## 6 Detection, Identification, and Differentiation Tools for Mycobacteria
Furthermore, cells can be sorted after flow cytometry analysis with several possible downstream applications, ranging from omics (e.g., genomics), cultivation, antimicrobial sensitivity testing, among other processes, enabling a better understanding of the NTM role in the environmental community, the viability analysis of NTM community, etc. [PERSON] and coworkers (2018) reported that _M. smegmatis_ with 4-N,N-dimethylamino-1,8-nophthalimide-conjugated trehalose (DMN-Tre) labeling exhibits increased fluorescence over the background due to metabolic conversion within the mycomembrane, in sputum samples [336]. However, particularly in soil samples, methods like FISH and flow cytometry that depend on the hybridization of probes and fluorescence detection face additional challenges. Many times, particulate matter follows the processing of samples along with bacteria and reaches the endpoint. Those particles influence the optical acquisition and may present background fluorescence introducing confounding effects, as well as, diminishing the hybridization efficiency of the probe. In addition, the downsides of such techniques is their cost and the need of technical expertise. In addition to the culture-dependent approach, which requires the long-lasting process of isolation of mycobacteria, there is a wide variety of molecular techniques available for the detection and identification of NTM species. One of the most commonly applied techniques for bacterial identification is 16S rRNA gene sequence analysis. Conserved or variable regions of the 16S rDNA amplicon are amplified depending on the goal of detection or identification. Still, due to the high similarity found across mycobacterial sequences, this analysis is frequently coupled with _rpoB_, associated with rifampicin resistance, and also by analysis of _hsp65_ that together enhance discriminatory power [138; 337]. The single-use of the 16S rRNA gene may lead to misidentification of species, contrary to _rpoB_ sequencing which seems to achieve better resolution at the species level [338]. The use of _rpoB_ sequencing for identification is wider in NTM, including MAC and RGM [338], and enables identification of approximately 80% of isolates from these species to species level [338]. The attempt to establish a reliable identification database has been the target of many efforts over the last years to fulfill one of the major gaps in the application of this multi-locus approach [337]. In the context of drinking waters, the _rpoB_ sequencing was capable of successfully identifying approximately three thousand sequences per water sample, allowing the comparison of water with different ages [339]. Insertion sequences (IS) have been widely used for the identification of mycobacteria. Several methods of both conventional and real-time PCR have been developed based on those sequences. Within the MAC, several subspecies can be identified by the presence of IS, namely, _IS901_ in MAA [340] and _IS902_ in _M. avium_ subsp. _Silvaticum_[341], amongst other ISs that enable differentiation besides identification. In the case of _M. ulcerans_, identification is possible through analysis of _IS2404_ and _IS2606_[342]. Some other species have been described to have IS useful for identification purposes, like _IS1407_ in _M. chelatum_[343], _IS1395_ in _M. xenopi_[344], _IS1511/1512_ in _M. gordone_[345], _IS1652_ in _M. kansasii_[346], and _IS6120_ in _M. smegmatis_[347]. More recently, [PERSON] et al. (2015) proposed a novel tetraplex real-time PCR that permits identification of _Mycobacterium_ genus by ITS amplification, as well as identification as _M. avium_ by _IS1311_ amplification and/or as MTC by _devR_ amplification, coupled with an internal amplification control [315]. This protocol was initially tested for animal samples and milk but has great potential for the identification of mycobacteria in environmental samples [315]. DNA probe commercial tests are also widely used for the identification of NTM.