Genetic affinities within a large global collection of pathogenic <i>Leptospira</i>: implications for strain identification and molecular epidemiology

Leptospirosis is an important zoonosis with widespread human health implications. The non-availability of accurate identification methods for the individualization of different Leptospira for outbreak investigations poses bountiful problems in the disease control arena. We harnessed fluorescent amplified fragment length polymorphism analysis (FAFLP) for Leptospira and investigated its utility in establishing genetic relationships among 271 isolates in the context of species level assignments of our global collection of isolates and strains obtained from a diverse array of hosts. In addition, this method was compared to an in-house multilocus sequence typing (MLST) method based on polymorphisms in three housekeeping genes, the rrs locus and two envelope proteins. Phylogenetic relationships were deduced based on bifurcating Neighbor-joining trees as well as median joining network analyses integrating both the FAFLP data and MLST based haplotypes. The phylogenetic relationships were also reproduced through Bayesian analysis of the multilocus sequence polymorphisms. We found FAFLP to be an important method for outbreak investigation and for clustering of isolates based on their geographical descent rather than by genome species types. The FAFLP method was, however, not able to convey much taxonomical utility sufficient to replace the highly tedious serotyping procedures in vogue. MLST, on the other hand, was found to be highly robust and efficient in identifying ancestral relationships and segregating the outbreak associated strains or otherwise according to their genome species status and, therefore, could unambiguously be applied for investigating phylogenetics of Leptospira in the context of taxonomy as well as gene flow. For instance, MLST was more efficient, as compared to FAFLP method, in clustering strains from the Andaman island of India, with their counterparts from mainland India and Sri Lanka, implying that such strains share genetic relationships and that leptospiral strains might be frequently circulating between the islands and the mainland

Comparison of Two Multilocus Sequence Based Genotyping Schemes for Leptospira Species

Two independent multilocus sequence based genotyping schemes (denoted here as 7L and 6L for schemes with 7 and 6 loci, respectively) are in use for Leptospira spp., which has led to uncertainty as to which should be adopted by the scientific community. The purpose of this study was to apply the two schemes to a single collection of pathogenic Leptospira, evaluate their performance, and describe the practical advantages and disadvantages of each scheme. We used a variety of phylogenetic approaches to compare the output data and found that the two schemes gave very similar results. 7L has the advantage that it is a conventional multi-locus sequencing typing (MLST) scheme based on housekeeping genes and is supported by a publically accessible database by which genotypes can be readily assigned as known or new sequence types by any investigator, but is currently only applicable to L. interrogans and L. kirschneri. Conversely, 6L can be applied to all pathogenic Leptospira spp., but is not a conventional MLST scheme by design and is not available online. 6L sequences from 271 strains have been released into the public domain, and phylogenetic analysis of new sequences using this scheme requires their download and offline analysis

Genetic Affinities within a Large Global Collection of Pathogenic Leptospira: Implications for Strain Identification and Molecular Epidemiology

Leptospirosis is an important zoonosis with widespread human health implications. The non-availability of accurate identification methods for the individualization of different Leptospira for outbreak investigations poses bountiful problems in the disease control arena. We harnessed fluorescent amplified fragment length polymorphism analysis (FAFLP) for Leptospira and investigated its utility in establishing genetic relationships among 271 isolates in the context of species level assignments of our global collection of isolates and strains obtained from a diverse array of hosts. In addition, this method was compared to an in-house multilocus sequence typing (MLST) method based on polymorphisms in three housekeeping genes, the rrs locus and two envelope proteins. Phylogenetic relationships were deduced based on bifurcating Neighbor-joining trees as well as median joining network analyses integrating both the FAFLP data and MLST based haplotypes. The phylogenetic relationships were also reproduced through Bayesian analysis of the multilocus sequence polymorphisms. We found FAFLP to be an important method for outbreak investigation and for clustering of isolates based on their geographical descent rather than by genome species types. The FAFLP method was, however, not able to convey much taxonomical utility sufficient to replace the highly tedious serotyping procedures in vogue. MLST, on the other hand, was found to be highly robust and efficient in identifying ancestral relationships and segregating the outbreak associated strains or otherwise according to their genome species status and, therefore, could unambiguously be applied for investigating phylogenetics of Leptospira in the context of taxonomy as well as gene flow. For instance, MLST was more efficient, as compared to FAFLP method, in clustering strains from the Andaman island of India, with their counterparts from mainland India and Sri Lanka, implying that such strains share genetic relationships and that leptospiral strains might be frequently circulating between the islands and the mainland

Discriminatory ability of two genotyping schemes and their respective loci.

#<p>p distances were estimated based on the Kimura Two Parameter nucleotide substitution model.</p><p>*dN/dS were estimated based on the Modified Nei-Gojobori Method with Jukes Cantor correction using MEGA 4. The values shown represent a combined value for <i>L. interrogans</i> and <i>L. kirschneri</i>. dN/dS was not estimated for <i>rrs2</i> as this does not encode a protein.</p

Sliding window analysis of concatenated sequence of all 13 loci.

<p>Sliding window analysis of concatenated sequence of all 13 loci, carried out using DNAsp v 5 using a window size of 400-bp, a step size of 50-bp, and points based on the mid-point of each window (i.e. the first point is at position 200). The names of the individual loci are shown. Three plots are given to represent the level of polymorphism within each of the two species, and the level of diversity between them. In terms of the within species variation, there is little difference between the two schemes and both point to generally higher levels of variation within <i>L. kirschneri</i> than <i>L. interrogans</i>. However, there are two loci used in the 6L scheme that are highly conserved between species (<i>lipL32</i> and <i>rrs2</i>), which means that in general the 7L scheme provides better between-species resolution.</p

Primers for 6 locus genotyping scheme used during this study [39].

<p>Primers for 6 locus genotyping scheme used during this study <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001374#pntd.0001374-Thaipadungpanit1" target="_blank">[39]</a>.</p

Neighbor joining trees of the 7L scheme and the 6 loci scheme.

<p>Neighbor joining trees reconstructed based on concatenated sequences of the 7L scheme (3,165 bp) (A), and the 6 loci scheme (2,844 bp) (B). Each bacterial strain is labeled by the following string: abbreviation of species name (Lint- <i>L. interrogans</i>, Lkir- <i>L. kirschneri</i>), strain name, and (for the 7L scheme only) sequence type (ST).</p