## 7 Conclusions  
The NTM background as environmental bacteria enables mycobacteria persistence in a gradient of ecological niches, from natural to anthropogenic environments, posing challenges to the identification and differentiation of these opportunistic pathogens when specifically involved in clinical settings and, consequently, to the adoption of the most effective control and treatment strategies. The identification and differentiation of NTM is a growing research field, with MALDI-Tof, FISH, flow cytometry, and WGS progressively assuming a steady relevance.  
The striking ecology of NTM results from the combination of several biological features, namely slow growth, a hydrophobic and lipid-rich impermeable cell wall, genetic variability associated with plasmid-mediated horizontal gene transfer and recombination, and transcriptional regulation signatures. Biofilm formation ability, symbiosis with protozoa, resistance to extreme pH stress, survival under anoxic or anaerobic conditions, and a remarkable metabolic activity of recalcitrant carbon compounds, also contribute to the adaptation and persistence of NTM in a huge variety of environments.  
NTM represent an increasing concern in the medical field, emerging as opportunistic pathogens, especially in immunocompromised and elderly individuals, with _M. ulcerans_, MAC, _M. kansasii_, _M. mulmoense_, _M. abscessus_, _M. chelonae_, _M. fortuitum_, _M. xenopi_, and _M. marinum_ representing the most clinically relevant species. This increased concern results not only by the increasing number of infections but also for the NTM natural and acquired capacity to survive antimycobacterial therapy. Moreover, NTM are also disease agents for domestic, livestock, and wildlife animals, raising apprehension related to their putative zoonotic potential. Alternative, non-conventional therapies have thus become hotspots of investigation.  
This review gathers up to date biological and ecological information of NTM, identifying knowledge marks but also research gaps that need to be addressed, stressing out that decision-makers need to recognize NTM as a public health concern that needs to be tackled in an increasingly susceptible elderly and immunocompromised population, as well as in low- or middle-income countries, where NTM infections are still highly misdiagnosed as tuberculosis. Antimicrobial resistance of NTM to several drugs limits therapeutic options and showcases the need for research investment in alternative approaches.  
Conceptualization, M.V.C.; investigation, A.C.P., B.R., and A.C.R.; funding acquisition, M.V.C.; writing--original draft preparation, A.C.P., B.R., and A.C.R.; writing--review and editing, M.V.C. All authors have read and agreed to the published version of the manuscript.  
This work was partially funded by Programa Operacional de Competitividade e Internacionalizacao (POCI) (FEDER component), Programa Operacional Regional de Lisboa, and Fundacao para a Ciencia e a Tecnologia (FCT), Portugal, in the scope of the project \"Colossus: Control Of tubercuLOSiS at the wildlife/livestock interface uSing innovative natUre-based Solutions\" (ref. POCI-01-0145- FEDER029783), project MyPATH (ref. PTDC/CVI7/117/794/2010) and strategic funding to cE3c and BioIS Research Units (UIDB/00329/2020 and UID/Multi/04046/2020). ACR and ACP were supported by FCT through doctoral grants (PD/BD/128031/2016 and SFRH/BD/136557/2018).  
The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.