title: Prospecting environmental mycobacteria: combined molecular approaches reveal unprecedented diversity.
abstract: 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 2.3×10(7) to 2.7×10(8) gene targets g(-1); slow growers prevalence from 2.9×10(5) to 1.2×10(7) 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.
authors.affiliation: School of Life Sciences, University of Warwick, Coventry, United Kingdom. alessandra.pontiroli@gmail.com
authors.forename: Alessandra
authors.initials: A
authors.lastname: Pontiroli
authors.forename: Tanya T
authors.initials: TT
authors.lastname: Khera
authors.forename: Brian B
authors.initials: BB
authors.lastname: Oakley
authors.forename: Sam
authors.initials: S
authors.lastname: Mason
authors.forename: Scot E
authors.initials: SE
authors.lastname: Dowd
authors.forename: Emma R
authors.initials: ER
authors.lastname: Travis
authors.forename: Girum
authors.initials: G
authors.lastname: Erenso
authors.forename: Abraham
authors.initials: A
authors.lastname: Aseffa
authors.forename: Orin
authors.initials: O
authors.lastname: Courtenay
authors.forename: Elizabeth M H
authors.initials: EM
authors.lastname: Wellington
mesh_terms: D001483:Base Sequence; D044822:Biodiversity; D002980:Climate; D019295:Computational Biology; D017931:DNA Primers / Q000235:genetics; D058645:Denaturing Gradient Gel Electrophoresis; D005002:Ethiopia; D008969:Molecular Sequence Data; D009161:Mycobacterium / Q000235:genetics*; D010802:Phylogeny; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods; D017422:Sequence Analysis, DNA; D012988:Soil Microbiology; D013045:Species Specificity
chemical_list: D017931:DNA Primers; D012336:RNA, Ribosomal, 16S