Molecular and biochemical insights into the in vivo evolution of AmpC-mediated resistance to ceftolozane/tazobactam during treatment of an MDR Pseudomonas aeruginosa infection.

BACKGROUND: Pseudomonas aeruginosa may develop resistance to novel cephalosporin/ß-lactamase inhibitor combinations during therapy through the acquisition of structural mutations in AmpC. OBJECTIVES: To describe the molecular and biochemical mechanisms involved in the development of resistance to ceftolozane/tazobactam in vivo through the selection and overproduction of a novel AmpC variant, designated PDC-315. METHODS: Paired susceptible/resistant isolates obtained before and during ceftolozane/tazobactam treatment were evaluated. MICs were determined by broth microdilution. Mutational changes were investigated through WGS. Characterization of the novel PDC-315 variant was performed through genotypic and biochemical studies. The effects at the molecular level of the Asp245Asn change were analysed by molecular dynamics simulations using Amber. RESULTS: WGS identified mutations leading to modification (Asp245Asn) and overproduction of AmpC. Susceptibility testing revealed that PAOΔC producing PDC-315 displayed increased MICs of ceftolozane/tazobactam, decreased MICs of piperacillin/tazobactam and imipenem and similar susceptibility to ceftazidime/avibactam compared with WT PDCs. The catalytic efficiency of PDC-315 for ceftolozane was 10-fold higher in relation to the WT PDCs, but 3.5- and 5-fold lower for piperacillin and imipenem. IC50 values indicated strong inhibition of PDC-315 by avibactam, but resistance to cloxacillin inhibition. Analysis at the atomic level explained that the particular behaviour of PDC-315 is linked to conformational changes in the H10 helix that favour the approximation of key catalytic residues to the active site. CONCLUSIONS: We deciphered the precise mechanisms that led to the in vivo emergence of resistance to ceftolozane/tazobactam in P. aeruginosa through the selection of the novel PDC-315 enzyme. The characterization of this new variant expands our knowledge about AmpC-mediated resistance to cephalosporin/ß-lactamase inhibitors in P. aeruginosa.