Prospecting Environmental Mycobacteria: Combined Molecular Approaches Reveal Unprecedented Diversity

Abstract

BackgroundEnvironmental mycobacteria (EM) include species commonly found in various terrestrial and aquatic environments, encompassing animal and human pathogens in addition to saprophytes. Approximately 150 EM species can be separated into fast and slow growers based on sequence and copy number differences of their 16S rRNA genes. Cultivation methods are not appropriate for diversity studies; few studies have investigated EM diversity in soil despite their importance as potential reservoirs of pathogens and their hypothesized role in masking or blocking M. bovis BCG vaccine.MethodsWe report here the development, optimization and validation of molecular assays targeting the 16S rRNA gene to assess diversity and prevalence of fast and slow growing EM in representative soils from semi tropical and temperate areas. New primer sets were designed also to target uniquely slow growing mycobacteria and used with PCR-DGGE, tag-encoded Titanium amplicon pyrosequencing and quantitative PCR.ResultsPCR-DGGE and pyrosequencing provided a consensus of EM diversity; for example, a high abundance of pyrosequencing reads and DGGE bands corresponded to M. moriokaense, M. colombiense and M. riyadhense. As expected pyrosequencing provided more comprehensive information; additional prevalent species included M. chlorophenolicum, M. neglectum, M. gordonae, M. aemonae. Prevalence of the total Mycobacterium genus in the soil samples ranged from 2.3×107 to 2.7×108 gene targets g−1; slow growers prevalence from 2.9×105 to 1.2×107 cells g−1.ConclusionsThis combined molecular approach enabled an unprecedented qualitative and quantitative assessment of EM across soil samples. Good concordance was found between methods and the bioinformatics analysis was validated by random resampling. Sequences from most pathogenic groups associated with slow growth were identified in extenso in all soils tested with a specific assay, allowing to unmask them from the Mycobacterium whole genus, in which, as minority members, they would have remained undetected.

A global survey of mycobacterial diversity in soil

“…These results contrast to a comparable survey of mycobacteria in household plumbing that used the same hsp65 marker gene sequencing approach, where the majority of the mycobacterial lineages included described taxa (7). The high proportion of undescribed mycobacterial lineages we recovered from soil is slightly higher than that reported previously in soils (20) and lakes (12) via 16S rRNA gene analyses. Of the small subset of soil mycobacterial clades that included described isolates, we found lineages that included M. rutilum , and M. avium complex, taxa that have previously been identified from cultivation-dependent analyses of soil mycobacterial communities (26, 53).…”

“…As such, it is likely that many environmental mycobacteria have remained uncharacterized (7, 36). Second, although PCR-based methods can be used to more broadly survey soil mycobacterial diversity (20, 27, 37, 38), most of these cultivation-independent studies have relied on the PCR amplification and sequencing of regions of the 16S rRNA gene, which often does not provide sufficient resolution to differentiate between distinct species or strains within the Mycobacterium genus (28, 37, 38). Alternate genetic markers such as the heat shock protein gene (hsp65) have proven useful for resolving closely-related mycobacterial species, but to date these marker gene sequencing approaches have been used predominantly for clinical isolate identification (39–42).…”

The Goldilocks model of immune symbiosis with Mycobacteria and Candida colonizers

“…NTM include species that colonize human epithelia, as well as species that are ubiquitous in soil and aquatic environments 20,21 . NTM spp that colonize human epithelia are rarely pathogenic, include constituents of the healthy microbiota, and can be found along the urogenital tract ( M. smegmatis , M. lentiflavum ) 22–24 , gastrointestinal tract ( M. lentiflavum ) 24 , mouth or respiratory tract ( M. confluentis , M. brander i , M. bohemicum , M. interjectum , M. intermedium, M. conspicuum ) 25–31 , and skin ( M. smegmatis , M. bohemicum , M. intermedium ) 32–34 .…”

The Many Lives of Nontuberculous Mycobacteria

“…The NTM species that colonize human epithelia are largely nonpathogenic and can be found on skin (2)(3)(4) and along the genitourinary (5)(6)(7), gastrointestinal (7), and respiratory (8)(9)(10)(11)(12)(13)(14) tracts. The NTM species that are found in soil and aquatic environments include Mycobacterium vaccae, the Mycobacterium avium complex (MAC), and the Mycobacterium abscessus complex (MABSC) (15,16). Although most NTM are traditionally considered to be opportunistic pathogens, M. vaccae is unique in that it is also a transient human colonizer (17) and benefits the host in a manner that resembles ecological mutualism: M. vaccae inhibits pulmonary allergic inflammation in mice (18), as well as decreases anxiety in both mice (19) and humans (20), via an as-yet-undefined gut-brain-microbiota axis (21).…”