Prospecting environmental mycobacteria: combined molecular approaches reveal unprecedented diversity.
<h4>Background</h4>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 sequenc...
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Public Library of Science (PLoS)
2013-01-01
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| Series: | PLoS ONE |
| Online Access: | https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0068648&type=printable |
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| author | Alessandra Pontiroli Tanya T Khera Brian B Oakley Sam Mason Scot E Dowd Emma R Travis Girum Erenso Abraham Aseffa Orin Courtenay Elizabeth M H Wellington |
| author_facet | Alessandra Pontiroli Tanya T Khera Brian B Oakley Sam Mason Scot E Dowd Emma R Travis Girum Erenso Abraham Aseffa Orin Courtenay Elizabeth M H Wellington |
| author_sort | Alessandra Pontiroli |
| collection | DOAJ |
| description | <h4>Background</h4>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.<h4>Methods</h4>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.<h4>Results</h4>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).<h4>Conclusions</h4>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. |
| format | Article |
| id | doaj-art-c151d4d3037d450faf4909d2fdc8efd7 |
| institution | OA Journals |
| issn | 1932-6203 |
| language | English |
| publishDate | 2013-01-01 |
| publisher | Public Library of Science (PLoS) |
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| series | PLoS ONE |
| spelling | doaj-art-c151d4d3037d450faf4909d2fdc8efd72025-08-20T02:05:31ZengPublic Library of Science (PLoS)PLoS ONE1932-62032013-01-0187e6864810.1371/journal.pone.0068648Prospecting environmental mycobacteria: combined molecular approaches reveal unprecedented diversity.Alessandra PontiroliTanya T KheraBrian B OakleySam MasonScot E DowdEmma R TravisGirum ErensoAbraham AseffaOrin CourtenayElizabeth M H Wellington<h4>Background</h4>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.<h4>Methods</h4>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.<h4>Results</h4>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).<h4>Conclusions</h4>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.https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0068648&type=printable |
| spellingShingle | Alessandra Pontiroli Tanya T Khera Brian B Oakley Sam Mason Scot E Dowd Emma R Travis Girum Erenso Abraham Aseffa Orin Courtenay Elizabeth M H Wellington Prospecting environmental mycobacteria: combined molecular approaches reveal unprecedented diversity. PLoS ONE |
| title | Prospecting environmental mycobacteria: combined molecular approaches reveal unprecedented diversity. |
| title_full | Prospecting environmental mycobacteria: combined molecular approaches reveal unprecedented diversity. |
| title_fullStr | Prospecting environmental mycobacteria: combined molecular approaches reveal unprecedented diversity. |
| title_full_unstemmed | Prospecting environmental mycobacteria: combined molecular approaches reveal unprecedented diversity. |
| title_short | Prospecting environmental mycobacteria: combined molecular approaches reveal unprecedented diversity. |
| title_sort | prospecting environmental mycobacteria combined molecular approaches reveal unprecedented diversity |
| url | https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0068648&type=printable |
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