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United States Department of Agriculture

Agricultural Research Service

Research Project: Pre-Harvest Interventions For Application During Poultry Production To Reduce Food-Borne Bacterial Pathogens

Location: Poultry Microbiological Safety Research

Title: Prospecting Environmental Mycobacteria: combined molecular approaches reveal unprecedented diversity

Authors
item Pontiroli, Alesandra -
item Khera, Tanya -
item Oakley, Brian
item Mason, Sam -
item Dowd, Scot -
item Erenso, Girum -
item Aseffa, Abraham -
item Courtenay, Orin -
item Wellington, Elizabeth -

Submitted to: PLoS One
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: May 31, 2013
Publication Date: N/A

Interpretive Summary: Many foodborne bacteria that cause illnesses in people (human pathogens) have environmental reservoirs, or natural sources from which they can infect people. Determining the numbers and types of pathogens in the environment is the first step in designing strategies to prevent their transmission from the environment to people. Mycobacterium is an important human and agricultural pathogens. Mycobacterium is usually acquired directly from the environment and can serve as a useful model to understand the importance of environmental reservoirs for other pathogens, including foodborne pathogens. Distinguishing between environmental sources and food sources of infection is an important task for food safety microbiology. For all pathogens, direct environmental surveys are the only accurate approach to properly measure diversity as only a small subset of pathogen types can be grown in the lab. In this study, the authors used state-of-the-art DNA sequencing methods to demonstrate a large diversity and high numbers of pathogens in environments in which human and agricultural uses are mixed, demonstrating that environmental reservoirs can be important sources of infection. This result has important implications for preventing human infection by foodborne pathogens, nearly all of which have large environmental reservoirs.

Technical Abstract: Environmental mycobacteria (EM) include species commonly found in a variety of terrestrial and aquatic environments and encompass 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. Few studies have investigated EM diversity in soil despite their importance as a potential reservoir of pathogens and their hypothesized role in masking or blocking vaccine, which is made from the M. bovis BCG strain. Cultivation methods are not appropriate for diversity studies; hence 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 quantative PCR (qPCR). 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 and M. aemonae. Prevalence of the total Mycobacterium genus in the soil samples ranged from 2.3 x 107 to 2.7 x 108 gene targets g-1 and for the slow growers from 2.9 x 105 to 1.2 x 107 gene targets g-1. This combined molecular approach enabled qualitative and quantitative assessment of EM across samples.

Last Modified: 9/22/2014
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