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A Genotype-Phenotype Correlation Study of SHV ?-Lactamases Offers New Insight into SHV Resistance Profiles.

ABSTRACT: The SHV ?-lactamases (BLs) have undergone strong allele diversification that has changed their substrate specificities. Based on 147 NCBI entries for SHV alleles, in silico mathematical models predicted 5 positions as relevant for the ?-lactamase inhibitor (BLI)-resistant (2br) phenotype, 12 positions as relevant for the extended-spectrum BL (ESBL) (2be) phenotype, and 2 positions as related solely to the narrow-spectrum (2b) phenotype. These positions and six additional positions described in other studies (including one promoter mutation) were systematically substituted and investigated for their substrate specificities in a BL-free Escherichia coli background, representing, to our knowledge, the most comprehensive substrate and substitution analysis for SHV alleles to date. An in vitro analysis confirmed the essentiality of positions 238 and 179 for the 2be phenotype and of position 69 for the 2br phenotype. The E240K and E240R substitutions, which do not occur alone in known 2br SHV variants, led to a 2br phenotype, indicating a latent BLI resistance potential of these substitutions. The M129V, A234G, S271I, and R292Q substitutions conferred latent resistance to cefotaxime. In addition, seven positions that were found not always to be associated with the ESBL phenotype resulted in increased resistance to ceftaroline. We also observed that coupling of a strong promoter (IS26) to an A146V mutant with the 2b phenotype resulted in highly increased resistance to BLIs, cefepime, and ceftaroline but not to third-generation cephalosporins, indicating that SHV enzymes represent an underestimated risk for empirical therapies that use piperacillin-tazobactam or cefepime to treat different infectious diseases caused by Gram-negative bacteria.

SUBMITTER: Neubauer S

PROVIDER: S-EPMC7318035 | biostudies-literature | 2020 Jun

REPOSITORIES: biostudies-literature

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A Genotype-Phenotype Correlation Study of SHV β-Lactamases Offers New Insight into SHV Resistance Profiles.

Neubauer Svetlana S Madzgalla Sara S Marquet Mike M Klabunde Anne A Büttner Bernd B Göhring Alexander A Brandt Christian C Feller Karl-Heinz KH Pletz Mathias W MW Makarewicz Oliwia O

Antimicrobial agents and chemotherapy 20200623 7

The SHV β-lactamases (BLs) have undergone strong allele diversification that has changed their substrate specificities. Based on 147 NCBI entries for SHV alleles, <i>in silico</i> mathematical models predicted 5 positions as relevant for the β-lactamase inhibitor (BLI)-resistant (2br) phenotype, 12 positions as relevant for the extended-spectrum BL (ESBL) (2be) phenotype, and 2 positions as related solely to the narrow-spectrum (2b) phenotype. These positions and six additional positions describ ...[more]

PMID: 32284385

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Project description:β-Lactamase enzymes (EC 3.5.2.6) are a significant threat to the continued use of β-lactam antibiotics to treat infections. A novel non-β-lactam β-lactamase inhibitor with activity against many class A and C and some class D β-lactamase variants, avibactam, is now available in the clinic in partnership with ceftazidime. Here, we explored the activity of avibactam against a variety of characterized isogenic laboratory constructs of β-lactamase inhibitor-resistant variants of the class A enzyme SHV (M69I/L/V, S130G, K234R, R244S, and N276D). We discovered that the S130G variant of SHV-1 shows the most significant resistance to inhibition by avibactam, based on both microbiological and biochemical characterizations. Using a constant concentration of 4 mg/liter of avibactam as a β-lactamase inhibitor in combination with ampicillin, the MIC increased from 1 mg/liter for blaSHV-1 to 256 mg/liter for blaSHV S130G expressed in Escherichia coli DH10B. At steady state, the k2/K value of the S130G variant when inactivated by avibactam was 1.3 M(-1) s(-1), versus 60,300 M(-1) s(-1) for the SHV-1 β-lactamase. Under timed inactivation conditions, we found that an approximately 1,700-fold-higher avibactam concentration was required to inhibit SHV S130G than the concentration that inhibited SHV-1. Molecular modeling suggested that the positioning of amino acids in the active site of SHV may result in an alternative pathway of inactivation when complexed with avibactam, compared to the structure of CTX-M-15-avibactam, and that S130 plays a role in the acylation of avibactam as a general acid/base. In addition, S130 may play a role in recyclization. As a result, we advance that the lack of a hydroxyl group at position 130 in the S130G variant of SHV-1 substantially slows carbamylation of the β-lactamase by avibactam by (i) removing an important proton acceptor and donator in catalysis and (ii) decreasing the number of H bonds. In addition, recyclization is most likely also slow due to the lack of a general base to initiate the process. Considering other inhibitor-resistant mechanisms among class A β-lactamases, S130 may be the most important amino acid for the inhibition of class A β-lactamases, perhaps even for the novel diazabicyclooctane class of β-lactamase inhibitors.

Dataset Information

A Genotype-Phenotype Correlation Study of SHV ?-Lactamases Offers New Insight into SHV Resistance Profiles.

Publications

A Genotype-Phenotype Correlation Study of SHV β-Lactamases Offers New Insight into SHV Resistance Profiles.

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