## 3 Mycobacterium Ecology: The Underlying Resilience Biology
### Biofilm Formation and Quorum-Sensing
smegmatis_ cells in different stages of biofilm formation [104]. There was an overexpression of mycobacteria biosynthesis genes, exochelin biosynthetic genes, and the putative iron ATP-binding cassette (ABC) transporter in the initial biofilm stages, showing iron uptake as a key aspect in _M. smegmatis_ biofilm development [104]. Many trivial deficiencies of metabolism could affect biofilm formation by indirect means. For instance, metabolic deficiencies that directly or indirectly decrease the abundance and/or composition of cell wall components involved in the triggering, attachment, maturation, and/or maintenance stages of the biofilm structure may hamper the biofilm formation process and thus result in lower adaptability and survival capacity of NTM, indirectly causing lower virulence and pathogenicity in opportunistic infection scenarios. Several intrinsic mechanisms of biofilm resistance to antimicrobials comprise physical or chemical diffusion barriers, slow bacterial growth, general stress response activation, and the rise of a biofilm-specific phenotype [105]. NTM cells grown in biofilms are transiently more resistant to disinfectants [69] and antibiotics [31]. Antibiotic-film molecules, such as N-acetylcysteine and Tween 80, can be used to increase the antimicrobial effect of molecules against NTM biofilms. Biofilm environments also facilitate HGT between mycobacterial cells, since increased genetic competence, accumulation of genetic elements (e.g., antibiotic resistance genes), and high cell densities occur in those environments [106]. Conjugation is, until now, the only known mechanism for horizontal transfer of resistance genes in biofilms [106]. Moreover, a study in _M. smgematis_ showed that the recipient  
Figure 3: Biofilm formation of _Mycobacterium smegmatis. M. smegmatis_ cells can be present in suspension on the environment (**I**), but under several environmental conditions, they aggregate in microcolonies (**II**), followed by an increase in cell density and formation of an extracellular matrix (**III**), leading to the formation of a mature biofilm (**IV**). In _M. smegmatis_, biofilm formation is iron (Fe\({}^{3+}\))-dependent, producing siderophores (e.g., exochelin) responsible for chelating and transporting iron into the mycobacterial cell wall transporters (e.g., iron ATP-binding cassette (ABC) transporters or mycobactin). Monomeromycylyl-diacylglycerol (MMDAG) are important cell wall components involved in biofilm formation, with their secretion being dependent of Lsr2 and GroEL1 proteins regulation of the KasA protein synthesis and MMDAG secretion by mmpl11 efflux pump. Additionally, the _Rv0024_ gene promotes biofilm formation, while the _glmM_ gene inhibits it. Mycolic acids suffer a transformation from long mycolic acids (C\({}_{70}\)-C\({}_{90}\)) into short mycolic acids (C\({}_{56}\)-C\({}_{68}\)) when mycobacteria enter the biofilm state. The formation of those short mycolic acids is regulated by the ability of KasA to synthesize trehalose dimycolate, the ability of serine esterase to hydrolyze trehalose dimycolate into short mycolic acids, and by the mce1 operon. Additionally, short mycolic acids are secreted into the extracellular matrix. Mycobacteria cells in the biofilm phenotype have a reinforcing capacity to survive oxidative stress by expressing the redox homeostatic system (RHOCS). In this system, increasing concentration of intracellular NADH promotes the synthesis of PknG protein, which phosphorylates the L13 protein, increasing the binding capacity of L13 with RenU, leading to the oxidation of NADH into NAD\({}^{+}\) and H\({}^{+}\). strain is incapable of forming biofilms, needing to be actively recruited to the biofilm structure by the DNA donor strain for conjugation to occur [106]. Mycobacteria possess a quorum-sensing-like mechanism, up to now described as being mainly involved in biofilm formation, however, the majority of accumulated knowledge on this topic is based on indirect research (e.g., bioinformatic analyses) and molecular and physiological research in _M.