Characterization of OXA-258 enzymes and AxyABM efflux pump from Achromobacter ruhlandii

PAPALIA, MARIANA; TRAGLIA, GERMÁN; RUGGIERO, MELINA; ALMUZARA, MARISA; VAY, CARLOS; GUTKIND, GABRIEL; RAMÍREZ, SOLEDAD MARÍA; RADICE, MARCELA

Journal of Global Antimicrobial Resistance

Elsevier

Año: 2018

2213-7165

OBJECTIVES: The aim of this study was to characterize OXA-258 variants and other features that may contribute to the carbapenem resistant 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 antibiotics. 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 resistant determinants was conducted. Substrates of AxyABM efflux pump were investigated by inhibition assays using phenylalanine (Phe)-arginine (Arg)-β-naphthylamide. Outer membrane proteins profiles were resolved by 12% 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 coding genes in A. ruhlandii 38 and 319, respectively. Complete sequences for AxyABM and AxyXY efflux pumps, previously described in Achromobacter xylosoxidans, were detected. Decrease in the MIC values for chloramphenicol, nalidixic acid, trimethoprim-sulfamethoxazole was observed in the presence of inhibitor suggesting that these antibiotics are substrates of AxyABM. AxyXY coding genes of A. ruhlandii 38 and A. ruhlandii 319 displayed 99% identity between them. No differences were observed in the outer membrane proteins profiles.CONCLUSIONS: The contribution of OXA-258a 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.