Temocillin Resistance in the Enterobacter cloacae Complex Is Conferred by a Single Point Mutation in BaeS, Leading to Overexpression of the AcrD Efflux Pump

• Published in: Antimicrobial agents and chemotherapy 2023-06, Vol.67 (6), p.e0035823-e0035823
• Main Authors: Guérin, François, Gravey, François, Reissier, Sophie, Penven, Malo, Michaux, Charlotte, Le Hello, Simon, Cattoir, Vincent
• Format: Article
• Language: English
• Subjects: Anti-Bacterial Agents - metabolism; Anti-Bacterial Agents - pharmacology; Antimicrobial Chemotherapy; Enterobacter cloacae - genetics; Enterobacter cloacae - metabolism; Escherichia coli - genetics; Life Sciences; Mechanisms of Resistance; Membrane Transport Proteins - genetics; Microbial Sensitivity Tests; Microbiology and Parasitology; Point Mutation
• ISSN: 0066-4804; 1098-6596
• DOI: 10.1128/aac.00358-23

The Enterobacter cloacae complex (ECC) has become a major opportunistic pathogen with antimicrobial resistance issues. Temocillin, an "old" carboxypenicillin that is remarkably stable toward β-lactamases, has been used as an alternative for the treatment of multidrug-resistant ECC infections. Here, we aimed at deciphering the never-investigated mechanisms of temocillin resistance acquisition in . By comparative genomic analysis of two clonally related ECC clinical isolates, one susceptible (Temo_S [MIC of 4 mg/L]) and the other resistant (Temo_R [MIC of 32 mg/L]), we found that they differed by only 14 single-nucleotide polymorphisms, including one nonsynonymous mutation (Thr175Pro) in the two-component system (TCS) sensor histidine kinase BaeS. By site-directed mutagenesis in Escherichia coli CFT073, we demonstrated that this unique change in BaeS was responsible for a significant (16-fold) increase in temocillin MIC. Since the BaeSR TCS regulates the expression of two resistance-nodulation-cell division (RND)-type efflux pumps (namely, AcrD and MdtABCD) in E. coli and Salmonella, we demonstrated by quantitative reverse transcription-PCR that , , and genes were significantly overexpressed (15-, 11-, and 3-fold, respectively) in Temo_R. To confirm the role of each efflux pump in this mechanism, multicopy plasmids harboring or were introduced into either Temo_S or the reference strain E. cloacae subsp. ATCC 13047. Interestingly, only the overexpression of conferred a significant increase (from 8- to 16-fold) of the temocillin MIC. Altogether, we have shown that temocillin resistance in the ECC can result from a single BaeS alteration, likely resulting in the permanent phosphorylation of BaeR and leading to AcrD overexpression and temocillin resistance through enhanced active efflux.