Phylogenomic exploration of the relationships between strains of Mycobacterium avium subspecies paratuberculosis

Abstract

BackgroundMycobacterium avium subspecies paratuberculosis (Map) is an infectious enteric pathogen that causes Johne’s disease in livestock. Determining genetic diversity is prerequisite to understanding the epidemiology and biology of Map. We performed the first whole genome sequencing (WGS) of 141 global Map isolates that encompass the main molecular strain types currently reported. We investigated the phylogeny of the Map strains, the diversity of the genome and the limitations of commonly used genotyping methods.ResultsSingle nucleotide polymorphism (SNP) and phylogenetic analyses confirmed two major lineages concordant with the former Type S and Type C designations. The Type I and Type III strain groups are subtypes of Type S, and Type B strains are a subtype of Type C and not restricted to Bison species.We found that the genome-wide SNPs detected provided greater resolution between isolates than currently employed genotyping methods. Furthermore, the SNP used for IS1311 typing is not informative, as it is likely to have occurred after Type S and C strains diverged and does not assign all strains to the correct lineage. Mycobacterial Interspersed Repetitive Unit-Variable Number Tandem Repeat (MIRU-VNTR) differentiates Type S from Type C but provides limited resolution between isolates within these lineages and the polymorphisms detected do not necessarily accurately reflect the phylogenetic relationships between strains.WGS of passaged strains and coalescent analysis of the collection revealed a very high level of genetic stability, with the substitution rate estimated to be less than 0.5 SNPs per genome per year.ConclusionsThis study clarifies the phylogenetic relationships between the previously described Map strain groups, and highlights the limitations of current genotyping techniques. Map isolates exhibit restricted genetic diversity and a substitution rate consistent with a monomorphic pathogen. WGS provides the ultimate level of resolution for differentiation between strains. However, WGS alone will not be sufficient for tracing and tracking Map infections, yet importantly it can provide a phylogenetic context for affirming epidemiological connections.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-2234-5) contains supplementary material, which is available to authorized users.

Novel Single Nucleotide Polymorphism-Based Assay for Genotyping Mycobacterium avium subsp. paratuberculosis

“…A phylogenetic tree was generated based on the SNP analysis, and distinct phylogenetic groups were identified ( Fig. 1 and 2), which conformed to the broadly recognized phylogenetic structure of M. avium subspecies paratuberculosis (10). By using an adaptation of the set cover problem, the minimum number of SNPs required to discriminate between all the isolates was calculated to be 93.…”

“…Genome sequences from 133 M. avium subspecies paratuberculosis isolates generated in a previous study (10) were selected for SNP and phylogenetic analyses. This panel was chosen to maximize genetic diversity and reduce phylogenetic discovery bias (2), since it comprised isolates with different genotypes (determined by multiplex PFGE and MIRU-VNTR), which were selected from 17 different countries and isolated from 8 different host species.…”

“…Genetic diversity among M. avium subspecies paratuberculosis strains has been investigated using molecular techniques, such as restriction fragment length polymorphism (RFLP) and IS900 analysis (IS900 RFLP) (3), pulsed-field gel electrophoresis (PFGE) (4), amplified fragment length polymorphism (AFLP) analysis (5), random amplified polymorphic DNA (RAPD) analysis (6), mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) analysis (7), and short-sequence repeat (SSR) analysis (8). However, these techniques have limited discriminatory power when applied to M. avium subspecies paratuberculosis; although this power can be increased by combining complementary genotyping techniques, it is often insufficient for accurately determining relationships among isolates or global epidemiological studies (9, 10).Whole-genome sequencing (WGS) provides the ultimate resolution of isolates, and, unlike the techniques listed above, it can provide a phylogenetic context to facilitate global epidemiology studies and affirm epidemiological connections (10,11,12). Although WGS is becoming cheaper, it is still too expensive to be used for routine genotyping of M. avium subspecies paratuberculosis isolates and requires robust data handling and analysis processes.…”

“…Whole-genome sequencing (WGS) provides the ultimate resolution of isolates, and, unlike the techniques listed above, it can provide a phylogenetic context to facilitate global epidemiology studies and affirm epidemiological connections (10,11,12). Although WGS is becoming cheaper, it is still too expensive to be used for routine genotyping of M. avium subspecies paratuberculosis isolates and requires robust data handling and analysis processes.…”

“…Genetic diversity among M. avium subspecies paratuberculosis strains has been investigated using molecular techniques, such as restriction fragment length polymorphism (RFLP) and IS900 analysis (IS900 RFLP) (3), pulsed-field gel electrophoresis (PFGE) (4), amplified fragment length polymorphism (AFLP) analysis (5), random amplified polymorphic DNA (RAPD) analysis (6), mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) analysis (7), and short-sequence repeat (SSR) analysis (8). However, these techniques have limited discriminatory power when applied to M. avium subspecies paratuberculosis; although this power can be increased by combining complementary genotyping techniques, it is often insufficient for accurately determining relationships among isolates or global epidemiological studies (9,10).…”