A Single Multilocus Sequence Typing (MLST) Scheme for Seven Pathogenic Leptospira Species

Abstract

BackgroundThe 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 FindingsWe 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.ConclusionThe 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.

Characterization of Leptospira interrogans serovar Pomona isolated from swine in Brazil

“…This is also observed with the new MLST protocol [25] in which serovar Pomona can be assigned to STs 38 and 140 together with serovars Gem and Guaratuba, respectively. Although MLST techniques enable Leptospira species differentiation [9,25], serovar assignment still needs improvement. Only when molecular methods ensure species and serovar identification, will they completely replace serological methods.…”

“…As MLST does not always specify Leptospira serovars [9], the same ST 37 also encompasses serovar Canicola when using the scheme previously proposed [19,9,11]. This is also observed with the new MLST protocol [25] in which serovar Pomona can be assigned to STs 38 and 140 together with serovars Gem and Guaratuba, respectively. Although MLST techniques enable Leptospira species differentiation [9,25], serovar assignment still needs improvement.…”

Multiple-locus variable-number tandem repeat analysis and clinical characterization of Leptospira interrogans canine isolates

“…PFGE is useful for identifying leptospiral serovars (Herrmann et al, 1992), but the resulting complex patterns may be challenging for interlaboratory pattern comparisons. MLST has also been used for Leptospira species genotyping (Boonsilp et al, 2013). However, our previous study demonstrated that MLST was unsuitable for serovar (or serogroup) identification and had less discriminatory power than PFGE .…”

Genetic stability of vaccine strains by multilocus sequence typing and pulsed-field gel electrophoresis analysis: Implications for quality control of the leptospiral vaccine

“…[17][18][19] In addition, multilocus sequence typing (MLST), the most widespread genotyping methodology for bacterial pathogens, is used to describe the molecular epidemiology of leptospirosis in some countries or regions. 20,21 These approaches have potential to confirm the identity and genetic stability of bacterial strains over multiple vaccine seed passages. Therefore, the aims of the present study were to assess the genetic stability of leptospiral vaccine strains by using PFGE and MLST.…”

“…MLST was performed on 7 housekeeping genes (including glmU, pntA, sucA, tpiA, pfkB, mreA, and caiB) as previously described. 20 PCR amplification of MLST target genes was performed in a volume of 50 ml and contained a final concentration of 1.5-3.5 mM MgCl 2 , 0.2 mM dNTP, 1 unit Taq DNA polymerase (Takara, Dalian, China), 0.25 mM of each primer, and 20-50 ng of Leptospira DNA. Amplification was performed under the following thermocycler conditions: one cycle of 95 C for 2 min, 30 cycles each of 95 C for 10 s; 46 C for 15 s; and 72 C for 30 s, followed by a final period of 72 C for 7 min.…”