Identification of a CTX-M-255 β-lactamase containing a G239S substitution selectively conferring resistance to penicillin/β-lactamase inhibitor combinations

Minna Rud Andreasen, Tim Rick, Nicolai R. Alexandersen, Katrine Hartung Hansen, Martin Schou Pedersen, Jakob K Warweitsky, Carolina M Botelho, Susanne Häussler, Lotte Jelsbak, Kristian Schønning*

*Corresponding author

Publikation: Bidrag til tidsskriftTidsskriftartikelForskningpeer review

Abstract

Objectives
An Escherichia coli isolate, WGS1363, showed resistance to piperacillin/tazobactam but susceptibility to cephalosporins and contained a previously unrecognized β-lactamase, CTX-M-255, as the only acquired β-lactamase. CTX-M-255 was identical to CTX-M-27 except for a G239S substitution. Here, we characterize the hydrolytic spectrum of CTX-M-255 and a previously reported β-lactamase, CTX-M-178, also containing a G239S substitution and compare it to their respective parental enzymes, CTX-M-27 and CTX-M-15.

Methods
All β-lactamase genes were expressed in E. coli TOP10 and MICs to representative β-lactam-antibiotics were determined. Furthermore, blaCTX-M-15, blaCTX-M-27, blaCTX-M-178 and blaCTX-M-255 with C-terminal His-tag fusions were affinity purified for enzyme kinetic assays determining Michaelis–Menten kinetic parameters against representative β-lactam-antibiotics and IC50s of clavulanate, sulbactam, tazobactam and avibactam.

Results
TOP10-transformants expressing blaCTX-M-178 and blaCTX-M-255 showed resistance to penicillin/β-lactamase combinations and susceptibility to cephalothin and cefotaxime in contrast to transformants expressing blaCTX-M-15 and blaCTX-M-27. Determination of enzyme kinetic parameters showed that CTX-M-178 and CTX-M-255 both lacked hydrolytic activity against cephalosporins and showed impaired hydrolytic efficiency against penicillin antibiotics compared to their parental enzymes. Both enzymes appeared more active against piperacillin compared to benzylpenicillin and ampicillin. Compared to their parental enzymes, IC50s of β-lactamase-inhibitors were increased more than 1000-fold for CTX-M-178 and CTX-M-255.

Conclusions
CTX-M-178 and CTX-M-255, both containing a G239S substitution, conferred resistance to piperacillin/tazobactam and may be characterized as inhibitor-resistant CTX-M β-lactamases. Inhibitor resistance was accompanied by loss of activity against cephalosporins and monobactams. These findings add to the necessary knowledge base for predicting antibiotic susceptibility from genotypic data.
OriginalsprogEngelsk
TidsskriftJournal of Antimicrobial Chemotherapy
Vol/bind79
Udgave nummer4
Sider (fra-til)810-814
Antal sider5
ISSN0305-7453
DOI
StatusUdgivet - 1 apr. 2024

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