is that tazobactam greatly extends the activity of piperacillin, notably against enterobacteria, but also against staphylococci and anaerobes. The survey confirmed the very broad spectrum of activity of piperacillin/tazobactam. Resistance occurred in about 17% of the Enterobacter, Citrobacter, Serratia group, where we believe it to have been caused by derepressed Class I enzymes since these strains were cross-resistant to third-generation cephalosporins. Otherwise, resistance was largely confined to such organisms as E. faecium and methicillin-resistant staphylococci, which have piperacillin insensitive penicillin-binding proteins. Finally, some question remains on the antistaphylococcal activity of piperacillin/tazobactam, where MIC tests gave a more favorable impression than disc tests. Nevertheless, early clinical results against staphylococcal infection appear good, with a response rate of nearly 90% [15].
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Comparative in-vitro activity of biapenem against enterobacteria with β-lactamase-mediated antibiotic resistance
The Journal of antimicrobial chemotherapy, 1994Co-Authors: H Y Chen, D M LivermoreAbstract:The effects of enterobacterial Beta-Lactamases were studied for biapenem (L627), a new carbapenem. Susceptibility tests were performed for isogenic mutant series of Citrobacter freundii, Enterobacter cloacae, Morganella morganii, Serratia marcescens and Proteus vulgaris which varied only in chromosomal Beta-Lactamase expression. Beta-Lactamase-derepressed organisms in these series were as susceptible as Beta-Lactamase-inducible strains to biapenem; Beta-Lactamase-basal mutants were up to eight-fold more susceptible. Similar patterns of relative activity against the different expression types were noted for imipenem and biapenem. These data were related to direct induction and hydrolysis assays: biapenem, like imipenem, was a strong inducer of several Class I enzymes and of the P. vulgaris cefuroximase and, like the other carbapenems, was only very slowly hydrolysed by these enzymes. Moreover, like meropenem, biapenem reversibly deactivated these Beta-Lactamases. Piperacillin and the cephalosporins, tested as comparators, were more labile than carbapenems to the Class I enzymes, were weak inducers below their MICs and lacked deactivator function. In consequence their MICs were higher for derepressed organisms than for those with inducible or basal Beta-Lactamase expression. Unlike the carbapenems, they selected derepressed mutants from inducible populations. Biapenem, like imipenem and meropenem, retained full activity against most transconjugants of Escherichia coli K-12 that produced plasmid-mediated Beta-Lactamases, including extended-spectrum TEM mutants. Only production of OXA-10 (previously PSE-2) enzyme gave a slight reduction in susceptibility to the new carbapenem. Biapenem resistance (MIC 16 mg/L) did, however, occur in S. marcescens S6, which produced a chromosomal carbapenemase. This enzyme hydrolysed biapenem. Overall, our findings indicate that biapenem shares the favourable properties of imipenem and meropenem in its interactions with the most important Beta-Lactamases of enterobacteria.
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Determinants of the activity of Beta-Lactamase inhibitor combinations.
The Journal of antimicrobial chemotherapy, 1993Co-Authors: D M LivermoreAbstract:Inhibitor combinations provide one strategy to overcome Beta-Lactamase-mediated resistance. Their success depends, obviously, on the inhibitor being able to bind and inactivate the Beta-Lactamase molecules. Clavulanate, sulbactam and tazobactam are irreversible inactivators of many Beta-Lactamases, forming covalent complexes which resist hydrolysis. 'Suicide' kinetics are seen with some, but not all, enzymes. All three compounds inactivate staphylococcal penicillinase, the chromosomal Beta-Lactamases of Proteus vulgaris and Bacteroides spp., and the Class IV Beta-Lactamases present in some klebsiellae. Tazobactam, but not the other compounds, has moderate activity against some Class I (AmpC) chromosomal Beta-Lactamases, notably that of Morganella morganii, but not that of Enterobacter cloacae. Both clavulanate and tazobactam are strong inhibitors of the widely distributed TEM and SHV plasmid-mediated Beta-Lactamases; sulbactam is a weaker inhibitor. Other factors, aside from the affinity of the inhibitor for the enzyme, co-determine the success or failure of inhibition. Potentiation is most readily achieved if little enzyme is produced, and if the organism is very permeable to the inhibitor. Thus, resistance to inhibitor combinations is rare in strains of Haemophilus influenzae and Neisseria gonorrhoeae that produce TEM-Beta-Lactamase, but is commoner in enterobacteria that produce this enzyme, since these are less permeable and sometimes manufacture very large amounts of enzyme. The partner beta-lactam agent is also important. Irrespective of the inhibitor used, piperacillin is easier to protect against TEM Beta-Lactamases and the M. morganii Class I enzyme than are ampicillin, amoxycillin or ticarcillin. This may relate to the lower affinity of piperacillin for these enzymes, or to its greater affinity for the bacterial penicillin-binding proteins. Finally, pH can affect the degree of inhibition achieved with sulphones for some Beta-Lactamases, notably TEM-1.
Jean-marie Frère - One of the best experts on this subject based on the ideXlab platform.
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CENTA as a chromogenic substrate for studying Beta-Lactamases.
Antimicrob Agents Chemother, 2001Co-Authors: Carine Bebrone, F. Mahy, S. Rival, Jd Docquier, Gm Rossolini, Rf Pratt, Jean-marie Frère, Catherine Moali, Jacques Fastrez, Moreno GalleniAbstract:CENTA, a chromogenic cephalosporin, is readily hydrolyzed by Beta-Lactamases of all classes except for the Aeromonas hydrophila metalloenzyme. Although it cannot practically be used for the detection of Beta-Lactamase-producing strains on agar plates, it should be quite useful for kinetic studies and the detection of the enzymes in crude extracts and chromatographic fractions.CENTA, a chromogenic cephalosporin, is readily hydrolyzed by Beta-Lactamases of all classes except for the Aeromonas hydrophila metalloenzyme. Although it cannot practically be used for the detection of Beta-Lactamase-producing strains on agar plates, it should be quite useful for kinetic studies and the detection of the enzymes in crude extracts and chromatographic fractions.
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Catalytic properties of class A Beta-Lactamases: efficiency and diversity.
The Biochemical journal, 1998Co-Authors: André Matagne, Josette Lamotte-brasseur, Jean-marie FrèreAbstract:Beta-Lactamases are the main cause of bacterial resistance to penicillins, cephalosporins and related beta-lactam compounds. These enzymes inactivate the antibiotics by hydrolysing the amide bond of the beta-lactam ring. Class A Beta-Lactamases are the most widespread enzymes and are responsible for numerous failures in the treatment of infectious diseases. The introduction of new beta-lactam compounds, which are meant to be 'Beta-Lactamase-stable' or Beta-Lactamase inhibitors, is thus continuously challenged either by point mutations in the ubiquitous TEM and SHV plasmid-borne Beta-Lactamase genes or by the acquisition of new genes coding for Beta-Lactamases with different catalytic properties. On the basis of the X-ray crystallography structures of several class A Beta-Lactamases, including that of the clinically relevant TEM-1 enzyme, it has become possible to analyse how particular structural changes in the enzyme structures might modify their catalytic properties. However, despite the many available kinetic, structural and mutagenesis data, the factors explaining the diversity of the specificity profiles of class A Beta-Lactamases and their amazing catalytic efficiency have not been thoroughly elucidated. The detailed understanding of these phenomena constitutes the cornerstone for the design of future generations of antibiotics.
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Interactions between Active-Site-Serine Beta-Lactamases and Mechanism-Based Inactivators: A Kinetic Study and an Overview
Biochemical Journal, 1993Co-Authors: André Matagne, M. F. Ghuysen, Jean-marie FrèreAbstract:The interactions between three class A Beta-Lactamases and three Beta-Lactamase inactivators (clavulanic acid, sulbactam and olivanic acid MM13902) were studied. Interestingly, the interaction between the Streptomyces cacaoi Beta-Lactamase and clavulanate indicated little irreversible inactivation. With sulbactam, irreversible inactivation was found to occur with the three studied enzymes, but no evidence for transiently inactivated adducts was found. Irreversible inactivation of the S. albus G and S. cacaoi enzymes was particularly slow. With olivanate, irreversible inactivation was also observed with the three enzymes, but with the S. cacaoi enzyme, no hydrolysis could be detected. A tentative summary of the results found in the literature is also presented (including 6 beta-halogenopenicillanates), and the general conclusions underline the diversity of the mechanisms and the wide variations of the rate constants observed when class A Beta-Lactamases interact with Beta-Lactamase inactivators, in agreement with the behaviours of the same enzymes towards their good and poor substrates.
M Menendez - One of the best experts on this subject based on the ideXlab platform.
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Interaction of Beta-Lactamases I and II from Bacillus cereus with semisynthetic cephamycins. Kinetic studies.
The Biochemical journal, 1991Co-Authors: J Martin Villacorta, Patrícia Arriaga, José Laynez, M MenendezAbstract:The influence of C-6 alpha- or C-7 alpha-methoxylation of the beta-lactam ring in the catalytic action of class A and B Beta-Lactamases has been investigated. For this purpose the kinetic behaviour of Beta-Lactamases I (class A) and II (class B) from Bacillus cereus was analysed by using several cephamycins, moxalactam, temocillin and related antibiotics. These compounds behaved as poor substrates for Beta-Lactamase II, with high Km values and very low catalytic efficiencies. In the case of Beta-Lactamase I, the substitution of a methoxy group for a H atom at C-7 alpha or C-6 alpha decreased the affinity of the substrates for the enzyme. Furthermore, the acylation of cephamycins was completely blocked, whereas that of penicillins was slowed down by a factor of 10(4)-10(5), acylation being the rate-determining step of the process.
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Interaction of β-lactamases I and II from Bacillus cereus with semisynthetic cephamycins. Kinetic studies
Biochemical Journal, 1991Co-Authors: J Martin Villacorta, Patrícia Arriaga, José Laynez, M MenendezAbstract:The influence of C-6 alpha- or C-7 alpha-methoxylation of the beta-lactam ring in the catalytic action of class A and B Beta-Lactamases has been investigated. For this purpose the kinetic behaviour of Beta-Lactamases I (class A) and II (class B) from Bacillus cereus was analysed by using several cephamycins, moxalactam, temocillin and related antibiotics. These compounds behaved as poor substrates for Beta-Lactamase II, with high Km values and very low catalytic efficiencies. In the case of Beta-Lactamase I, the substitution of a methoxy group for a H atom at C-7 alpha or C-6 alpha decreased the affinity of the substrates for the enzyme. Furthermore, the acylation of cephamycins was completely blocked, whereas that of penicillins was slowed down by a factor of 10(4)-10(5), acylation being the rate-determining step of the process.
Christopher R Bethel - One of the best experts on this subject based on the ideXlab platform.
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design synthesis and crystal structures of 6 alkylidene 2 substituted penicillanic acid sulfones as potent inhibitors of acinetobacter baumannii oxa 24 carbapenemase
Journal of the American Chemical Society, 2010Co-Authors: Elena Santillana, Alejandro Beceiro, Anne M Distler, Anjaneyulu Sheri, Jared M Sampson, Matthew Kalp, Sundar Ram Reddy Pagadala, Sarah M Drawz, Christopher R Bethel, Focco Van Den AkkerAbstract:Class D {beta}-lactamases represent a growing and diverse class of penicillin-inactivating enzymes that are usually resistant to commercial {beta}-lactamase inhibitors. As many such enzymes are found in multi-drug resistant (MDR) Acinetobacter baumannii and Pseudomonas aeruginosa, novel {beta}-lactamase inhibitors are urgently needed. Five unique 6-alkylidene-2{prime}-substituted penicillanic acid sulfones (1-5) were synthesized and tested against OXA-24, a clinically important {beta}-lactamase that inactivates carbapenems and is found in A. baumannii. Based upon the roles Tyr112 and Met223 play in the OXA-24 {beta}-lactamase, we also engineered two variants (Tyr112Ala and Tyr112Ala,Met223Ala) to test the hypothesis that the hydrophobic tunnel formed by these residues influences inhibitor recognition. IC{sub 50} values against OXA-24 and two OXA-24 {beta}-lactamase variants ranged from 10 {+-} 1 (4 vs WT) to 338 {+-} 20 nM (5 vs Tyr112Ala, Met223Ala). Compound 4 possessed the lowest K{sub i} (500 {+-} 80 nM vs WT), and 1 possessed the highest inactivation efficiency (k{sub inact}/K{sub i} = 0.21 {+-} 0.02 {micro}M{sup -1}s{sup -1}). Electrospray ionization mass spectrometry revealed a single covalent adduct, suggesting the formation of an acyl-enzyme intermediate. X-ray structures of OXA-24 complexed to four inhibitors (2.0-2.6 {angstrom}) reveal the formation of stable bicyclic aromatic intermediates with their carbonyl oxygen in the oxyanionmore » hole. These data provide the first structural evidence that 6-alkylidene-2{prime}-substituted penicillin sulfones are effective mechanism-based inactivators of class D {beta}-lactamases. Their unique chemistry makes them developmental candidates. Mechanisms for class D hydrolysis and inhibition are discussed, and a pathway for the evolution of the BlaR1 sensor of Staphylococcus aureus to the class D {beta}-lactamases is proposed.« less
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clavulanic acid inactivation of shv 1 and the inhibitor resistant s130g shv 1 beta lactamase insights into the mechanism of inhibition
Journal of Biological Chemistry, 2005Co-Authors: Deley Sulton, Christopher R Bethel, Andrea M. Hujer, Doritza Paganrodriguez, Xiang Zhou, Yiding Liu, Marion S Helfand, Jodi M Thomson, Vernon E Anderson, John D BuynakAbstract:Clavulanic acid is a potent mechanism-based inhibitor of TEM-1 and SHV-1Beta-Lactamases, enzymes that confer resistance to beta-lactams in many gram-negative pathogens. This compound has enjoyed widespread clinical use as part of beta-lactam Beta-Lactamase inhibitor therapy directed against penicillin-resistant pathogens. Unfortunately, the emergence of clavulanic acid-resistant variants of TEM-1 and SHV-1 Beta-Lactamase significantly compromise the efficacy of this combination. A single amino acid change at Ambler position Ser130 (Ser --> Gly) results in resistance to inactivation by clavulanate in the SHV-1 and TEM-1Beta-Lactamases. Herein, we investigated the inactivation of SHV-1 and the inhibitor-resistant S130G variant Beta-Lactamases by clavulanate. Using liquid chromatography electrospray ionization mass spectrometry, we detected multiple modified proteins when SHV-1 Beta-Lactamase is inactivated by clavulanate. Matrix-assisted laser desorption ionization-time of flight mass spectrometry was used to study tryptic digests of SHV-1 and S130GBeta-Lactamases (+/- inactivation with clavulanate) and identified peptides modified at the active site Ser70. Ultraviolet (UV) difference spectral studies comparing SHV-1 and S130GBeta-Lactamases inactivated by clavulanate showed that the formation of reaction intermediates with absorption maxima at 227 and 280 nm are diminished and delayed when S130GBeta-Lactamase is inactivated. We conclude that the clavulanic acid inhibition of the S130G Beta-Lactamase must follow a branch of the normal inactivation pathway. These findings highlight the importance of understanding the intermediates formed in the inactivation process of inhibitor-resistant Beta-Lactamases and suggest how strategic chemical design can lead to novel ways to inhibit Beta-Lactamases.
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Molecular analysis of the simultaneous production of two SHV-type extended-spectrum Beta-Lactamases in a clinical isolate of Enterobacter cloacae by using single-nucleotide polymorphism genotyping.
Antimicrobial Agents and Chemotherapy, 2005Co-Authors: Dóra Szabó, Christopher R Bethel, Melissa A. Melan, Andrea M. Hujer, Robert A. Bonomo, Kristine M. Hujer, Katalin Kristóf, David L. PatersonAbstract:Bacteria that simultaneously produce multiple extended-spectrum Beta-Lactamases are frequently isolated. We report an Enterobacter cloacae isolate, ES24, producing four different Beta-Lactamases (AmpC type Beta-Lactamase, TEM-1, SHV-7, and a novel extended-spectrum Beta-Lactamase, SHV-30). Direct sequencing of bla(SHV) gene products gave a "double peak" at position 703, suggesting the presence of more than one allele. Using fluorescence resonance energy transfer real-time PCR to detect single-nucleotide polymorphisms, we were able to distinguish two different bla(SHV) genes in a single isolate. This may prove to be a useful technique in surveys of Beta-Lactamase production in contemporary clinical isolates.
David J. Payne - One of the best experts on this subject based on the ideXlab platform.
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Beta-Lactamase epidemiology and the utility of established and novel Beta-Lactamase inhibitors
Expert opinion on investigational drugs, 2000Co-Authors: David J. Payne, John H. BatesonAbstract:Beta-Lactamase inhibitor:beta-lactam combinations remain one of the most successful strategies for the treatment of bacterial infections. Over the last 20 years the number and diversity of serine and metallo active site Beta-Lactamases has increased dramatically. This review highlights some of the new additions to the Beta-Lactamase arena and discusses how the commercially available Beta-Lactamase inhibitors are keeping pace with the changing epidemiology of Beta-Lactamases. In addition, we survey the progress with the design of novel inhibitors of serine and metallo-Beta-Lactamases. Focus is given to the recent advances in the design of metallo-Beta-Lactamase inhibitors as these enzymes pose a serious emerging threat to the use of all beta-lactam based therapies.
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Rapid identification of metallo- and serine Beta-Lactamases.
Antimicrobial agents and chemotherapy, 1994Co-Authors: David J. Payne, John H. Bateson, R. Cramp, Jane E. Neale, D. J. C. KnowlesAbstract: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.