Resumo

BACKGROUND Klebsiella variicola is a new species from Klebsiella genus. It was firstly identified in plants, but its association with hospital infections has already been reported. Due to the close genetic relatedness between K. variicola and K. pneumoniae, biochemical tests fail to differentiate them. Four chromosomal class A β-lactamase genes were recognized in Klebsiella: bla_SHV, bla_OKP and bla_LEN in K. pneumoniae; and bla_OXY in K. oxytoca. The bla_SHV is a chromosomal- and plasmid-borne gene encoding narrow and extended spectrum β-lactamases. Conversely, LEN enzymes have a restrict activity against β-lactams, conferring resistance only to penicillins. Considering K. variicola, there is a lack of information concerning its intrinsic resistance determinants. OBJECTIVE We aimed to characterize and determine the functionality of the class A β-lactamase gene in K. variicola by in silico and in vitro analysis. METHODS Using the Biocyc software, the unique complete genome of K. variicola available was analyzed for the presence of β-lactamase homologs. The conserved motifs of class A β-lactamase family were verified in the complete K. variicola genome using the Conserved Domain Database. Primers targeting the entire coding region of the putative β-lactamase were designed and used to amplify and sequence this gene in the clinical and environmental K. variicola strains from Brazil. The PCR products were cloned in expression vector and used to transform Escherichia coli. The transformants were submitted to susceptibility tests to evaluate the putative β-lactamase activity. RESULTS AND DISCUSSION It was identified nine putative β-lactamases in the K. variicola AT-22 complete genome. One of these, Kvar_2736, presented 91% amino acid identity with SHV and 99% with LEN. Moreover, it presented the three functional motifs of class A β-lactamases: SXXK, SDN and KTG, at amino acid positions 70, 130 and 234, respectively. Three different bla_LEN alleles were also identified in the Brazilian K. variicola strains. One of them was identical to that found in the complete K. variicola genome, while the others corresponded to new and not yet described alleles. Both the clinical Brazilian K. variicola isolates and their transformants were resistant to ampicillin, amoxicillin and ticarcillin, a resistance profile compatible with that evoked by LEN β-lactamase. CONCLUSION These findings provided the first evidences of the bla_LEN as the chromosomal class β-lactamase from K. variicola.