Molecular Characterization of a Novel Class 1 Integron Containing bla(GES-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 β-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 β-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.