Molecular characterization of a novel class 1 integron containing blaGES-1 and a fused product of aac(3)-ib/Aac(6 ')-Ib ' gene cassettes in Pseudomonas aeruginosa

As seen by the disk diffusion method, the clinical strain of Pseudomonas aeruginosa Pa695, resistant to all extended-spectrum cephalosporins and aminoglycosides, exhibited an unusual synergistic effect between ceftazidime and imipenem. This isolate produced an extended-spectrum beta-lactamase (ESBL) with a pI of 5.8 that appeared to be chromosomally encoded. Cloning experiments revealed that this ESBL was encoded by bla(GES-1), previously described in an integron from Klebsiella pneumoniae. In P. aeruginosa Pa695, a higher level of resistance to ceftazidime than to ticarcillin was observed, and no synergy between the beta-lactamase inhibitors and extended-spectrum cephalosporins was detected, in contrast to the resistance pattern observed in K. pneumoniae. Further sequence analysis demonstrated that the bla(GES-1) gene cassette was located in a class 1 integron, which contained another sequence corresponding to the fused aac(3)-Ib and aac(6')-Ib' gene cassettes. The fusion product was functional, as was the product of each gene cloned separately: AAC(3)-I, despite the deletion of the four last amino acids, and AAC(6'), which carried three amino acid changes compared with the most homologous sequence. The AAC(3)-I protein conferred an expected gentamicin and fortimicin resistance, and the AAC(6'), despite the Leu-119-->Ser substitution, yielded resistance to kanamycin, tobramycin, and dibekacin, but slightly affected netilmicin and amikacin, and had no apparent effect on gentamicin. The fusion product conveyed a large profile of resistance, combining the AAC(6') activity with a higher level of gentamicin resistance without accompanying fortimicin resistance.