Molecular mechanisms driving the in vivo development of OXA-10-mediated resistance to ceftolozane/tazobactam and ceftazidime/avibactam during treatment of XDR Pseudomonas aeruginosa infections.

The development of resistance to ceftolozane/tazobactam and ceftazidime/avibactam during treatment of Pseudomonas aeruginosa infections is concerning. 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. 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. 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. Modification of OXA-10 is a mechanism involved in the in vivo acquisition of resistance to cephalosporin/β-lactamase inhibitor combinations in P. aeruginosa.

Arca-Suárez J ，Lasarte-Monterrubio C ，Rodiño-Janeiro BK ，Cabot G ，Vázquez-Ucha JC ，Rodríguez-Iglesias M ，Galán-Sánchez F ，Beceiro A ，González-Bello C ，Oliver A ，Bou G ... -  《-》

Selection of AmpC β-Lactamase Variants and Metallo-β-Lactamases Leading to Ceftolozane/Tazobactam and Ceftazidime/Avibactam Resistance during Treatment of MDR/XDR Pseudomonas aeruginosa Infections.

Ruedas-López A ，Alonso-García I ，Lasarte-Monterrubio C ，Guijarro-Sánchez P ，Gato E ，Vázquez-Ucha JC ，Vallejo JA ，Fraile-Ribot PA ，Fernández-Pérez B ，Velasco D ，Gutiérrez-Urbón JM ，Oviaño M ，Beceiro A ，González-Bello C ，Oliver A ，Arca-Suárez J ，Bou G ... -  《-》

Activity of cefiderocol, imipenem/relebactam, cefepime/taniborbactam and cefepime/zidebactam against ceftolozane/tazobactam- and ceftazidime/avibactam-resistant Pseudomonas aeruginosa.

To evaluate the activity of cefiderocol, imipenem/relebactam, cefepime/taniborbactam and cefepime/zidebactam against a clinical and laboratory collection of ceftolozane/tazobactam- and ceftazidime/avibactam-resistant Pseudomonas aeruginosa β-lactamase mutants. The activity of cefiderocol, imipenem/relebactam, cefepime/taniborbactam, cefepime/zidebactam and comparators was evaluated against a collection of 30 molecularly characterized ceftolozane/tazobactam- and/or ceftazidime/avibactam-resistant P. aeruginosa isolates from patients previously treated with cephalosporins. To evaluate how the different β-lactamases in the clinical isolates affected the resistance to these agents, a copy of each blaPDC, blaOXA-2 and blaOXA-10 ancestral and mutant allele from the clinical isolates was cloned in pUCp24 and expressed in dual blaPDC-oprD (for blaPDC-like genes) or single oprD (for blaOXA-2-like and blaOXA-10-like genes) PAO1 knockout mutants. MICs were determined using reference methodologies. For all isolates, MICs were higher than 4 and/or 8 mg/L for ceftolozane/tazobactam and ceftazidime/avibactam, respectively. Cefiderocol was the most active agent, showing activity against all isolates, except one clinical isolate that carried an R504C substitution in PBP3 (MIC = 16 mg/L). Imipenem/relebactam was highly active against all isolates, except two clinical isolates that carried the VIM-20 carbapenemase. Cefepime/zidebactam and cefepime/taniborbactam displayed activity against most of the isolates, but resistance was observed in some strains with PBP3 amino acid substitutions or that overexpressed mexAB-oprM or mexXY efflux pumps. Evaluation of transformants revealed that OXA-2 and OXA-10 extended-spectrum variants cause a 2-fold increase in the MIC of cefiderocol relative to parental enzymes. Cefiderocol, imipenem/relebactam, cefepime/taniborbactam and cefepime/zidebactam show promising and complementary in vitro activity against ceftolozane/tazobactam- and ceftazidime/avibactam-resistant P. aeruginosa. These agents may represent potential therapeutic options for ceftolozane/tazobactam- and ceftazidime/avibactam-resistant P. aeruginosa infections.

Lasarte-Monterrubio C ，Fraile-Ribot PA ，Vázquez-Ucha JC ，Cabot G ，Guijarro-Sánchez P ，Alonso-García I ，Rumbo-Feal S ，Galán-Sánchez F ，Beceiro A ，Arca-Suárez J ，Oliver A ，Bou G ... -  《-》

Simultaneous and divergent evolution of resistance to cephalosporin/β-lactamase inhibitor combinations and imipenem/relebactam following ceftazidime/avibactam treatment of MDR Pseudomonas aeruginosa infections.

To describe and characterize the emergence of resistance to ceftolozane/tazobactam, ceftazidime/avibactam and imipenem/relebactam in a patient receiving ceftazidime/avibactam treatment for an MDR Pseudomonas aeruginosa CNS infection. One baseline (PA1) and two post-exposure (PA2 and PA3) isolates obtained before and during treatment of a nosocomial P. aeruginosa meningoventriculitis were evaluated. MICs were determined by broth microdilution. Mutational changes were investigated through WGS. The impact on β-lactam resistance of mutations in blaPDC and mexR was determined through cloning experiments and complementation assays. Isolate PA1 showed baseline resistance mutations in DacB (I354A) and OprD (N142fs) conferring resistance to conventional antipseudomonals but susceptibility to ceftazidime/avibactam, ceftolozane/tazobactam and imipenem/relebactam. Post-exposure isolates showed two divergent ceftazidime/avibactam-resistant phenotypes associated with distinctive mutations affecting the intrinsic P PDC β-lactamase (S254Ins) (PA2: ceftolozane/tazobactam and ceftazidime/avibactam-resistant) or MexAB-OprM negative regulator MexR in combination with modification of PBP3 (PA3: ceftazidime/avibactam and imipenem/relebactam-relebactam-resistant). Cloning experiments demonstrated the role of PDC modification in resistance to ceftolozane/tazobactam and ceftazidime/avibactam. Complementation with a functional copy of the mexR gene in isolate PA3 restored imipenem/relebactam susceptibility. We demonstrated how P. aeruginosa may simultaneously develop resistance and compromise the activity of new β-lactam/β-lactamase inhibitor combinations when exposed to ceftazidime/avibactam through selection of mutations leading to PDC modification and up-regulation of MexAB-OprM-mediated efflux.

Alonso-García I ，Vázquez-Ucha JC ，Lasarte-Monterrubio C ，González-Mayo E ，Lada-Salvador P ，Vela-Fernández R ，Aja-Macaya P ，Guijarro-Sánchez P ，Rumbo-Feal S ，Muíño-Andrade M ，Fernández-González A ，Martínez-Guitián M ，Beceiro A ，Rodríguez-Iglesias M ，Oliver A ，Arca-Suárez J ，Galán-Sánchez F ，Bou G ... -  《-》

Challenging Antimicrobial Susceptibility and Evolution of Resistance (OXA-681) during Treatment of a Long-Term Nosocomial Infection Caused by a Pseudomonas aeruginosa ST175 Clone.

Selection of extended-spectrum mutations in narrow-spectrum oxacillinases (e.g., OXA-2 and OXA-10) is an emerging mechanism for development of in vivo resistance to ceftolozane-tazobactam and ceftazidime-avibactam in Pseudomonas aeruginosa Detection of these challenging enzymes therefore seems essential to prevent clinical failure, but the complex phenotypic plasticity exhibited by this species may often lead to their underestimation. The underlying resistance mechanisms of two sequence type 175 (ST175) P. aeruginosa isolates showing multidrug-resistant phenotypes and recovered at early and late stages of a long-term nosocomial infection were evaluated. Whole-genome sequencing (WGS) was used to investigate resistance genomics, whereas molecular and biochemical methods were used for characterization of a novel extended-spectrum OXA-2 variant selected during therapy. The metallo-β-lactamase blaVIM-20 and the narrow-spectrum oxacillinase blaOXA-2 were present in both isolates, although they differed by an inactivating mutation in the mexB subunit, present only in the early isolate, and in a mutation in the blaOXA-2 β-lactamase, present only in the final isolate. The new OXA-2 variant, designated OXA-681, conferred elevated MICs of the novel cephalosporin-β-lactamase inhibitor combinations in a PAO1 background. Compared to OXA-2, kinetic parameters of the OXA-681 enzyme revealed a substantial increase in the hydrolysis of cephalosporins, including ceftolozane. We describe the emergence of the novel variant OXA-681 during treatment of a nosocomial infection caused by a Pseudomonas aeruginosa ST175 high-risk clone. The ability of OXA-681 to confer cross-resistance to ceftolozane-tazobactam and ceftazidime-avibactam together with the complex antimicrobial resistance profiles exhibited by the clinical strains harboring this new enzyme argue for maintaining active surveillance on emerging broad-spectrum resistance in P. aeruginosa.

Arca-Suárez J ，Fraile-Ribot P ，Vázquez-Ucha JC ，Cabot G ，Martínez-Guitián M ，Lence E ，González-Bello C ，Beceiro A ，Rodríguez-Iglesias M ，Galán-Sánchez F ，Bou G ，Oliver A ... -  《-》