Characterization of Pseudomonas aeruginosa resistance to ceftolozane-tazobactam due to ampC and/or ampD mutations observed during treatment using semi-mechanistic PKPD modeling

A double ampC (AmpC(G183D)) and ampD (AmpD(H157Y)) genes mutations have been identified by whole genome sequencing in a Pseudomonas aeruginosa (PaS) that became resistant (PaR) in a patient treated by ceftolozane/tazobactam (C/T). To precisely characterize the respective contributions of these mutations on the decreased susceptibility to C/T and on the parallel increased susceptibility to imipenem (IMI), mutants were generated by homologous recombination in PAO1 reference strain (PAO1- AmpC(G183D), PAO1-AmpD(H157Y), PAO1-AmpC(G183D)/AmpD(H157Y)) and in PaR (PaR-AmpC(PaS)/AmpD(PaS)). Sequential time-kill curve experiments were conducted on all strains and analyzed by semi-mechanistic PKPD modeling. A PKPD model with adaptation successfully described the data, allowing discrimination between initial and time-related (adaptive resistance) effects of mutations. With PAO1 and mutant-derived strains, initial EC(50) values increased by 1.4, 4.1, and 29-fold after AmpC(G183D) , AmpD(H157Y) and AmpC(G183D)/AmpD(H157Y) mutations, respectively. EC(50) values were increased by 320, 12.4, and 55-fold at the end of the 2 nd experiment. EC(50) of PAO1-AmpC(G183D)/AmpD(H157Y) was higher than that of single mutants at any time of the experiments. Within the PaR clinical background, reversal of AmpC(G183D), and AmpD(H157Y) mutations led to an important decrease of EC(50) value, from 80.5 mg/L to 6.77 mg/L for PaR and PaR-AmpC(PaS)/AmpD(PaS), respectively. The effect of mutations on IMI susceptibility mainly showed that the AmpC(G183D) mutation prevented the emergence of adaptive resistance. The model successfully described the separate and combined effect of AmpC(G183D) and AmpD(H157Y) mutations against C/T and IMI, allowing discrimination and quantification of the initial and time-related effects of mutations. This method could be reproduced in clinical strains to decipher complex resistance mechanisms.