Molecular characterization of a novel class 1 integron containing bla(GES-1) and a fused product of aac3-Ib/aac6'-Ib' gene cassettes in Pseudomonas aeruginosa

Abstract

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 aac3-Ib and aac6'-Ib' gene cassettes. The fusion product was functional, as was the product of each gene cloned separately: AAC3-I, despite the deletion of the four last amino acids, and AAC6', which carried three amino acid changes compared with the most homologous sequence. The AAC3-I protein conferred an expected gentamicin and fortimicin resistance, and the AAC6', 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 AAC6' activity with a higher level of gentamicin resistance without accompanying fortimicin resistance.

FIG. 1.

Schematic representations of the different bla_GES-1- and bla_IBC-1-containing integrons. (1) Part of the structure of the recombinant plasmid pC23 encoding bla_GES-1 in P. aeruginosa Pa695. The horizontal arrows indicate the translation orientation. The solid lines represent the sequenced fragment from P. aeruginosa Pa695 with the different genes boxed, and the dotted lines indicate the unanalyzed sequence. The conserved core and inverse core sites are boxed, and the cassette boundaries are represented by vertical arrows. Dashes are used to indicate where the reported sequence was identical to already-published sequences. (2) Structure of the bla_GES-1 gene cassette-containing integron from K. pneumoniae ORI-1 (33). (3) Structure of the bla_IBC-1 gene cassette-containing integron from E. cloacae HT9 (12).

FIG. 2.

Sequence comparison of the fusion point of the aac(3)-Ib/aac(6")-Ib" fused cassette in Pa695 and the ends of the original aac(3)-Ib and aac(6")-Ib" gene cassettes. The data were compiled from the sequences obtained from plasmids pSCH6006 (37), pTK1 (32), pLMM562 (5), pMT222 (41), and the one analyzed here. The possible start codons are underlined, and the stop codons are indicated by asterisks. The deleted region of the aac(3)-Ib gene is shown in boldface type. The conserved core sites are boxed, and the vertical arrow indicates the site of fusion between the aac(3)-Ib and aac(6")-Ib" genes. The deduced amino acid sequence is designated in single-letter code below the nucleotide sequence.

FIG. 3.

Schematic representation of cloning experiments of the integron and its aac(3)-Ib, aac(6")-Ib" and aac(3)-Ib/aac(6")-Ib" genes performed in pGEM-T vector after PCR amplification. Arrowheads represent primer positions and their orientations, and M and ∗ indicate the location of the start and the stop codon, respectively.