In vivo evolution of ceftazidime–avibactam resistance in blaOXA-244-positive E. coli potentially linked to PBP3 insertion and mutations in acrB and PBP2

Abstract

Background: Ceftazidime/avibactam has been introduced as a promising treatment option against multidrug-resistant Gram-negative bacteria. Objectives: To investigate the development of ceftazidime/avibactam resistance in a blaOXA-48-like-carrying Escherichia coli strain with YRIK insertion in penicillin-binding protein 3 (PBP3). Methods: Eight clinical isolates were recovered from a single patient treated with ceftazidime/avibactam. The isolates were analysed using antimicrobial susceptibility testing, and WGS to identify potential resistance mechanisms. In vitro serial passage experiments with increasing ceftazidime/avibactam exposure were performed to model the in vivo resistance development. Quantitative RT–PCR was used to assess acrB mRNA expression. Results: Ceftazidime/avibactam resistance emerged during treatment, accompanied by significant increases in aztreonam/avibactam and avibactam MICs. All isolates, including those susceptible to ceftazidime/avibactam, had a YRIK insertion in PBP3. Additional mutations were identified in the AcrB efflux pump component and, in most cases in its regulatory genes and PBP2 in the resistant isolates. No significant differences in acrB expression levels were found between susceptible and resistant isolates, suggesting that structural changes in AcrB, rather than overexpression, are likely to contribute to resistance. Serial passage experiments confirmed these findings by demonstrating the emergence of mutations in the same genes under increasing ceftazidime/avibactam pressure. Conclusions: This study shows a complex resistance mechanism involving a YRIK insertion in PBP3, combined with mutations in AcrB and PBP2, as drivers of ceftazidime/avibactam resistance. These findings highlight the importance of monitoring E. coli isolates with YRIK insertions during ceftazidime/avibactam treatment and warrant further investigation into efflux pump-mediated resistance in Enterobacterales.