Biochemical Characterization of β-Lactamases fromMycobacterium abscessusComplex and Genetic Environment of the β-Lactamase-Encoding Gene

Abstract

The objectives of this study were to determine the kinetic parameters of purified recombinant Bla and Bla by spectrophotometry, analyze the genetic environment of the bla and bla genes in both species by polymerase chain reaction and sequencing, furthermore, in silico models of both enzymes in complex with imipenem were obtained by modeling tools. Our results showed that Bla and Bla have a similar hydrolysis behavior, displaying high catalytic efficiencies toward penams, cephalothin, and nitrocefin; none of the enzymes are well inhibited by clavulanate. Bla hydrolyzes imipenem at higher efficiency than cefotaxime and aztreonam. Bla and Bla showed that their closest structural homologs are KPC-2 and SFC-1, which correlate to the mild carbapenemase activity toward imipenem observed at least for BlaMmas. They also seem to differ from other class A β-lactamases by the presence of a more flexible Ω loop, which could impact in the hydrolysis efficiency against some antibiotics. A -35 consensus sequence (TCGACA) and embedded at the 3' end of MAB_2874, which may constitute the bla and bla promoter. Our results suggest that the resistance mechanisms in fast-growing mycobacteria could be probably evolving toward the production of β-lactamases that have improved catalytic efficiencies against some of the drugs commonly used for the treatment of mycobacterial infections, endangering the use of important drugs like the carbapenems.

Effect of β-lactamase production and β-lactam instability on MIC testing results for Mycobacterium abscessus

“…Cefoxitin MIC values of 32 mg/L (day 3) for both M. abscessus ATCC 19977 and the Dbla Mab mutant (Table 1) agree with earlier reported data, 27 corroborating previous molecular modelling and structural analyses, 42 which delineated that substitutions of the b-lactam ring by a methoxy group in cefoxitin inhibits the activity of class A b-lactamases. 30 As expected, amikacin MICs were independent of the bla genotype.…”

“…27,29 Determination of the kinetic parameters of this enzyme revealed that it can slowly hydrolyse imipenem and meropenem, while cefoxitin hydrolysis by Bla_Mab is immensely slow, as the methoxy group at cefoxitin's b-lactam ring was predicted to block the activity of class A b-lactamases. 27,29,30 As imipenem, meropenem and cefoxitin are known to have limited in vitro stability, 31 we assessed by LC high-resolution MS (LC-HRMS) the in vitro stability of these b-lactams and, by exploiting a bla Mab deletion mutant that we generated, we addressed the biological effect of b-lactam stability and b-lactamase production on MIC testing results after different periods of incubation. The direct impact of b-lactam stability on AST testing was further addressed by MIC determination of fresh and pre-incubated b-lactam antibiotics for the ampicillin-susceptible Escherichia coli XL1-Blue MRF 0 strain.…”

Mycobacterium abscessus and β-Lactams: Emerging Insights and Potential Opportunities

“…The observation that these BLIs do not reduce the minimum inhibitory concentration (MIC) of β-lactams against Mab in a whole-cell assay (Kaushik et al, 2017 ) is additional confirmation that β-lactamase activity in Mab is more robust than in Mtb . Subspecies massiliense harbors an additional β-lactamase, Bla Mmas (Ramirez et al, 2017 ).…”

“…This study also observed that β-lactams plus avibactam exhibited similar efficacy against the parental Mab strain as compared to each drug alone against Δ bla Mab , suggesting that avibactam fully inhibits Bla Mab. While Bla Mab and Bla Mmas hydrolyze penicillins and cephalosporins with similar efficacy, Bla Mmas also exhibits mild carbapenemase activity, a potential concern as it suggests an evolutionary movement toward β-lactamases with extended spectra (Ramirez et al, 2017 ). This study also noted that Bla Mmas is structurally similar to other acquired carbapenemases normally found in gram negative bacteria, such as KPC-2 and SFC-1.…”

In Vitro Activity of the New β-Lactamase Inhibitors Relebactam and Vaborbactam in Combination with β-Lactams against Mycobacterium abscessus Complex Clinical Isolates

“…isms possess a constitutively expressed, broad-spectrum ␤-lactamase, Bla Mab , which contributes to the intrinsic resistance of MABC members to most ␤-lactam antibiotics (8)(9)(10)(11)(12). Several studies have indicated that Bla Mab is not significantly inhibited by ␤-lactam-based ␤-lactamase inhibitors, namely clavulanate, tazobactam, and sulbactam (9,(13)(14)(15). In contrast, the non-␤-lactam-based ␤-lactamase diazabicyclooctane (DBO) inhibitor avibactam does inhibit Bla Mab , thereby reducing the MIC of many ␤-lactams for MABC, especially carbapenems and cephalosporins, to clinically achievable concentrations (16)(17)(18)(19)(20).…”