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.
Highlights
Environmental mycobacteria (EM), known as nontuberculous, opportunistic or atypical mycobacteria, belong to the genus Mycobacterium but are generally considered distinct from the M. tuberculosis complex, especially in terms of the impact on public health [1]
These studies aimed either at the whole genus or at slow growers, but through targeting the whole genus, they risk underrepresentation of the low prevalence slow growers which have a single rrn operon [31] copy. To address this issue we report here the development of a combined approach to determine the diversity of EM which includes slow growers and the M. tuberculosis and the M. avium complex too, using the 16S rRNA gene as target
A method was devised which exploited a particular signature in the Mycobacterium genus 16S rRNA gene, known as the long helix 18 [6], which is associated with pathogenicity and is harboured by the majority of slow growers known to date
Summary
Environmental mycobacteria (EM), known as nontuberculous, opportunistic or atypical mycobacteria, belong to the genus Mycobacterium but are generally considered distinct from the M. tuberculosis complex, especially in terms of the impact on public health [1]. Direct diversity analysis of EM in soil has focused on 16S rRNA genes using PCR-DGGE, clone libraries and T-RFLP [24,25,26,27,28,29,30] These studies aimed either at the whole genus or at slow growers, but through targeting the whole genus, they risk underrepresentation of the low prevalence slow growers which have a single rrn operon [31] copy. To address this issue we report here the development of a combined approach to determine the diversity of EM which includes slow growers and the M. tuberculosis and the M. avium complex too, using the 16S rRNA gene as target. 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
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
