Molecular mechanisms driving thein vivodevelopment of OXA-10-mediated resistance to ceftolozane/tazobactam and ceftazidime/avibactam during treatment of XDRPseudomonas aeruginosainfections

Abstract

Background The development of resistance to ceftolozane/tazobactam and ceftazidime/avibactam during treatment of Pseudomonas aeruginosa infections is concerning. Objectives Characterization of the mechanisms leading to the development of OXA-10-mediated resistance to ceftolozane/tazobactam and ceftazidime/avibactam during treatment of XDR P. aeruginosa infections. Methods Four paired ceftolozane/tazobactam- and ceftazidime/avibactam-susceptible/resistant isolates were evaluated. MICs were determined by broth microdilution. STs, resistance mechanisms and genetic context of β-lactamases were determined by genotypic methods, including WGS. The OXA-10 variants were cloned in PAO1 to assess their impact on resistance. Models for the OXA-10 derivatives were constructed to evaluate the structural impact of the amino acid changes. Results The same XDR ST253 P. aeruginosa clone was detected in all four cases evaluated. All initial isolates showed OprD deficiency, produced an OXA-10 enzyme and were susceptible to ceftazidime, ceftolozane/tazobactam, ceftazidime/avibactam and colistin. During treatment, the isolates developed resistance to all cephalosporins. Comparative genomic analysis revealed that the evolved resistant isolates had acquired mutations in the OXA-10 enzyme: OXA-14 (Gly157Asp), OXA-794 (Trp154Cys), OXA-795 (ΔPhe153-Trp154) and OXA-824 (Asn143Lys). PAO1 transformants producing the evolved OXA-10 derivatives showed enhanced ceftolozane/tazobactam and ceftazidime/avibactam resistance but decreased meropenem MICs in a PAO1 background. Imipenem/relebactam retained activity against all strains. Homology models revealed important changes in regions adjacent to the active site of the OXA-10 enzyme. The blaOXA-10 gene was plasmid borne and acquired due to transposition of Tn6746 in the pHUPM plasmid scaffold. Conclusions Modification of OXA-10 is a mechanism involved in the in vivo acquisition of resistance to cephalosporin/β-lactamase inhibitor combinations in P. aeruginosa.

Functional and Structural Characterization of OXA-935, a Novel OXA-10-family β-lactamase from Pseudomonas aeruginosa

“…Compared to strains expressing a vector control, the expression of OXA-14 resulted in a 16-fold increase in the ceftazidime MIC in PAO1 and an 8-fold increase in PA14, consistent with previous reports (6,7,17). Expression of OXA-935 resulted in a 16-fold increase in ceftazidime MIC in PAO1 and a 32-fold increase in PA14, providing evidence that both OXA-14 and OXA-935 confer resistance to ceftazidime, but that expression of OXA-935 has a greater impact on MIC.…”

“…W154, in addition to stabilizing K70, helps orient K70 so that it has optimal interactions with other residues in the active site (21). Critically, mutations in W154 (W154C), deletion of F153-W154, and duplications of negatively charged residues in this region have been identified in P. aeruginosa clinical isolates resistant to ceftazidime-avibactam and ceftolozane-tazobactam (17, 30). In addition, the Ω-loop of OXA-935 was considerably more flexible than in OXA-14.…”

“…The copyright holder for this preprint this version posted September 23, 2021. ; https://doi.org/10.1101/2021.09.20.461173 doi: bioRxiv preprint confers ceftazidime resistance without an accompanying G157D substitution (17). Phylogenetic analysis of these OXA-10 family proteins showed that they are divided into two major groups classified by their first identified and earliest-named members, OXA-7 and OXA-10.…”

“…S1). OXA-795 also confers ceftazidime resistance without an accompanying G157D substitution (17). Phylogenetic analysis of these OXA-10 family proteins showed that they are divided into two major groups classified by their first identified and earliest-named members, OXA-7 and OXA-10.…”

“…Finally, OXA-10-family -lactamases are often found in mobile transmissible elements such as plasmids (17). OXA-935 is no exception in that it is located within a plasmid-borne integron that harbors additional AMR elements (1).…”