Dataset Information

The Lys234Arg substitution in the enzyme SHV-72 is a determinant for resistance to clavulanic acid inhibition.

ABSTRACT: The new beta-lactamase SHV-72 was isolated from clinical Klebsiella pneumoniae INSRA1229, which exhibited the unusual association of resistance to the amoxicillin-clavulanic acid combination (MIC, 64 microg/ml) and susceptibility to cephalosporins, aztreonam, and imipenem. SHV-72 (pI 7.6) harbored the three amino acid substitutions Ile8Phe, Ala146Val, and Lys234Arg. SHV-72 had high catalytic efficiency against penicillins (k(cat)/K(m), 35 to 287 microM(-1) x s(-1)) and no activity against oxyimino beta-lactams. The concentration of clavulanic acid necessary to inhibit the enzyme activity by 50% was 10-fold higher for SHV-72 than for SHV-1. Molecular-dynamics simulation suggested that the Lys234Arg substitution in SHV-72 stabilized an atypical conformation of the Ser130 side chain, which moved the O gamma atom of Ser130 around 3.5 A away from the key O gamma atom of the reactive serine (Ser70). This movement may therefore decrease the susceptibility to clavulanic acid by preventing cross-linking between Ser130 and Ser70.

SUBMITTER: Mendonca N

PROVIDER: S-EPMC2346665 | biostudies-literature | 2008 May

REPOSITORIES: biostudies-literature

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The Lys234Arg substitution in the enzyme SHV-72 is a determinant for resistance to clavulanic acid inhibition.

Mendonça Nuno N Manageiro Vera V Robin Frédéric F Salgado M José MJ Ferreira Eugénia E Caniça Manuela M Bonnet Richard R

Antimicrobial agents and chemotherapy 20080303 5

The new beta-lactamase SHV-72 was isolated from clinical Klebsiella pneumoniae INSRA1229, which exhibited the unusual association of resistance to the amoxicillin-clavulanic acid combination (MIC, 64 microg/ml) and susceptibility to cephalosporins, aztreonam, and imipenem. SHV-72 (pI 7.6) harbored the three amino acid substitutions Ile8Phe, Ala146Val, and Lys234Arg. SHV-72 had high catalytic efficiency against penicillins (k(cat)/K(m), 35 to 287 microM(-1) x s(-1)) and no activity against oxyimi ...[more]

PMID: 18316518

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Project description:Amoxicillin-clavulanic acid (AMC) is one of the most frequently prescribed antibiotic formulations in the Western world. Extensive oral use of this antimicrobial combination influences the gut microbiota. One of the most abundant early colonizers of the human gut microbiota is represented by different taxa of the Bifidobacterium genus, which include many members that are considered to bestow beneficial effects upon their host. In the current study, we investigated the impact of AMC administration on the gut microbiota composition, comparing the gut microbiota of 23 children that had undergone AMC antibiotic therapy to that of 19 children that had not been treated with antibiotics during the preceding 6 months. Moreover, we evaluated AMC sensitivity by MIC test of 261 bifidobacterial strains, including reference strains for the currently recognized 64 bifidobacterial (sub)species, as well as 197 bifidobacterial isolates of human origin. These assessments allowed the identification of four bifidobacterial strains that exhibit a high level of AMC insensitivity, which were subjected to genomic and transcriptomic analyses to identify the putative genetic determinants responsible for this AMC insensitivity. Furthermore, we investigated the ecological role of AMC-resistant bifidobacterial strains by in vitro batch cultures.IMPORTANCE Based on our results, we observed a drastic reduction in gut microbiota diversity of children treated with antibiotics, which also affected the abundance of Bifidobacterium, a bacterial genus commonly found in the infant gut. MIC experiments revealed that more than 98% of bifidobacterial strains tested were shown to be inhibited by the AMC antibiotic. Isolation of four insensitive strains and sequencing of their genomes revealed the identity of possible genes involved in AMC resistance mechanisms. Moreover, gut-simulating in vitro experiments revealed that one strain, i.e., Bifidobacterium breve PRL2020, is able to persist in the presence of a complex microbiota combined with AMC antibiotic.

Project description:Tackling the ever-looming threat of antibiotic resistance remains a challenge for clinicians and microbiologists across the globe. Sulfhydryl variable (SHV) is a known bacterial enzyme associated with antibiotic resistance. The SHV enzyme has many variants. The present article describes identification and molecular interaction of a putative inhibitor with the bacterial SHV enzyme as a step towards novel antibacterial drug discovery. The MCULE-platform was used for screening a collection of 5 000 000 ligand molecules to evaluate their binding potential to the bacterial SHV-1 enzyme. Estimation of pharmacokinetic features was realized with the aid of the 'SWISS ADME' tool. Toxicity-checks were also performed. The docked complex of 'the top screened out ligand' and 'the bacterial SHV-1 protein' was subjected to molecular dynamics simulation of 101 ns. The obtained ligand molecule, 1,1'-(4H,8H-Bis[1,2,5]oxadiazolo[3,4-b:3',4'-e]pyrazine-4,8-diyl)diethanone, displayed the most favourable binding interactions with bacterial SHV-1. A total of 15 amino acid residues were found to hold the ligand in the binding site of SHV-1. Noticeably, 12 of the 15 residues were found as common to the binding residues of the reference (PDB ID: 4ZAM). The RMSD values plotted against the simulation time showed that nearby 11 ns, equilibrium was reached and, thenceforth, the 'SHV-1-Top ligand' complex remained typically stable. Starting from around 11 ns and straight to 101 ns, the backbone RMSD fluctuations were found to be confined inside a range of 1.0-1.6 Å. The ligand, 1,1'-(4H,8H-Bis[1,2,5]oxadiazolo[3,4-b:3',4'-e]pyrazine-4,8-diyl)diethanone, satisfied ADMET criteria. Furthermore, the practicability of the described 'SHV-1-Top ligand' complex was reinforced by a comprehensive molecular dynamics simulation of 101 ns. This ligand hence can be considered a promising lead for antibiotic design against SHV-1 producing resistant bacteria, and thus warrants wet laboratory evaluation.

Dataset Information

The Lys234Arg substitution in the enzyme SHV-72 is a determinant for resistance to clavulanic acid inhibition.

Publications

The Lys234Arg substitution in the enzyme SHV-72 is a determinant for resistance to clavulanic acid inhibition.

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