Characterisation of OXA-258 enzymes and AxyABM efflux pump in Achromobacter ruhlandii

Abstract

Objectives: The aim of this study was to characterise OXA-258 variants and other features that may contribute to carbapenem resistance in Achromobacter ruhlandii. Methods: Kinetic parameters for purified OXA-258a and OXA-258b were determined measuring the rate of hydrolysis of a representative group of antimicrobial agents. Whole-genome shotgun sequencing was performed on A. ruhlandii 38 (producing OXA-258a) and A. ruhlandii 319 (producing OXA-258b), and in silico analysis of antimicrobial resistance determinants was conducted. Substrates of the AxyABM efflux pump were investigated by inhibition assays using phenylalanine-arginine β-naphthylamide (PAβN). Outer membrane protein profiles were resolved by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Results: Kinetic measurements of purified OXA-258 variants displayed an overall weak catalytic efficiency toward β-lactams. A detectable hydrolysis of imipenem was observed. In silico genomic analysis confirmed the presence of 32 and 35 putative efflux pump-encoding genes in A. ruhlandii strains 38 and 319, respectively. Complete sequences for AxyABM and AxyXY efflux pumps, previously described in Achromobacter xylosoxidans, were detected. Decreases in the MICs for chloramphenicol, nalidixic acid and trimethoprim/sulfamethoxazole were observed in the presence of the inhibitor PAβN, suggesting that these antibiotics are substrates of AxyABM. AxyXY-encoding genes of A. ruhlandii 38 and A. ruhlandii 319 displayed 99% identity. No differences were observed in the outer membrane protein profiles. Conclusions: The contribution of OXA-258 enzymes to the final β-lactam resistance profile may be secondary. Further studies on other putative resistance markers identified in the whole-genome analysis should be conducted to understand the carbapenem resistance observed in A. ruhlandii.