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1.
Article in English | MEDLINE | ID: mdl-31712200

ABSTRACT

Nocardia farcinica, one of the most frequent pathogenic species responsible for nocardiosis, is characterized by frequent brain involvement and resistance to ß-lactams mediated by a class A ß-lactamase. Kinetic parameters for hydrolysis of various ß-lactams by FARIFM10152 from strain IFM 10152 were determined by spectrophotometry revealing a high catalytic activity (kcat/Km ) for amoxicillin, aztreonam, and nitrocefin. For cephems, kcat/Km was lower but remained greater than 104 M-1 s-1 A low catalytic activity was observed for meropenem, imipenem, and ceftazidime hydrolysis. FARIFM10152 inhibition by avibactam and clavulanate was compared using nitrocefin as a reporter substrate. FARIFM10152 was efficaciously inhibited by avibactam with a carbamoylation rate constant (k2/Ki ) of (1.7 ± 0.3) × 104 M-1 s-1 The 50% effective concentrations (EC50s) of avibactam and clavulanate were 0.060 ± 0.007 µM and 0.28 ± 0.06 µM, respectively. Amoxicillin, cefotaxime, imipenem, and meropenem MICs were measured for ten clinical strains in the presence of avibactam and clavulanate. At 4 µg/ml, avibactam and clavulanate restored amoxicillin susceptibility in all but one of the tested strains but had no effect on the MICs of cefotaxime, imipenem, and meropenem. At 0.4 µg/ml, amoxicillin susceptibility (MIC ≤ 8 µg/ml) was restored for 9 out of 10 strains by avibactam but only for 4 out of 10 strains by clavulanate. Together, these results indicate that avibactam was at least as potent as clavulanate, suggesting that the amoxicillin-avibactam combination could be considered as an option for the rescue treatment of N. farcinica infections if clavulanate cannot be used.


Subject(s)
Anti-Bacterial Agents/pharmacology , Azabicyclo Compounds/pharmacology , Nocardia/drug effects , Nocardia/enzymology , beta-Lactamase Inhibitors/pharmacology , beta-Lactamases/metabolism , Amoxicillin-Potassium Clavulanate Combination/pharmacology , Anti-Bacterial Agents/metabolism , Enzyme Inhibitors/pharmacology , Hydrolysis , Kinetics , Microbial Sensitivity Tests , Nocardia/metabolism , beta-Lactamases/drug effects
2.
Diagn Microbiol Infect Dis ; 90(3): 186-189, 2018 Mar.
Article in English | MEDLINE | ID: mdl-29217419

ABSTRACT

Infections due to Mycobacterium abscessus carry a poor prognosis since this rapidly growing mycobacterium is intrinsically resistant to most antibiotics. Here, we evaluate the in vitro activity of the new oxazolidinone tedizolid against a collection of 44M. abscessus clinical isolates. The MIC50s and MIC90s of tedizolid (2 and 8µg/mL, respectively) were 2- to 16-fold lower than those of linezolid. There was no difference between the 3M. abscessus subspecies. Time-kill assays did not show any bactericidal activity at 4- and 8-fold the MIC. Combination of tedizolid with clarithromycin was synergistic against 1 out of 6 isolates, while indifferent interactions were observed for tedizolid combined with tigecycline, ciprofloxacin, and amikacin.


Subject(s)
Anti-Bacterial Agents/pharmacology , Clarithromycin/pharmacology , Linezolid/pharmacology , Mycobacterium abscessus/drug effects , Oxazolidinones/pharmacology , Tetrazoles/pharmacology , Amikacin/pharmacology , Ciprofloxacin/pharmacology , Drug Combinations , Drug Resistance, Multiple, Bacterial , Humans , Microbial Sensitivity Tests , Minocycline/analogs & derivatives , Minocycline/pharmacology , Mycobacterium Infections, Nontuberculous/drug therapy , Mycobacterium abscessus/isolation & purification , Tigecycline
3.
J Antimicrob Chemother ; 72(4): 1081-1088, 2017 04 01.
Article in English | MEDLINE | ID: mdl-28039278

ABSTRACT

Objectives: Mycobacterium tuberculosis and Mycobacterium abscessus produce broad-spectrum class A ß-lactamases, BlaC and Bla Mab , which are inhibited by clavulanate and avibactam, respectively. BlaC differs from Bla Mab at Ambler position 132 in the conserved motif SDN (SDG versus SDN, respectively). Here, we investigated whether this polymorphism could account for the inhibition specificity of ß-lactamases from slowly and rapidly growing mycobacteria. Methods: Enzyme kinetics were determined to assess the impact of the substitutions G 132 N in BlaC and N 132 G in Bla Mab on ß-lactamase inhibition by clavulanate and avibactam. The stability of acylenzymes was evaluated by MS. The impact of the substitutions on the antibacterial activity of drug combinations was determined based on production of the ß-lactamases in Escherichia coli . Results: The substitution G 132 N increased 140-fold the efficacy of BlaC inhibition by avibactam and abolished clavulanate inhibition due to acylenzyme hydrolysis. Bla Mab efficiently hydrolysed clavulanate, but the substitution N 132 G led to a 5600-fold reduction in the hydrolysis rate constant k cat due to stabilization of Bla Mab -clavulanate covalent adducts. The N 132 G substitution also led to a 610-fold reduction in the efficacy of Bla Mab carbamylation by avibactam. Testing resistance to the amoxicillin/clavulanate and amoxicillin/avibactam combinations revealed that modifications in the catalytic properties of the ß-lactamases resulted in opposite shifts from susceptibility to resistance and vice versa. Conclusions: G 132 N and N 132 G had opposite effects on the inhibition of BlaC and Bla Mab , indicating that these substitutions might lead to acquisition of resistance to either of the ß-lactamase inhibitors, but not to both of them.


Subject(s)
Azabicyclo Compounds/metabolism , Clavulanic Acid/metabolism , Mycobacterium/enzymology , beta-Lactamase Inhibitors/metabolism , beta-Lactamases/metabolism , Amino Acid Substitution , Enzyme Stability , Escherichia coli/genetics , Escherichia coli/metabolism , Mass Spectrometry , Mutant Proteins/genetics , Mutant Proteins/metabolism
4.
Article in English | MEDLINE | ID: mdl-28069651

ABSTRACT

The substitution N132G in the SDN motif of class A ß-lactamases from rapidly growing mycobacteria was previously shown to impair their inhibition by avibactam but to improve the stability of acyl-enzymes formed with clavulanate. The same substitution was introduced in KPC-2 and CTX-M-15 to assess its impact on ß-lactamases from Enterobacteriaceae and evaluate whether it may lead to resistance to the ceftazidime-avibactam combination. Kinetic parameters for the inhibition of the ß-lactamases by avibactam and clavulanate were determined by spectrophotometry using nitrocefin as the substrate. The substitution N132G impaired (>1,000-fold) the efficacy of carbamylation of KPC-2 and CTX-M-15 by avibactam. The substitution improved the inhibition of KPC-2 by clavulanate due to reduced deacylation, whereas the presence or absence of N132G resulted in the inhibition of CTX-M-15 by clavulanate. The hydrolysis of amoxicillin and nitrocefin by KPC-2 and CTX-M-15 was moderately affected by the substitution N132G, but that of ceftazidime, ceftaroline, and aztreonam was drastically reduced. Isogenic strains producing KPC-2 and CTX-M-15 were constructed to assess the impact of the substitution N132G on the antibacterial activities of ß-lactam-inhibitor combinations. For amoxicillin, the substitution resulted in resistance and susceptibility for avibactam and clavulanate, respectively. For ceftazidime, ceftaroline, and aztreonam, the negative impact of the substitution on ß-lactamase activity prevented resistance to the ß-lactam-avibactam combinations. In conclusion, the N132G substitution has profound effects on the substrate and inhibition profiles of class A ß-lactamases, which are largely conserved in distantly related enzymes. Fortunately, the substitution does not lead to resistance to the ceftazidime-avibactam combination.


Subject(s)
Amino Acid Substitution , Anti-Bacterial Agents/pharmacology , Azabicyclo Compounds/pharmacology , Clavulanic Acid/pharmacology , Escherichia coli/genetics , beta-Lactamases/genetics , Amino Acid Motifs , Amoxicillin/pharmacology , Aztreonam/pharmacology , Ceftazidime/pharmacology , Cephalosporins/chemistry , Cephalosporins/pharmacology , Cloning, Molecular , Drug Combinations , Escherichia coli/metabolism , Gene Expression , Kinetics , Mutation , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Reverse Genetics , Substrate Specificity , beta-Lactamases/metabolism , Ceftaroline
5.
J Med Chem ; 59(7): 3427-38, 2016 Apr 14.
Article in English | MEDLINE | ID: mdl-26937999

ABSTRACT

Combinations of ß-lactams of the carbapenem class, such as meropenem, with clavulanate, a ß-lactamase inhibitor, are being evaluated for the treatment of drug-resistant tuberculosis. However, carbapenems approved for human use have never been optimized for inactivation of the unusual ß-lactam targets of Mycobacterium tuberculosis or for escaping to hydrolysis by broad-spectrum ß-lactamase BlaC. Here, we report three routes of synthesis for modification of the two side chains carried by the ß-lactam and the five-membered rings of the carbapenem core. In particular, we show that the azide-alkyne Huisgen cycloaddition reaction catalyzed by copper(I) is fully compatible with the highly unstable ß-lactam ring of carbapenems and that the triazole ring generated by this reaction is well tolerated for inactivation of the L,D-transpeptidase LdtMt1 target. Several of our new carbapenems are superior to meropenem both with respect to the efficiency of in vitro inactivation of LdtMt1 and reduced hydrolysis by BlaC.


Subject(s)
Carbapenems/chemical synthesis , Carbapenems/pharmacology , Peptidyl Transferases/antagonists & inhibitors , beta-Lactamase Inhibitors/pharmacology , beta-Lactamases/metabolism , Bacterial Proteins/antagonists & inhibitors , Bacterial Proteins/metabolism , Humans , Hydrolysis , Kinetics , Mycobacterium tuberculosis/drug effects , Mycobacterium tuberculosis/enzymology , Peptidyl Transferases/metabolism , Tuberculosis/drug therapy , Tuberculosis/microbiology , beta-Lactamase Inhibitors/chemical synthesis
6.
Antimicrob Agents Chemother ; 59(9): 5714-20, 2015 Sep.
Article in English | MEDLINE | ID: mdl-26149997

ABSTRACT

Combinations of ß-lactams with clavulanate are currently being investigated for tuberculosis treatment. Since Mycobacterium tuberculosis produces a broad spectrum ß-lactamase, BlaC, the success of this approach could be compromised by the emergence of clavulanate-resistant variants, as observed for inhibitor-resistant TEM variants in enterobacteria. Previous analyses based on site-directed mutagenesis of BlaC have led to the conclusion that this risk was limited. Here, we used a different approach based on determination of the crystal structure of ß-lactamase BlaMAb of Mycobacterium abscessus, which efficiently hydrolyzes clavulanate. Comparison of BlaMAb and BlaC allowed for structure-assisted site-directed mutagenesis of BlaC and identification of the G(132)N substitution that was sufficient to switch the interaction of BlaC with clavulanate from irreversible inactivation to efficient hydrolysis. The substitution, which restored the canonical SDN motif (SDG→SDN), allowed for efficient hydrolysis of clavulanate, with a more than 10(4)-fold increase in k cat (0.41 s(-1)), without affecting the hydrolysis of other ß-lactams. Mass spectrometry revealed that acylation of BlaC and of its G(132)N variant by clavulanate follows similar paths, involving sequential formation of two acylenzymes. Decarboxylation of the first acylenzyme results in a stable secondary acylenzyme in BlaC, whereas hydrolysis occurs in the G(132)N variant. The SDN/SDG polymorphism defines two mycobacterial lineages comprising rapidly and slowly growing species, respectively. Together, these results suggest that the efficacy of ß-lactam-clavulanate combinations may be limited by the emergence of resistance. ß-Lactams active without clavulanate, such as faropenem, should be prioritized for the development of new therapies.


Subject(s)
Clavulanic Acid/metabolism , Mycobacterium tuberculosis/enzymology , beta-Lactamases/metabolism , Clavulanic Acid/pharmacology , Microbial Sensitivity Tests , Mycobacterium tuberculosis/drug effects , beta-Lactam Resistance/genetics , beta-Lactams/pharmacology
7.
Antimicrob Agents Chemother ; 59(5): 2938-41, 2015 May.
Article in English | MEDLINE | ID: mdl-25733512

ABSTRACT

The production of ß-lactamases Bla(Mab) and BlaC contributes to ß-lactam resistance in Mycobacterium abscessus and Mycobacterium tuberculosis, respectively. Ceftaroline was efficiently hydrolyzed by these enzymes. Inhibition of M. tuberculosis BlaC by clavulanate decreased the ceftaroline MIC from ≥ 256 to 16 to 64 µg/ml, but these values are clinically irrelevant. In contrast, the ceftaroline-avibactam combination should be evaluated against M. abscessus since it inhibited growth at lower and potentially achievable drug concentrations.


Subject(s)
Cephalosporins/pharmacology , Mycobacterium tuberculosis/drug effects , Mycobacterium tuberculosis/enzymology , Mycobacterium/drug effects , Azabicyclo Compounds/pharmacology , Mycobacterium/enzymology , beta-Lactamase Inhibitors/pharmacology , beta-Lactamases/metabolism , Ceftaroline
8.
J Antimicrob Chemother ; 69(3): 691-6, 2014 Mar.
Article in English | MEDLINE | ID: mdl-24132992

ABSTRACT

OBJECTIVES: Imipenem and cefoxitin are used to treat Mycobacterium abscessus infections and have moderate activity against this fast-growing mycobacterium (MIC50 of 16 and 32 mg/L, respectively). M. abscessus is highly resistant to most other ß-lactams, although the underlying mechanisms have not been explored. Here, we characterized M. abscessus class A ß-lactamase (Bla(Mab)) and investigated its role in ß-lactam resistance. METHODS: Hydrolysis kinetic parameters of purified Bla(Mab) were determined by spectrophotometry for various ß-lactams and compared with those of related BlaC from Mycobacterium tuberculosis. MICs of ß-lactams were determined for M. abscessus CIP104536 and for Escherichia coli producing Bla(Mab) and BlaC. RESULTS: Bla(Mab) had a broad hydrolysis spectrum, similar to that of BlaC, but with overall higher catalytic efficiencies, except for cefoxitin. As expected from its in vivo efficacy, cefoxitin was very slowly hydrolysed by Bla(Mab) (k(cat)/K(m) = 6.7 M(-1) s(-1)). Bla(Mab) hydrolysed imipenem more efficiently (k(cat)/K(m) = 3.0 × 10(4) M(-1) s(-1)), indicating that the in vivo activity of this drug might be improved by combination with a ß-lactamase inhibitor. ß-Lactamase inhibitors clavulanate, tazobactam and sulbactam did not inhibit Bla(Mab). This enzyme efficiently hydrolysed clavulanate, in contrast to BlaC, which is irreversibly acylated by this inhibitor. Bla(Mab) and BlaC were functional in E. coli and the resistance profiles mediated by these enzymes were in agreement with the kinetic parameters. CONCLUSIONS: M. abscessus produces a clavulanate-insensitive broad-spectrum ß-lactamase that limits the in vivo efficacy of ß-lactams.


Subject(s)
Mycobacterium/enzymology , beta-Lactam Resistance , beta-Lactamases/metabolism , Hydrolysis , Kinetics , Substrate Specificity
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