Recovery of Mycobacteria from Heavily Contaminated Environmental Matrices.

Vit Ulmann,Ross Tim Weston,Ivo Pavlik,Vladimir Babak,Helena Modrá

doi:10.3390/microorganisms9102178

Vit Ulmann, Ross Tim Weston + Show 3 more

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https://doi.org/10.3390/microorganisms9102178

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Journal: Microorganisms	Publication Date: Oct 19, 2021
Citations: 15	License type: CC BY 4.0

Affiliation: La Trobe University, Mendel University in Brno

Abstract

For epidemiology studies, a decontamination method using a solution containing 4.0% NaOH and 0.5% tetradecyltrimethylammonium bromide (TDAB) represents a relatively simple and universal procedure for processing heavily microbially contaminated matrices together with increase of mycobacteria yield and elimination of gross contamination. A contamination rate only averaging 7.3% (2.4% in Cluster S; 6.9% in Cluster R and 12.6% in Cluster E) was found in 787 examined environmental samples. Mycobacteria were cultured from 28.5% of 274 soil and water sediments samples (Cluster S), 60.2% of 251 samples of raw and processed peat and other horticultural substrates (Cluster R), and 29.4% of 262 faecal samples along with other samples of animal origin (Cluster E). A total of 38 species of slow and rapidly growing mycobacteria were isolated. M. avium ssp. hominissuis, M. fortuitum and M. malmoense were the species most often isolated. The parameters for the quantitative detection of mycobacteria by PCR can be significantly refined by treating the sample suspension before DNA isolation with PMA (propidium monoazide) solution. This effectively eliminates DNA residue from both dead mycobacterial cells and potentially interfering DNA segments present from other microbial flora. In terms of human exposure risk assessment, the potential exposure to live non-tuberculous mycobacteria can be more accurately determined.

Highlights

We propose an improved method involving a modified decontamination process and a novel quantitative polymerase chain reaction (qPCR) method incorporating a pre-treatment with propidium monoazide (PMA) to accurately detect the presence of viable mycobacteria in environmental samples
This method allows for the quantification of the level of mycobacteria present and subsequent identification of individual mycobacterial species
The addition of tetradecyltrimethylammonium bromide (TDAB) proved to be effective in increasing mycobacteria yield and eliminating gross contamination to 2–13% in three different kinds of heavily contaminated environmental matrices

Summary

Introduction

Mycobacteria are acid-fast, Gram-positive bacilli characterised as intracellular parasites. 195 mycobacterial species and subspecies have been identified (List of Prokaryotic Names with Standing in Nomenclature, LPSN) [1]. Similar to other groups of bacteria, the number of described mycobacterial species has undergone an expansion due to the high discriminatory nature of whole genome sequencing [2]. The group includes obligate pathogenic mycobacteria causing tuberculosis (TB; members of the Mycobacterium tuberculosis complex, MTC) and leprosy (M. leprae), as well as non-tuberculous mycobacteria (NTM) occurring widely in aquatic and terrestrial environments [3,4].

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