Prospecting environmental mycobacteria: combined molecular approaches reveal unprecedented diversity

Background: Environmental 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. Methods: We 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. Results: PCR-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. Conclusions: This 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

OTU based diversity analysis.

<p>Rarefaction curves for EM (A) and slow growing EM (B) data sets.</p

Primers used to target mycobacteria for diversity analysis.

*<p>to these primers a GC clamp was added for DGGE; ¹ those that possess the long helix 18 (Leclerc et al., 2003).</p

Characteristics of the pyrosequenced libraries including number of reads pre and post quality control, OTU, EM counts as determined by qPCR, richness diversity indices and non parametric diversity estimators.

*<p>unique OTUs; NaN: not available; _R: random resampling; SD: standard deviation of the mean triplicate experiments.</p

Strains used in this study.

*<p>Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH Mascheroder Weg 1b, 38124 Braunschweig, Germany *** American Type Culture Collection (ATCC), P.O. Box 1549, Manassas, VA 20108, USA****Supplied by John Magee, Regional Centre for Mycobacteriology, Newcastle, UK.¹ Growth fashion: F =  fast; S =  slow.</p

DGGE EM profiling using 16S rRNA gene PCR amplicons.

<p>(A) Profiles of individual EM type strains obtained using the <i>Mycobacterium</i> genus specific primer set JSY16S. L is the reference ladder (TrackIt™ 50 bp DNA Ladder, Invitrogen, Ltd., Paisley, UK), lanes 1–12 are, respectively: <i>M. smegmatis</i>, <i>M. aichiense</i>, <i>M. aurum</i>, <i>M. gilvum</i>, <i>M. phlei</i>, <i>M. agri</i>, <i>M. peregrinum</i>, <i>M. duvalii, M. abscessus</i>, <i>M. fortuitum, M. vaccae</i>and a mixture of equimolar quantities of the above listed EM species. (B) Profiles obtained using the slow mycobacteria specific primer set APTK16S. L is a reference ladder (TrackIt™ 50 bp DNA Ladder, Invitrogen, Ltd., Paisley, UK), lanes 1–7 are, respectively: <i>M. intracellulare, M. marinum</i>, <i>M. kansasii</i>, <i>M. xenopi</i>, <i>M. aviumparatuberculosis</i>, <i>M. bovis BCG</i> and a mixture of equimolar quantities of the above listed EM species. (C) EM soil community profiling using the <i>Mycobacterium</i> genus specific primers (JSY16S). L is a reference sizing ladder (TrackIt™ 50 bp DNA Ladder, Invitrogen, Ltd., Paisley, UK), C is the negative PCR control, samples 1 – 4 are the four Ethiopian soils (1108, 1109,1110, 1111) and 5 is Cryfield. The arrows (1A–1I) indicate the bands that were excised and sequenced (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068648#pone-0068648-t004" target="_blank">Table 4</a>). (D) EM soil community profiling using the slow grower mycobacteria specific 16S rRNA gene specific primers (APTK16S). L is a reference sizing ladder (TrackIt™ 50 bp DNA Ladder, Invitrogen, Ltd., Paisley, UK), C is the negative PCR control, samples 1–4 are the four Ethiopian soils (1108, 1109, 1110, 1111) and 5 is Cryfield. The arrows (2A–2I) represent the bands that were excised and sequenced (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068648#pone-0068648-t004" target="_blank">Table 4</a>).</p

Visual representation of the beta diversity across sites.

<p>Proportion (%) of species in each sample after blasting the whole data sets containing the amplicons obtained with genus (A) and slow grower (B) specific primer sets.</p

Sequence comparisons of bands excised from DGGE gels (Fig. 1 C and D) and blasted against the <i>16S rRNA</i> gene database retrieved from SILVA.

<p>Sequence comparisons of bands excised from DGGE gels (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068648#pone-0068648-g001" target="_blank">Fig. 1</a> C and D) and blasted against the <i>16S rRNA</i> gene database retrieved from SILVA.</p