INVESTIGADORES
RUGGIERO Melina
congresos y reuniones científicas
Título:
INQ-1: Preliminary Characterization of a beta-lactamase from Inquilinus limosus
Autor/es:
M. PINO; P. POWER; J. DI CONZA; M. RUGGIERO; G. GUTKIND
Lugar:
San Francisco. California
Reunión:
Congreso; 52nd Interscience Conference on Antimicrobial Agents and Chemotherapy; 2012
Institución organizadora:
American Society for Microbiology
Resumen:
Introduction: Inquilinus limosus is increasingly isolated from cystic fibrosis patients (CFP). Our strain was isolated from a pediatric CFP. By previous in silico analysis, conserved motifs compatible with class A/C beta-lactamases were observed in the 360-aminoacid putative beta-lactamase INQ-1; no clear omega-loop could be assigned. Our goal was to biochemically characterize INQ-1 b-lactamase from I. limosus. Methods: The INQ-1 encoding gene was cloned in a suitable vector. The enzyme was overproduced and highly purified by conventional chromatographic methods. Main kinetic parameters of INQ-1 were determined for selected antibiotics. For poor substrates, apparent Km was determined as Ki using 110uM cephalothin as the reporter substrate. For inactivators, IC50 was calculated after competition experiments, with and without pre-incubation, using the same reporter substrate. Results: INQ-1 showed no detectable hydrolysis of cefuroxime, ceftazidime and cefotaxime. Both cephalothin and benzylpenicillin showed similar catalytic efficiencies (kcat/Km= 0,056 uM-1.s-1 and 0,048 uM-1.s-1, respectively). Inhibition by clavulanic acid was observed only after pre-incubation competitive assays (Indirect IC50 = 56 uM). On the other hand, 3-phenyl boronic acid showed enzymatic inhibition also in direct competitive assays at high concentrations (Direct IC50= 390 uM; Indirect IC50= 151 uM). Conclusion: Based on both in silico and partial biochemical analysis, INQ-1 could be a b-lactamase with mixed class A/C behavior, which could make a stakeout on the present classification necessary. Catalytic evaluations prevent to assign to this enzyme the full resistance pattern observed in the I. limosus isolate.