RESUMO
The two genetic groups (oxy-1 and oxy-2) previously identified in the Klebsiella oxytoca taxon are recognizable by four independent molecular markers: (i). ERIC-1R profiles, (ii). 16S ribosomal DNA (rDNA) signature sequences, (iii). singular nucleotides in a defined fragment of the rpoB gene, and (iv) the type of the strain's bla(OXY) gene (i.e., bla(OXY-1) or bla(OXY-2)). K. oxytoca strains SG266 and SG271 could not be classified into these genetic groups based on their ERIC-1R profile and bla(OXY) gene sequence. With regard to the gene identity percentages between the bla(OXY-1) and bla(OXY-2) gene groups (86.8% +/- 0.4%) and within a bla(OXY) gene group (>99%), it was concluded that the bla(OXY) gene of strain SG271 was representative of a new bla(OXY) gene group (bla(OXY-3)), since the mean identity percentages between it and the two bla(OXY) gene groups were 85.5% +/- 0.2% and 84.4% +/- 0.4%, respectively. Since the corresponding percentages were 95.0% +/- 0.4% and 86.2% +/- 0.3% for strain SG266, it was impossible to classify its bla(OXY) gene, which was therefore named bla(OXY-4). The 16S rDNA signature sequences of the two strains could be determined only after cloning experiments. The SG266 clones displayed the same signature sequence as that of the genetic group oxy-1, whereas the SG271 clones displayed three different 16S rDNA signature sequences that also differed from those of the two genetic groups. Singular nucleotides were found within the rpoB sequence of the two strains, allowing for their distinction from the two genetic groups. All of these results, combined with those previously obtained by the ERIC-1R PCR method, indicate that strain SG271 is representative of a new K. oxytoca genetic group (oxy-3), whereas strain SG266 could not be classified.
Assuntos
Klebsiella oxytoca/enzimologia , Klebsiella oxytoca/genética , beta-Lactamases/genética , Antibacterianos/farmacologia , Sequência de Bases , DNA Ribossômico/biossíntese , DNA Ribossômico/genética , RNA Polimerases Dirigidas por DNA/genética , Focalização Isoelétrica , Testes de Sensibilidade Microbiana , Dados de Sequência Molecular , Filogenia , Plasmídeos/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , beta-LactamasRESUMO
Whilst searching for a molecular method to identify the different species of Raoultella and Klebsiella oxytoca, it was observed that the OXY-1 and OXY-2 beta-lactamase-producing K. oxytoca isolates displayed two distinguishable enterobacterial repetitive intergenic consensus (ERIC)-1R profiles. It was hypothesized that the two groups of chromosomal beta-lactamases might correspond to two groups of strains in the K. oxytoca taxon. To confirm this hypothesis, clinical isolates and reference strains of K. oxytoca were studied by determination of the sequence of their bla(OXY) genes, and of a partial fragment of their 16S rRNA (387 bp) and rpoB (512 bp) genes. The sequence data were phylogenetically analysed by using the parsimony method. Four clinical isolates possessed a bla(OXY-1) gene and nine possessed a bla(OXY-2) gene. The mean percentage of rpoB and 16S rRNA gene identity was > 99% within each group of strains, whereas it was 96.56 +/- 0.24% for rpoB genes and 97.80 +/- 0.22% for 16S rRNA genes between the group of strains harbouring the bla(OXY-1) gene and the group harbouring the bla(OXY-2) gene. The phylogenetic tree resulting from combined analysis of the 16S rRNA and rpoB datasets showed that the K. oxytoca isolates were monophyletic and separated into two clades; these clades included strains with either the bla(OXY-1) gene or the bla(OXY-2) gene. This result was supported with high bootstrap values of 97 and 99%, respectively. Moreover, the two groups of strains displayed distinct ERIC-1R profiles, with bands characteristic of each profile. Thus, the chromosomal bla(OXY) gene sequence is able to delineate not only two groups of beta-lactamases in K. oxytoca, but also two clades in the K. oxytoca taxon, in a manner similar to the sequence of housekeeping genes. These results suggest that K. oxytoca should be divided into two genetic groups, group OXY-1 represented by K. oxytoca strain SL781 (=CIP 104963) and group OXY-2 by K. oxytoca strain SL91l (= CIP 106098).
Assuntos
RNA Polimerases Dirigidas por DNA/genética , Klebsiella oxytoca/classificação , Klebsiella oxytoca/genética , Reação em Cadeia da Polimerase/métodos , RNA Ribossômico 16S/genética , beta-Lactamases/genética , Técnicas de Tipagem Bacteriana , Cromossomos Bacterianos/genética , DNA Intergênico/análise , DNA Ribossômico/análise , Genes de RNAr , Humanos , Dados de Sequência Molecular , Filogenia , Sequências Repetitivas de Ácido Nucleico , Análise de Sequência de DNA , Especificidade da EspécieRESUMO
The enterobacterial repetitive intergenic consensus 1R PCR method, which provided recognizable profiles for reference strains of the three species of Raoultella and the two genetic groups of Klebsiella oxytoca, was applied to 19 clinical isolates identified as K. oxytoca. By this method, as confirmed by species-specific gene sequencing, two Raoultella ornithinolytica and two unclassifiable K. oxytoca isolates were identified.
