Table 4.

Main factors that affect the NTM drug development

Challenges	Comments	Ref
Hydrophobicity and innate resistance	• Hydrophobic, lipid-rich double membrane cell envelope (major permeability barrier) • Non-polar cell surface (prevents adherence or binding of antibiotics charged positive or negative) • Reversible colony morphology switch (variability in drug resistance) • Efflux pumps (prevent intracellular accumulation of drugs	[315–317, 319]
	• Polymorphism in the target gene (natural resistance to drugs—i.e. preventing drug binding) • Modification of the target binding site (bacterial gene expression upon drug exposure) • Enzymes (metabolizes drugs to a less active form)
Acquired drug resistance	• Genomic mutations (mutations in the target or other related genes to confer high-level resistance after long-course treatment) • Lateral gene transfer of drug resistance genes (less frequent but possible)	[315, 321, 323]
Lack of bactericidal activity	• Current drugs-base regimens are bacteriostatic or weakly bactericidal at high concentration:     o High metabolic rate and slow division of bacteria	[315, 325]
Poor correlation between in vitro MIC determination and clinical outcomes	• Mycobacteria growth conditions for MIC are very different from NTM pulmonary disease:     o MIC       - Exponential growth       - Suspension in aerated nutrient-rich broth     o Lung       - Different type of complex and dynamic lesions       - Stress appearance       - Drug tolerance or “phenotypic drug resistance”       - Growth in airways mucus and as biofilms       - Effect of local microenvironments on drug penetration	[315, 318]
Intracellular growth and residence in phagocytic cells	• NTM can grow, survive and persist extra and intracellularly:     o Escape macrophage apoptosis mechanism (possibility to spread and infect other cells)     o Restriction of intra-phagosomal acidification     o Decrease apoptosis and block autophagy flux	[315, 316, 319, 322]
	• Found within phagocytic cells and in granulomas in infected organs (lung and spleen)
Caseum, mucus and biofilm growth	• Capability of maintaining long-term viability:     o Mycobacteria change to a non-replicative state under nutrient starvation or oxygen deprivation) • Drug resistance:     o Antibiotics do not actively destroy cell components • High drug-tolerance under non-replicative conditions:     o Molecular mechanisms—“phenotypic drug resistance” • High production of mucus in NTM pulmonary disease (bacteria evasion of the immune system and affected drug susceptibility)	[315, 316, 324, 326]