Class 2 integron with a novel cassette array in a Burkholderia cenocepacia isolate.

Burkholderia cepacia complex (BCC) organisms are gramnegative opportunistic emerging pathogens associated with a poor prognosis for patients with cystic fibrosis (CF) (10). Carbapenems and ceftazidime are administered to patients suffering from BCC infections, and trimethoprim-sulfamethoxazole has historically been the drug of choice (6). Recently, the acquisition of resistance determinants to sulfamethoxazole located in the 3 conserved region of class 1 integrons in BCC isolates from CF patients has been described (2). The goal of this study was to determine the presence of class 1, 2, and 3 integrons in a Burkholderia cenocepacia strain (BC1) isolated from the sputum of a 14-year-old CF patient in a surgery and transplant center in Buenos Aires, Argentina. The phenotypic analysis was performed using biochemical reactions (4) and led us to identify the isolate as BCC. The genotypic identification by the recA PCR-restriction fragment length polymorphism method (9) resulted in a IIIB recA lineage, corresponding to Burkholderia cenocepacia. A search for the presence of the cable pilin subunit gene (cblA) and the B. cepacia epidemic strain marker, by PCR with specific primers (9), yielded negative results. The BC1 strain was susceptible to trimethoprim-sulfamethoxazole (MIC 1 g/ml), meropenem (MIC 0.012 g/ml), ceftazidime (MIC 0.19 g/ml), and minocycline (MIC 1 g/ml) according to CLSI (formerly NCCLS) (1, 7). By analysis of the class 1, 2, and 3 integrons present in the BC1 strain by PCR with specific primers (Table 1), we found only a class 2 integron (8). In order to identify the inserted gene cassettes, PCR cartography was performed with primers described in Table 1. Two PCR products, obtained with the 125 CS and satR primers and the Inti2R and satR primers (Fig. 1) were sequenced on both strands using an ABI 373 sequencer and analyzed using the Genetics Computer Group (GCG) software (Wisconsin Package, version 10.3). Only a sat2 gene cassette was found in the variable region, and it is noteworthy that this novel rearrangement did not have the orfX cassette always present in Tn7-like transposons (Fig. 1). Also, the analysis of the sequence led us to determine that the internal stop codon in the class 2 integrase gene was present as it has been previously described (3). Therefore, this novel rearrangement could likely have arisen from the action of a class 1 integrase from another element in trans (3). We also detected the presence of tnsE and tnsD genes located in the 3 conserved sequence region and involved in Tn7 transposition (11) (Table 1 and Fig. 1). We have named it Tn7::In2-1 (accession number DQ082896) in order to identify novel rearrangements of class 2 integrons in a simple manner. Our results provide evidence that class 2 integrons located in the Tn7 family of transposons are able to be incorporated in the genome of BCC, as described for class 1 integrons recently (2).