Dados do Trabalho

Título

Acinetobacter-derived cephalosporinases: Identification of new motifs involved with resistance to carbapenems and other beta-lactams

Introdução

Acinetobacter baumannii is an opportunistic bacterium involved with healthcare-associated infections. Antimicrobial resistance in A. baumannii arises through various mechanisms, including the production of antibiotic-inactivating enzymes. Acinetobacter-derived cephalosporinases (ADC) are primarily associated with beta-lactam resistance. These enzymes can hydrolyze cephalosporins but not carbapenems, although changes in their hydrolytic capacity have been linked to amino acid alterations. Recently, the ADC-68 was characterized with activity also against carbapenems, in addition to the third and fourth generation cephalosporins. In a study developed at Aggeu Magalhães Institute, ADCs with amino acid substitutions like those described in ADC-68 were identified in A. baumannii isolates from infections in hospitalized patients in Pernambuco.

Objetivo (s)

To investigate the ability of the different ADC variants to hydrolyze carbapenems, as well as to identify the main motifs involved with resistance to carbapenems and other beta-lactams.

Material e Métodos

The enzymatic variants were cloned into the pET28a(+) and expressed in E. coli BL21(DE3). The sensitivity profile of transformed cells to beta-lactams was evaluated by microdilution and enzymatic hydrolysis tests. To investigate additional amino acid substitutions that expand the hydrolytic spectrum of ADC, the variants were modified with specific motifs from ADC-68 (T179I, P194A, D220G, G320R) using site-directed mutagenesis.

Resultados e Conclusão

The ADCs evaluated in this study have ESBL and carbapenemase activity, in addition to the identification of isolated and simultaneous substitutions of amino acids that partially or completely alter its hydrolysis profile to imipenem and other beta-lactams. The insertion of Pro-Ala-Ser (A221_P222insPAS) in the Ω-loop of ADC significantly expands the active site cavity and may enhance enzymatic affinity for imipenem. Individual and simultaneous substitutions of D220G and G320R greatly influence the hydrolysis profile of ADC, reversing it towards broad-spectrum penicillin, third/fourth-generation cephalosporins, and carbapenems. Substitutions of T179I and P194A alter the hydrolysis rate of ADC towards imipenem without affecting the hydrolysis spectrum of the enzymatic variants.

Palavras-chave

Drug resistance; Beta-lactamases; Healthcare-associated infections.

Agradecimentos

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq grant #400767/2019-8).