TY - JOUR

T1 - Identification of a CTX-M-255 β-lactamase containing a G239S substitution selectively conferring resistance to penicillin/β-lactamase inhibitor combinations

AU - Andreasen, Minna Rud

AU - Rick, Tim

AU - Alexandersen, Nicolai Riff

AU - Hansen, Katrine Hartung

AU - Pedersen, Martin Schou

AU - Warweitzky, Jakob K

AU - Botelho, Carolina Mastella

AU - Häussler, Susanne

AU - Jelsbak, Lotte

AU - Schønning, Kristian

N1 - © The Author(s) 2024. Published by Oxford University Press on behalf of British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

PY - 2024

Y1 - 2024

N2 - 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.

AB - 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.

KW - beta-Lactamase Inhibitors/pharmacology

KW - Anti-Bacterial Agents/pharmacology

KW - Escherichia coli

KW - beta-Lactamases/genetics

KW - Penicillins/pharmacology

KW - Cephalosporins/pharmacology

KW - Tazobactam/pharmacology

KW - Piperacillin/pharmacology

KW - Monobactams

KW - Piperacillin, Tazobactam Drug Combination

KW - Microbial Sensitivity Tests

U2 - 10.1093/jac/dkae033

DO - 10.1093/jac/dkae033

M3 - Journal article

C2 - 38366372

VL - 79

SP - 810

EP - 814

JO - Journal of Antimicrobial Chemotherapy

JF - Journal of Antimicrobial Chemotherapy

SN - 0305-7453

IS - 4

ER -