## 3 Mycobacterium Ecology: The Underlying Resilience Biology
### Biofilm formation and quorum sensing  
The MM is composed mainly of mycolic acids, with a heterogeneous composition depending on the leaflet side [51]. The inner leaflet is composed of mycolic acids covalently linked to AG, which is consequently covalently linked to PG [51]. The mycoloyl chains of mycolic acids are intercalated in a zipper-like manner, with the long mycoloyl chains being folded in an \(\omega\)-shape [52]. Fatty acids,  
Figure 2: The resilient biology of non-tuberculous mycobacteria. (**A**) Non-tuberculous mycobacteria (NTM) possess a unique outer membrane with several biolayers that increase cell hydrophobicity and environmental resistance. In particular, several classes of lipids are extremely important for mycobacteria sliding motility and cell attachment to the surface, triggering early biofilm formation. (**B**) Biofilm formation is quorum-sensing dependent with oxidative stress (ROS/NOS) and divalent cations (A\({}^{2+}\)) promoting whiB3 and luxR gene expression, with the consequent increase production of autoinducer-2 (AI-2), leading to the increase in cyclic diguanylate (c-di-GMP) activation and biofilm formation-associated gene expression. Contrary, humic acids inhibit gene expression of biofilm formation-related genes. Additionally, eDNA is secreted into the extracellular matrix by FtsK/SpoIIIE secretion system. (**C**) NTM are also resistant to recalcitrant compounds, such as polycyclic aromatic hydrocarbons (PAH) and heavy metals. The degradation of PAHs is achieved by a dioxygenase system composed of a dehydrogenase, the dioxygenase small (beta)-subunit, and the dioxygenase large (alpha)-subunit, while mercury (Hg\({}^{2+}\)) triggers the synthesis of mercuric reductases and copper (Cu\({}^{2+}\)) and cadmium (Cd\({}^{2+}\)) are chelated into the mycobacterial cell wall as sulfides. (**D**) NTM can resist antimicrobials by several mechanisms, including: the intrinsic cell wall provides a physical barrier towards the entrance of antimicrobials; the erm genes that cause the methylation of rRNA, resulting in resistance to macrolides, limcosamide, and streptomycin B; and the expression of efflux pumps of different superfamilies, namely multidrug and toxic compound extrusion (MATE), resistance-modulation-cell division (RND), ATP-binding cassette (ABC), major facilitator (MFS), and small multidrug resistance (SMR), that actively secrete several antimicrobial compounds. The information present in this figure was combined from data reported by several studies regarding various non-tuberculous mycobacteria species. the base of mycolic acids, are produced by two fatty acid synthases: FAS-I, encoded by the _fas_ gene, and FAS-II. The first produces acyl-CoA, while the second produces \(\beta\)-ketoacyl-ACP (MtFabD and MtFabH) followed by fatty acid elongation and maturation (HadA, HadB, HadC, InhA, KasA, and KasB) [53]. The mycolic acids are then activated (_FadD32_ and _Rv3801c_ genes), condensed (_Pks13_ and _Rv3800c_ genes), and reduced (_CmrA_ and _Rv2509_ genes) [53]. The outer leaflet of the MM is composed of various lipids, namely phospholipids, trehalose mycolates, glycopeptidolipids, and lipopolysaccharides [51]. The PG is composed of both \(N\)-acetylmuramic acid (MurNAc) and \(N\)-glycoylmuramic acid (MurNGlyc) in most mycobacteria, with _M. leprae_ showing a PG exclusively composed of MurNAc [54]. The general presence of MurNGlyc increases mycobacterial resistance to lysozyme, with the \(namH\) gene encoding the hydroxylase involved in \(N\)-glycosylation so that the deletion of this gene leads to lysozyme sensitivity [55]. Around 10% of the MurNAc of PG are covalently linked to AG by the phosphotransferase of the LytR-CpsA-Psr family named Lcp1 (_Rv3267_ gene) [56]. The glycosylated PG fragments induce the production of TNF-\(\alpha\), which induces the recruitment and activation of phagocytic cells, potentiating the ecological niche within the host [57].