## 6 Detection, Identification, and Differentiation Tools for Mycobacteria  
Environmental matrices are mostly studied in the context of NTM infection for which person-to-person transmission is very difficult to demonstrate. Both culture-dependent and independent approaches rely on the intrinsic characteristics of the type of matrices tested. The isolation of mycobacteria, namely the slow-growing, is very difficult from microbially rich environments, for which harsh decontamination is recommended [317]. In addition, the intrinsic characteristics of several species of _Mycobacterium_ require specific micronutrients (e.g., _M. haemophilum_), growth enhancers (e.g., _M. genavense_), optimal pH, temperature, and oxygen levels, which allied to a particular sensibility to decontamination (e.g., _M. ulcerans_) sets obstacles to bacteriological isolation [252; 318]. The composition of environmental samples is complex and influences the optimal application of the techniques. For water samples, both from natural water sources (e.g., surface waters or rivers) or anthropogenically-derived (e.g., drinking water systems or household water), most authors concentrate large volumes of water by filtration and/centrifugation [114; 319]. Membrane filtration is standardized. This membrane can be used for mycobacterial culture, however, the ratio of mycobacteria to other microorganisms in water samples is usually low, requiring decontamination for better performance, normally with cetylpyridinium chloride (CPC) [82]. CPC has been tested widely in different water samples and even in mycobacterial biofilms, being indicated as the most efficient decontamination for such samples. For soil samples, the biogeochemical composition of each type of soil (e.g., pH, ionic charge, presence of particulate matter) influences greatly the success of culturing mycobacteria and DNA extraction efficiency. Other intrinsic characteristics of mycobacteria, like the ability to adhere to soil particles further hamper isolation during bacteriological culture. Soil samples also present inhibitors (e.g., humic acids) that need to be eliminated to achieve mycobacteria DNA amplification from soil nucleic acid extracts. Several decontaminants have also been tested, but all of them seem to reduce the number of mycobacteria isolated [252]. Efforts have been made to improve the selective potential of media to isolate mycobacteria from soil samples, e.g., by adding 250 mg/L malachite green to 7H10 agar [320]. Additionally, cell detachment from soil particles can be accomplished using chemical and/or physical processes. Different classes of chemical reagents can be used for cell detachment, from non-ionic surfactants to salts and ionic dispersants [321]. These chemical reagents usually work by weakening intermolecular forces that attach the cell to the substrate [321]. Physical detaching can be more often accomplished by vortex, sonication, and ultrasounds [321]. These physical methods release bacteria that are entrapped in micropores or channels [321]. Standardized methods for isolation of mycobacteria from environmental matrices have thus been difficult to achieve. Phenotypic methods are still the most suited methods for the identification of mycobacteria in low-income countries, where often trained staff is scarce and/or uneven access to molecular biology techniques. In a review by [PERSON] and co-workers (2018), a set of 13 parallel phenotypic tests enables the identification of the most frequently isolated species of NTM, including niacin accumulation, arylsulfatase, nitrate reduction, thermostable, high catalase, low catalase, hydrolysis of Tween-80, citrate utilization, iron uptake, urea hydrolysis, growth in presence of 5% NaCl, growth in MacConkey agar without crystal violet, and tellurite reduction tests [322]. In a work by [PERSON] and coworkers (2017), a set of ten phenotypical assays enabled the identification of 89.7% of 98 clinical isolates, when compared to _rpoB_ sequence analysis [323].