## 3 Mycobacterium Ecology: The Underlying Resilience Biology
### Antimicrobial Resistance  
The outer membrane hydrophobicity and impenetrability of mycobacteria is the dominant key factor responsible for antimicrobial resistance and, thus, NTM are susceptible to a small fraction of known antibiotics only. They are intrinsically resistant to several antimicrobial agents, mostly the hydrophilic ones, as their hydrophobic outer membrane leads to low transport rates of hydrophilic compounds through the cell wall [76]. In parallel, the high mutation rate of the single 16S rRNA cistron, leads to the accumulation of resistance mutations towards ribosomal-targeting antibiotics [76]. Previous work has associated polymorphisms in the 16S rRNA encoding gene to amikacin resistance in _M. abscessus_[132]. Additionally, several other genomic mutations, plasmid transfer of resistance determinants, and the production of enzymes that metabolize drugs to a less active form have been described as resistance mechanisms [133; 134]. Furthermore, the intracellular growth of NTM [10] combined with survival in casein under a non-replicative state of bacterial persistence [70; 135], mucus growth [135], and biofilm growth [136], make antibiotic delivery and action particularly difficult towards intrinsically resistant NTM [137]. Although all NTM possess some level of intrinsic resistance to antimicrobials, differences across NTM species have been demonstrated, with, for example, _M. kansasii_ being susceptible to multiple antibiotics, while _M. avium_ is susceptible mainly to macrolides [138]. The selection pressure that NTM are subjected to in natural environments, particularly in soil upon the action exerted by the antimicrobials (acting as selective agents) produced by their neighbors, has driven these bacteria to develop a panoply of resistance mechanisms to survive under these hostile environments [139]. In addition to the thick cell wall, NTM also possess efflux pumps that prevent the intracellular accumulation of drugs, therefore contributing to intrinsic and acquired resistance [140]. Efflux pumps belonging to the five superfamilies, namely ABC superfamily, major facilitator (MPS) superfamily, small multidrug resistance (SMR) superfamily, resistance-modulation-cell division (RND) superfamily, and multidrug and toxic compound extrusion (MATE) superfamily were reported among NTM (Figure 2D) [140]. In MAC, ABC and MFS pumps are associated with macrolide resistance [141]; in _M. abscessus_, the RND pumps confer resistance to clofazimine and bedaquiline, while the overexpression of MFS pumps has been associated with clarithromycin resistance [142; 143]. The ribosomal RNA methylase genes, including _erm_(38) in _M. smgematis_[144], _erm_(39) in _M. fortutium_[145], _erm_(40) in _M. mageritense_ and _M. volinskyi_[146], and _ern_(41) of _M. abscessus_ and _M. bulletii_[147], have been commonly associated with intrinsic antibiotic-resistant phenotypes (Figure 2D). Moreover, in other mycobacteria, such as _M. boenickei_, _M. goodii_, _M. houstonense_, _M. neworleansne_, _M. porcinum_, _M. peregrinum_, and _M. volinskyi_, the _erm_ genes were also identified [146]. These genes are linked to two different phenotypes: high resistance to lincosamides and low to moderate resistance to macrolides and streptogramin B; or high resistance to lincosamides, macrolides, and streptogramin B, depending on the addition of a mono- or dimethyl group, respectively, to 23S rRNA, which decreases the binding of these antibiotics to ribosomes [148]. The expression of these genes can be induced upon drug exposure and a lack of correlation between phenotypic resistance and molecular detection of _erm_ is also described [146]. Indeed, recent works suggest _erm_ gene sequencing for accurate antibiotic susceptibility prediction [149]. Clinically acquired macrolide resistance in several mycobacteria, such as _M. avium_[150], _M. intracellular_[151], _M. abscessus_, and _M. chelonae_[152]; or _embB_ gene in _M. smegmatis_[153], has also been linked to polymorphisms in the 23S rRNA gene.