Diagnostic Sequences That Distinguish M. avium Subspecies Strains

Abstract

Over a decade ago Mycobacterium avium subspecies paratuberculosis (Map) specific genes were initially identified in a whole genome context by comparing draft genome sequences of Map strain K-10 with Mycobacterium avium subspecies hominissuis (Mah) strain 104. This resulted in identification of 32 Map specific genes, not including repetitive elements, based on the two-genome comparison. The goal of this study was to define a more complete catalog of M. avium subspecies-specific genes. This is important for obtaining additional diagnostic targets for Johne's disease detection and for understanding the unique biology, evolution and niche adaptation of these organisms. There are now over 28 complete genome sequences representing three M. avium subspecies, including avium (Maa), Mah, and Map. We have conducted a comprehensive comparison of these genomes using two independent pan genomic comparison tools, PanOCT and Roary. This has led to the identification of more than 250 subspecies defining genes common to both analyses. The majority of these genes are arranged in clusters called genomic islands. We further reduced the number of diagnostic targets by excluding sequences having high BLAST similarity to other mycobacterial species recently added to the National Center for Biotechnology Information database. Genes identified as diagnostic following these bioinformatic approaches were further tested by DNA amplification PCR on an additional 20 M. avium subspecies strains. This combined approach confirmed 86 genes as Map-specific, seven as Maa-specific and three as Mah-specific. A single-tube PCR reaction was conducted as a proof of concept method to quickly distinguish M. avium subspecies strains. With these novel data, researchers can classify isolates in their freezers, quickly characterize clinical samples, and functionally analyze these unique genes.

Global Phylogeny of Mycobacterium avium and Identification of Mutation Hotspots During Niche Adaptation

“…The largest subcategory of MAP-specific genes in the present study was associated with secondary metabolites biosynthesis, transport, and catabolism. A similar finding was also reported by another recent investigation (Bannantine et al, 2020b). Several genes were also annotated as mammalian cell entry (Mce) genes.…”

“…Use of whole genes for diagnostic tests may be more accurate than using insertion sequences or SNPs in particular genes. The recent publication by Bannantine and colleagues (Bannantine et al, 2020b) and the complementary results from the present investigation demonstrate there are a variety of subspecies-specific genes that could be used diagnostic targets. Further work would be required to determine the suitability of these marker genes in a larger dataset as candidates for diagnostic targets.…”

“…More recently, Bannantine and colleagues used a pan-genome approach on 29 closed M. avium genomes. They reported 86 genes specific to MAP, seven specific to MAA, and three that were specific to MAH (Bannantine et al, 2020b). Subspecies-specific loci and genes are likely to be associated with adaptation to human and porcine hosts by MAH or ruminant hosts by MAP.…”

Identification of Leuconostoc species based on novel marker genes identified using real-time PCR via computational pangenome analysis

“…Thus, molecular assays targeting genetic markers achieve higher resolution within closely related species. The previous researchers have successfully identified for species, subspecies, or serovar-specific marker genes using the presence/absence matrix with a script built into Roary (Bannantine et al, 2021;Shang et al, 2021;Kim et al, 2022c).…”