A Global Survey of Mycobacterial Diversity in Soil

Abstract

Mycobacteria are a diverse bacterial group ubiquitous in many soil and aquatic environments. Members of this group have been associated with human and other animal diseases, including the nontuberculous mycobacteria (NTM), which are of growing relevance to public health worldwide. Although soils are often considered an important source of environmentally acquired NTM infections, the biodiversity and ecological preferences of soil mycobacteria remain largely unexplored across contrasting climates and ecosystem types. Using a culture-independent approach by combining 16S rRNA marker gene sequencing with mycobacterium-specific hsp65 gene sequencing, we analyzed the diversity, distributions, and environmental preferences of soil-dwelling mycobacteria in 143 soil samples collected from a broad range of ecosystem types. The surveyed soils harbored highly diverse mycobacterial communities that span the full extent of the known mycobacterial phylogeny, with most soil mycobacteria (97% of mycobacterial clades) belonging to previously undescribed lineages. While mycobacteria tended to have higher relative abundances in cool, wet, and acidic soil environments, several individual mycobacterial clades had contrasting environmental preferences. We identified the environmental preferences of many mycobacterial clades, including the clinically relevant Mycobacterium avium complex that was more commonly detected in wet and acidic soils. However, most of the soil mycobacteria detected were not closely related to known pathogens, calling into question previous assumptions about the general importance of soil as a source of NTM infections. Together, this work provides novel insights into the diversity, distributions, and ecological preferences of soil mycobacteria and lays the foundation for future efforts to link mycobacterial phenotypes to their distributions.IMPORTANCE Mycobacteria are common inhabitants of soil, and while most members of this bacterial group are innocuous, some mycobacteria can cause environmentally acquired infections of humans and other animals. Human infections from nontuberculous mycobacteria (NTM) are increasingly prevalent worldwide, and some areas appear to be "hotspots" for NTM disease. While exposure to soil is frequently implicated as an important mode of NTM transmission, the diversity, distributions, and ecological preferences of soil mycobacteria remain poorly understood. We analyzed 143 soils from a range of ecosystems and found that mycobacteria and lineages within the group often exhibited predictable preferences for specific environmental conditions. Soils harbor large amounts of previously undescribed mycobacterial diversity, and lineages that include known pathogens were rarely detected in soil. Together, these findings suggest that soil is an unlikely source of many mycobacterial infections. The biogeographical patterns we documented lend insight into the ecology of this important group of soil-dwelling bacteria.

Keywords: Mycobacterium; mycobacteria; soil microbiology.

FIG 1

Distribution of mycobacterial relative abundances and clade richness across samples. Bars indicate either the number of samples containing the specified relative abundance of mycobacteria (% of all bacterial or archaeal 16S rRNA reads) (A) or the number of samples containing the specified number of mycobacterial clades (via hsp65 reads), which varies from 0 to 18 clades per sample (B).

FIG 2

Relative abundances of mycobacteria across the 143 soils included in this study. Random forest analyses of 8 soil and site characteristics that described 25% of the variation in mycobacterial 16S rRNA gene relative abundances identified the most important environmental predictors as aridity index (A), soil pH (B), and minimum annual temperature (C). Boxplots show quartile ranges for each environmental category, with black dots indicating exact sample values. Significant differences between groups (determined using pairwise Wilcoxon signed-rank tests) are denoted by the asterisks with corresponding P values. Spearman correlations suggest that mycobacteria are more abundant in wetter, more acidic, and cooler soils. Aridity categories were based on site aridity index values: arid, 0 to 0.2; semiarid, 0.201 to 0.4; semihumid, 0.401 to 0.7; and Humid, 0.701 to 2.5. Categories for minimum annual temperature determined as cool, −35.0 to −9.0°C; temperate, −9.01 to 1.0°C; and warm, 1.0 to 22.0°C. (D) Differences in mycobacterial abundances across general ecosystem categories.

FIG 3

Phylogenetic tree representing described and previously undescribed mycobacterial hsp65 sequences. (A) A total of 472 exact sequence variants (ESVs) were identified in soils sampled here, a majority of which represent novel and undescribed taxa. These ESVs span the known phylogenetic diversity of the genus. Colors indicate reference mycobacterial strains (blue) from Dai et al. (55) and sequences recovered from soils in this study (orange). (B) Closely related ESVs were grouped into 159 clades based on patristic distance. The top 20 most ubiquitous clades are highlighted in color, with yellow colors indicating higher ubiquity (clades found in more soil samples). Four of the clades included described members, as indicated by red symbols, namely, clade 10 (M. stomatepiae, M. genavense, M. florentinum, M. lentiflavum, M. montefiorense, and M. triplex), clade 12 (M. novocastrense and M. rutilum), clade 31 (including M. intracellulare, M. avium subsp. paratuberculosis, M. avium subsp. silvaticum, M. colombiense, M. chimaera, and M. avium subsp. avium), and clade 44 (M. doricum and M. monacense). Small black triangles mark sequences from the reference database. Both trees are rooted with the hsp65 sequence from Nocardia farcinica (DSM43665).

FIG 4

Environmental preferences of selected mycobacterial clades. The environmental conditions associated with the presence of each clade shown here are significantly different from conditions in which the clade was absent, as determined by both Wilcoxon signed-rank tests and Spearman correlations (P < 0.05). Environmental variables that were consistently the most important for predicting clade presence in random forest models were aridity index (A), soil pH (B), and minimum annual temperature (C). Clades with notably strong environmental preferences are visualized in Fig. 1, and a comprehensive list of all significant relationships with supporting statistics is provided in Table S4 and S5. Three of these mycobacterial clades, namely, clade 10, clade 12, and clade 31, included described members (for a detailed list of named species, see Fig. 3 or Table S3).