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

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.

背景：现有依托多位点序列分型（multilocus sequence typing, MLST）网站的钩端螺旋体MLST分型方案仅适用于问号钩端螺旋体（L. interrogans）与柯氏钩端螺旋体（L. kirschneri）。本研究旨在拓展该方案的应用范围，使其可覆盖共计7种致病性钩端螺旋体物种。

方法与结果：本研究对现有MLST分型方案进行了优化，替换7个分型位点中的1个（将fadD替换为caiB），原因是原基因在部分致病性钩端螺旋体物种中未被检出。利用问号钩端螺旋体与柯氏钩端螺旋体的数据集对原始方案与优化后的方案进行比较，结果显示二者的分型分辨力无显著差异。我们采用优化后的方案对325株钩端螺旋体分离株进行了分型鉴定，涉及的物种包括：亚历山大钩端螺旋体（L. alexanderi，n=5）、博氏钩端螺旋体（L. borgpetersenii，n=34）、问号钩端螺旋体（n=222）、柯氏钩端螺旋体（n=29）、野尻钩端螺旋体（L. noguchii，n=9）、圣塔罗萨钩端螺旋体（L. santarosai，n=10）以及威利钩端螺旋体（L. weilii，n=16）。基于7个位点串联序列的系统发育分析表明，每个物种均对应独立的进化枝，且无菌株在物种水平上出现错分。我们对254株分离株完成了基因型与血清型的关联分析：在至少包含2株分离株的31个序列型（Sequence Type, ST）中，18个ST包含了隶属于2~3种不同血清型的分离株；反之，14种血清型分别包含2~10个不等的ST。本研究为钩端螺旋体属的全球系统地理学分布提供了新的观测结果，并证实了MLST在关联人类疾病与特定储存宿主方面的应用价值。

结论：本研究更新后的MLST分型方案依托升级后的MLST网站，可实现与钩端螺旋体病相关的7种主要致病性钩端螺旋体分离株的分型鉴定与物种归属判定。