RESUMEN
Acinetobacter baumannii is now one of the most frequently encountered nosocomial pathogens in intensive therapy units, and is renowned for being difficult to treat because of resistance to most antibiotics. Carbapenems are the remaining drugs of choice in many centres, but carbapenem resistance is now emerging in strains worldwide. Two subgroups of carbapenem-hydrolysing beta-lactamases, which differ in their amino-acid homology, have been found in some resistant strains. This report describes the emergence and characterisation of a novel carbapenemase (OXA-51) in genetically distinct carbapenem-resistant A. baumannii strains from Argentina. Enzyme kinetics and inhibitor studies were performed spectrophotometrically with purified beta-lactamase. Amplification of the gene was achieved with a two-step PCR method employing arbitrary partially degenerate and gene-specific primers. Transfer of imipenem resistance was attempted with the use of broth and membrane filter methods. Attempts to produce plasmid-cured variants were made in ethidium bromide curing experiments. OXA-51 was identified in two clones of A. baumannii, and was found to have < 63% amino-acid identity with subgroups 1 and 2. Enzyme kinetic studies confirmed that OXA-51 was a molecular class D enzyme with carbapenemase activity, and that it displayed the highest affinity for imipenem (Km value 11 microM). Sequence analysis of the gene identified distinct differences within conserved class D motifs when compared with subgroups 1 and 2. Attempts to transfer imipenem resistance and to determine a plasmid location for the gene failed. OXA-51 is the first of a new subgroup of carbapenemases to emerge in multiresistant clinical isolates of A. baumannii.
Asunto(s)
Acinetobacter baumannii/clasificación , Acinetobacter baumannii/genética , Proteínas Bacterianas , Resistencia betalactámica , beta-Lactamasas , Infecciones por Acinetobacter/microbiología , Acinetobacter baumannii/efectos de los fármacos , Acinetobacter baumannii/enzimología , Secuencia de Aminoácidos , Antibacterianos/farmacología , Argentina , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/aislamiento & purificación , Proteínas Bacterianas/metabolismo , Carbapenémicos/farmacología , Humanos , Imipenem/farmacología , Cinética , Pruebas de Sensibilidad Microbiana , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , beta-Lactamasas/química , beta-Lactamasas/genética , beta-Lactamasas/aislamiento & purificación , beta-Lactamasas/metabolismoRESUMEN
BACKGROUND: Acinetobacter baumannii is an important etiological agent causing nosocomial infections. High level of resistance for different kind of antimicrobials has been observed, including beta-lactam antibiotics. This feature, chromosomal or plasmid encoded, has been associated to integrons harbouring antibiotic resistance gene cassettes. AIMS: To investigate the presence of integrons among clinical isolates resistant to third generation cephalosporins (3GC). MATERIAL AND METHODS: One hundred A. baumannii strains isolated from several Chilean hospitals were included in this study. Minimal inhibitory concentrations (MIC) of 3GC by an agar dilution method were carried out. Integrons class 1, 2 and 3 were investigated by colony blot hybridisation and confirmed by PCR. RESULTS: High level of resistance to all assayed 3GC was observed. On the other hand, integrón class 2 was the most prevalent (77% of isolates) followed by integron class 1 (52%). Forty six percent of isolates hybridised with probes for both of them. However, no positive hybridisation was detected for integron class 3. CONCLUSIONS: Nevertheless, most isolates harboured one or both class of integron; there was no direct relationship between the presence of these genetic structures and the resistance to this kind of beta-lactam antibiotics.