Analysis of the 16S-23S rRNA gene internal transcribed spacer region in Klebsiella species

Abstract

The 16S-23S rRNA gene internal transcribed spacer (ITS) regions of Klebsiella spp., including Klebsiella pneumoniae subsp. pneumoniae, Klebsiella pneumoniae subsp. ozaenae, Klebsiella pneumoniae subsp. rhinoscleromatis, Klebsiella oxytoca, Klebsiella planticola, Klebsiella terrigena, and Klebsiella ornithinolytica, were characterized, and the feasibility of using ITS sequences to discriminate Klebsiella species and subspecies was explored. A total of 336 ITS sequences from 21 representative strains and 11 clinical isolates of Klebsiella were sequenced and analyzed. Three distinct ITS types-ITS(none) (without tRNA genes), ITS(glu) [with a tRNA(Glu (UUC)) gene], and ITS(ile+ala) [with tRNA(Ile (GAU)) and tRNA(Ala (UGC)) genes]-were detected in all species except for K. pneumoniae subsp. rhinoscleromatis, which has only ITS(glu) and ITS(ile+ala). The presence of ITS(none) in Enterobacteriaceae had never been reported before. Both the length and the sequence of each ITS type are highly conserved within the species, with identity levels from 0.961 to 1.000 for ITS(none), from 0.967 to 1.000 for ITS(glu), and from 0.968 to 1.000 for ITS(ile+ala). Interspecies sequence identities range from 0.775 to 0.989 for ITS(none), from 0.798 to 0.997 for ITS(glu), and from 0.712 to 0.985 for ITS(ile+ala). Regions with significant interspecies variations but low intraspecies polymorphisms were identified; these may be targeted in the design of probes for the identification of Klebsiella to the species level. Phylogenetic analysis based on ITS regions reveals the relationships among Klebsiella species similarly to that based on 16S rRNA genes.

FIG. 1.

Amplification of Klebsiella ITS regions. Lanes: 1, K. pneumoniae subsp. pneumoniae ATCC 10031; 2, K. pneumoniae subsp. ozaenae ATCC 11297; 3, K. pneumoniae subsp. rhinoscleromatis ATCC 13884; 4, K. terrigena CCM3568; 5, K. planticola CCM4428; 6, K. oxytoca ATCC 49473; 7, K. ornithinolytica ATCC 31898; M, DL2000 ladder; N, no template.

FIG. 2.

Alignment of the nucleotide sequences of ITS^none, ITS^glu, and ITS^ile+ala from eight representative K. pneumoniae subsp. pneumoniae strains. Each sequence is labeled with the strain number and ITS type. Dots represent nucleotides conserved in all ITS sequences. Dashes indicate gaps that have been inserted to produce optimal sequence alignment. The antiterminator (boxA) sequences are boxed and shaded. Conserved regions are boxed and labeled with CR numbers.

FIG. 3.

Alignment of the nucleotide sequences of ITS^none (A), ITS^glu (B), and ITS^ile+ala (C) from representative strains of Klebsiella species or subspecies. Each sequence is labeled with the strain number and the length. The tRNA and boxA sequences are boxed and shaded. Variable regions are boxed and labeled with VR numbers. K. pne, K. pneumoniae subsp. pneumoniae; K. oza, K. pneumoniae subsp. ozaenae; K. rhi, K. pneumoniae subsp. rhinoscleromatis; K. oxy, K. oxytoca; K. ter, K. terrigena; K pla, K. planticola; K. orn, K. ornithinolytica.

FIG. 3.

Alignment of the nucleotide sequences of ITS^none (A), ITS^glu (B), and ITS^ile+ala (C) from representative strains of Klebsiella species or subspecies. Each sequence is labeled with the strain number and the length. The tRNA and boxA sequences are boxed and shaded. Variable regions are boxed and labeled with VR numbers. K. pne, K. pneumoniae subsp. pneumoniae; K. oza, K. pneumoniae subsp. ozaenae; K. rhi, K. pneumoniae subsp. rhinoscleromatis; K. oxy, K. oxytoca; K. ter, K. terrigena; K pla, K. planticola; K. orn, K. ornithinolytica.

FIG. 4.

Phylogenetic relationships of Klebsiella species inferred from the alignments of ITS^none (A), ITS^glu (B), and ITS^ile+ala (C) in comparison with those inferred from 16S rRNA gene sequences (D). E. coli K12 is employed as the outer group reference. For each of the species or subspecies, the representative strain identified in Table 1 is used. Species and subspecies are abbreviated as explained in the legend to Fig. 3. (A to C) For ITS^none, the ITS^glu sequence of E. coli K12 excluding the tRNA gene is used. The length of each selected sequence is given in parentheses. (D) GenBank accession numbers for the 16S rRNA gene sequences of K. pneumoniae subsp. pneumoniae, K. pneumoniae subsp. ozaenae, K. pneumoniae subsp. rhinoscleromatis, K. planticola, K. terrigena, K. oxytoca, and K. ornithinolytica are given to the right of the species or subspecies abbreviation. Bootstrap values were calculated from 1,000 trees. Each number on a branch indicates the percentage of trees in which the node was supported. Bar, percent sequence divergence.