Screening of antibiotic resistance genes isolates of acinetobacter baumannii, molecular characterization of variants of Blages-22 and Blaoxa-66

Genetic mechanisms of resistance were determined in clinical strains of 101 Acinetobacter baumannii in this PhD thesis. Multiplex PCR was used for detecting blaOXA-51-like, blaOXA-23-like, blaOXA-40-like and blaOXA-58-like genes. ISAba1, blaIMP, blaVIM, blaGES, blaVEB, blaPER-2, aac-3-Ia, aac-6′-Ib and blaNDM-1 genes were detected by PCR and sequencing. By multiplex PCR, all strains carried blaOXA-51, 79 strains carried blaOXA-23 and one strain carried blaOXA-40. blaOXA-51 and blaOXA-23 were found together in 79 strains. ISAba1 element was detected in 81 strains, and in all cases it was found upstream of blaOXA-51. GES-type beta lactamases were found in 24 strains (GES-11 in 16 strains and GES-22 in 8 strains). blaOXA-58, blaIMP, blaVIM, blaVEB, blaPER-2, and blaNDM-1 genes were not observed. aac-3-Ia and aac-6′Ib genes were detected in 13 and 15 strains, respectively. All strains were collected into 12 different groups with REP-PCR. It was first reported that GES-11 and OXA-40 in A. baumannii from Turkey. GES-22 differs from GES-11 by a single amino acid substitution (M169L). Because M169 is part of the omega loop, a structure that is known to have major effects on substrate selectivity in class A β-lactamases, GES-22 was expressed, purified and kinetically characterized. This study shows that compared to GES-11, GES-22 displays more efficient hydrolysis of penicillins, cefotaxime and aztreonam, but a loss of efficiency against ceftazidime. Additionally, the M169L substitution confers on GES-22 more efficient hydrolysis of the clavulanic acid. These effects are highly similar to other mutations at the homologous position in other class A β-lactamases, suggesting that this methionine plays a key structural role in aligning active site residues and in substrate selectivity across the class. OXA-66 mutants were expressed, purified and kinetically characterized. It was observed that clinical mutations W222L, I129V, P130Q can increase the hydrolytic activity against carbapenems in OXA-66.