Species-specific identification of Leptospiraceae by 16S rRNA gene sequencing

Abstract

The genus Leptospira is classified into 13 named species and 4 genomospecies based upon DNA-DNA reassociation studies. Phenotypic tests are unable to distinguish between species of Leptospira, and there is a need for a simplified molecular approach to the identification of leptospires. 16S rRNA gene sequences are potentially useful for species identification of Leptospira, but there are a large number of sequences of various lengths and quality in the public databases. 16S rRNA gene sequences of near full length and bidirectional high redundancy were determined for all type strains of the species of the Leptospiraceae. Three clades were identified within the genus Leptospira, composed of pathogenic species, nonpathogenic species, and another clade of undetermined pathogenicity with intermediate 16S rRNA gene sequence relatedness. All type strains could be identified by 16S rRNA gene sequences, but within both pathogenic and nonpathogenic clades as few as two or three base pairs separated some species. Sequences within the nonpathogenic clade were more similar, and in most cases < or =10 bp distinguished these species. These sequences provide a reference standard for identification of Leptospira species and confirm previously established relationships within the genus. 16S rRNA gene sequencing is a powerful method for identification in the clinical laboratory and offers a simplified approach to the identification of Leptospira species.

FIG. 1.

Unrooted tree of 43 Leptospiraceae 16S rRNA gene sequences. The scale bar equals the fraction of base pairs that are different.

FIG. 2.

Base differences in the 16S rRNA genes of type strains formatted to emphasize the differences within each of the three clades of Leptospira. Path, pathogenic clade; Int, intermediate clade; NonP, nonpathogenic clade. Vertical numbers show the relative positions in L. interrogans AY631894. Center dots indicate the same base as L. interrogans; a dash indicates a gap. The A at position 57 in L. alexanderi would be inserted between 56 and 57 in the L. interrogans AY631894 sequence. The sequences indicate differences within each clade but do not include all differences between clades. Note that positions 95, 154, 158, 222, and 952 appear twice, and position 1077 appears in all three clades. There are >160 other positions that can be used to differentiate between Leptospira clades.