## 3 Mycobacterium Ecology: The Underlying Resilience Biology
### Biofilm Formation and Quorum-Sensing  
NTM have the natural ability to form biofilm-like structures, mainly due to their high cell surface hydrophobicity [(51)], dependent upon quorum-sensing-like mechanisms [(79)]. NTM spread onto solid surfaces by a sliding mechanism [(80)]. Sliding motility is mainly dependent on the existence of glycopeptidolipids that play a central role in biofilm formation in _M. avium_, _M. abscessus_, and _M. snegmatis_[(81)]. Hydrophobic interactions between fatty acid tails of the glycopeptidolipids of NTM and the hydrophobic solid surface enable attachment to surfaces and biofilm formation [(80)]. NTM are thus often considered as the first colonizers of mixed biofilm communities, then followed by other environmental bacteria. The species _M. abscessus_, _M. aurum_, _M. avium_, _M. agordonae_, _M. haemophilum_, _M. intracellular_, _M. marinum_, _M. shinoidei_, _M. terrae_, and _M. ulcerans_ have been recovered from water biofilm samples [(82)]. In _M. fortuitum_ and _M. chelonae_, biofilm formation occurs under both high and low nutrient conditions [(71)]. In _M. abscessus_, deletion mutants of the _mmpL4b_ and _mab_3168c_ genes were shown to be impaired in biofilm formation and virulence [(83; 84)]. In this species, there are two morphologically distinct phenotypes controlled by the product of the _mab_3168c_ gene; when present, _M. abscessus_ shows a rough colony type, characterized by corded microcolonies with higher tendency to form biofilms, and exhibits higher virulence; when the _mab_3168c_ gene is absent, _M. abscessus_ bacteria show a smooth colony type, characterized by smooth, rounded, small colonies with lower tendency to form biofilms, and a diminished virulence phenotype [(84)]. In _M. avium_, the ability to produce biofilms is associated with increased virulence and colonization of the bronchial mucosa [(85)]. In this species, biofilm formation only occurs in the presence of divalent cations (e.g., Ca\({}^{2+}\), Mg\({}^{2+}\), and Zn\({}^{2+}\)), and is enhanced in copiotrophic (e.g., higher presence of glucose and peptone) or oligotrophic environments, while it is inhibited in the presence of high concentrations of humic acids (Figure 2B) [(86; 87)]. Additionally, exposure to oxidative stress leads to an increased release of autoinducer-2 that promotes biofilm formation, an adaptative strategy of mycobacteria to survive to extreme environments [(88)]. Another important component of biofilms is the presence of extracellular DNA that in _M. avium_ is quorum-sensing dependent and excreted by the _FtsK/SpoIIIE_ DNA transport system (Figure 2B) [(89)]. _M. avium_ biofilm formation is also dependent on the activities of enzymes of the tricarboxylic acid (TCA) cycle (e.g., 6-oxodehydrogenase [SucA]), enzymes of glycopeptidolipid synthesis, a protein synthetase (pstB), and Rv1565c (a hypothetical membrane protein) [(90)]. Moreover, the biofilm formation of _M. avium_ triggers TNF-\(\alpha\) release that leads to apoptotic cell death of the macrophages, improving immune system evasion [(91)]. In _M. marinum_, cording of mycobacterial cells is commonly found and associated with biofilm formation, with lipooligosaccharides being the propellant for cell motility and biofilm formation [(92)]. Phthiocerol dimyccoerosaes and phenolic glycolipids are also crucial for cell-surface properties but not for cell motility, influencing biofilm formation [(93)]. In _M. ulcerans_, the extracellular matrix is composed of mycobacterial cell aggregates in discrete clusters; vesicles containing mycolactone and its biosynthetic machinery and more than 80 proteins with distinct roles in stress response and respiratory and intermediary metabolism; lipids (e.g., phosphatidylinositolol mannosides, phospholipids, triacylglycerol, and phthiodiolone diphthioceranates); and carbohydrates (e.g., glucose) [94]. Mycolactone is a polyketide toxin that is considered a virulence factor responsible for dermonecrotic and immunosuppressive activities [94].