A single multilocus sequence typing (MLST) scheme for seven pathogenic Leptospira species

Background The available Leptospira multilocus sequence typing (MLST) scheme supported by a MLST website is limited to L. interrogans and L. kirschneri. Our aim was to broaden the utility of this scheme to incorporate a total of seven pathogenic species. Methodology and Findings We modified the existing scheme by replacing one of the seven MLST loci (fadD was changed to caiB), as the former gene did not appear to be present in some pathogenic species. Comparison of the original and modified schemes using data for L. interrogans and L. kirschneri demonstrated that the discriminatory power of the two schemes was not significantly different. The modified scheme was used to further characterize 325 isolates (L. alexanderi [n = 5], L. borgpetersenii [n = 34], L. interrogans [n = 222], L. kirschneri [n = 29], L. noguchii [n = 9], L. santarosai [n = 10], and L. weilii [n = 16]). Phylogenetic analysis using concatenated sequences of the 7 loci demonstrated that each species corresponded to a discrete clade, and that no strains were misclassified at the species level. Comparison between genotype and serovar was possible for 254 isolates. Of the 31 sequence types (STs) represented by at least two isolates, 18 STs included isolates assigned to two or three different serovars. Conversely, 14 serovars were identified that contained between 2 to 10 different STs. New observations were made on the global phylogeography of Leptospira spp., and the utility of MLST in making associations between human disease and specific maintenance hosts was demonstrated. Conclusion The new MLST scheme, supported by an updated MLST website, allows the characterization and species assignment of isolates of the seven major pathogenic species associated with leptospirosis.
Author Summary Leptospirosis is a common zoonotic disease worldwide. Genotyping of the causative organisms provides important insights into disease transmission and informs preventive strategies and vaccine development. Multilocus sequence typing (MLST) is the most widespread genotyping methodology for bacterial pathogens, but the Leptospira scheme supported by a public MLST database is currently only applicable to L. interrogans and L. kirschneri. The purpose of this study was to extend the scheme to a total of seven pathogenic Leptospira species. This was achieved through the development of a modified scheme in which one of the seven MLST loci was replaced, together with newly designed primers for the remaining 6 loci. Comparison of the original and modified scheme demonstrated that they were very similar, hence sequence type (ST) assignments were largely carried over to the modified scheme. Phylogenetic trees reconstructed from concatenated sequences of the seven loci of the modified scheme demonstrated perfect classification of isolates into seven pathogenic species, which resided in clearly distinct phylogenetic clusters. Congruence was low between STs and serovars. The MLST scheme was used to gain new insights into the population genetic structure of Leptospira species associated with clinical disease and maintenance hosts in Asia.