Characterization of OXA-258, the Species Specific Marker for Achromobacter ruhlandii

M. PAPALIA; M. RUGGIERO; G. TRAGLIA; G. GUTKIND; M. RAMIREZ; M. RADICE

San Diego, California.

Congreso; 55th Interscience Conference on Antimicrobial Agents and Chemotherapy; 2015

American Society for Microbiology

Abstract:Background: A. ruhlandii is an opportunistic pathogen known to beresistant to a wide range of antibiotics; however, the knowledge about theresistance mechanisms is limited. Previously we described a new class Dbeta-lactamase named OXA-258a from carbapenem resistant A. ruhlandii strains,and also, OXA-258b, differing in one aminoacid, from a carbapenem susceptiblestrain. OXA-258 constitutes a naturally occurring beta-lactamase in A.ruhlandii, which differed from OXA-114a of A. xylosoxidans by 40amino acids, displaying 85% identity.The aim of this study was to characterize the kinetic parameters for bothOXA-258 variantsMethods: The full length blaOXA-258 was amplified byPCR using custom designed primers containing the NdeI and BamHIrestriction sites and was cloned into pK19 and pET-28a. Their authenticity wasconfirmed by sequencing. Beta-lactam MICs were determined in Escherichiacoli TOP10 transformed with pK19-OXA-258 plasmids according to CLSI.OXA-258a and OXA-258b were purified by immobilized metal ion affinity chromatography.His tag could not be remove as thrombin cleavage sites are present within thecoding sequence. Beta-lactam kinetic parameters (kcat and Km)were determined in 25mM phosphate buffer (pH 7.5) containing NaHCO350 mM.Results: Kinetic measurements of both enzymes indicated an overall weakcatalytic efficiency toward beta-lactams. Kcat values (s-1 ) weredetermined for OXA-258a and OXA-258b (cephalexin:0.0344 and 0.063; cephazoline:0.009 and 0.008) showing an efficient hydrolysis towards these antibiotics. Nohydrolysis was detected for ceftazidime, cefotaxime and cefepime. A detectablehydrolysis of imipenem was observed. However, kcat valuescould not be obtained because of high Km values, indicating avery poor affinity for this substrate.Conclusions: The contribution of OXA-258-like enzymes to the finalbeta-lactam resistance profile observed in A. ruhlandii may besecondary, as previously demonstrated for naturally occurring OXA-114 in A.xylosoxidans. No significant differences were observed between OXA-258a andOXA-258b, despite A. ruhlandii clinical isolates showed differentimipenem resistance profiles.