Molecular characterization of a new class 3 integron in Klebsiella pneumoniae

Abstract

Klebsiella pneumoniae FFUL 22K was isolated in April 1999 from the urine of an intensive care unit patient in Portugal. The strain showed an extended-spectrum cephalosporin resistance profile. A typical synergistic effect between cefotaxime or cefepime and clavulanic acid was observed. An Escherichia coli transformant displayed a similar resistance phenotype and harbored a ca. 9.4-kb plasmid (p22K9). Cloning experiments revealed that the extended-spectrum beta-lactamase was encoded by bla(GES-1), previously described in class 1 integrons from K. pneumoniae ORI-1 and Pseudomonas aeruginosa Pa695. Further sequence analysis demonstrated that the bla(GES-1) gene cassette was located on a new class 3 integron. The integron was 2863 bp long and consisted of an intI3 integrase gene, an attI3 recombination site, two promoter regions, and two gene cassettes. The IntI3 integrase was 98.8% identical to that of Serratia marcescens AK9373. The bla(GES-1) gene cassette was inserted at the attI3 site. The second gene cassette was the result of a fusion event between bla(OXA-10)-type and aac(6')-Ib gene cassettes and conferred resistance to kanamycin. This is the second class 3 integron reported and the first time that the bla(GES-1) gene cassette has been found on an integron belonging to this class, highlighting the considerable heterogeneity of their genetic environment and the spread of gene cassettes among different classes of integrons.

FIG. 1.

Schematic representation of class 3 integron In3-p22K9 and its flanking region from p22K9. Open reading frames are indicated by arrows, and the 59-base elements are indicated by black solid circles.

FIG. 2.

Sequence alignment and comparison of fusion point of bla_OXA-10-type/aac(6′)-Ib fused gene cassette (shown in capital letters) in p22K9 with the initial regions of the bla_OXA-10 (24) and aac(6′)-Ib (39) gene cassettes. The nucleotide sequences of the bla_OXA-10 and aac(6′)-Ib gene cassettes presenting 100% homology with the fused cassette are also shown in capital letters, where colons indicate identical nucleotides. The putative ribosome-binding sites are underlined, and the translational start codons are double underlined.