## 4 Drug Resistance Mechanisms in TB
### Complexity of TB Granulomas  
Long lasting therapies are also attributable to the complex pathology of TB. In the lungs of patients with active and latent TB, a spectrum of heterogeneous granulomatous lesions coexist, ranging from well-vascularized cellular granulomas, in which a rim of lymphocytes surrounds macrophages and neutrophils, to avascular caseous granulomas, characterized by a necrotic center with a cheese-like aspect (caseum) formed by the lysis of host cells and bacteria [29; 30]. In these lesions, tubercle bacilli range from actively replicating (AR) stages, particularly in cellular granulomas, to dormant, slowly-replicating or NR stages, typical of hypoxic caseous granulomas [31]. In Mtb-infected rabbits, the fraction unbound of a drug penetrates the caseum via passive diffusion, and caseum binding of a drug is proportional to hydrophobicity (cLogP) and aromatic ring count [32].  
The current 4-drugs therapeutic regimen (RIF-INH-PZA-EMB) is effective against AR intracellular bacilli in cellular granulomas, while NR extracellular bacilli localized in pH-neutral, caseous granulomas are refractory to drug action [17; 33; 34; 35; 36]. The necrotic center of caseous granulomas contains NR bacilli phenotypically resistant to several drugs (drug-tolerant persisters), with the exception of rifamycins, which are known to sterilize caseum in ex-vivo assays [35; 36]. Spatial and temporal differences in drug distribution and the kinetics of accumulation of drugs in specific lesion compartments may create local windows of monotherapy that increase the risk of the emergence of drug-resistance [17; 37]. This is in keeping with the knowledge that genetically resistant mutants of Mtb may emerge from the persistence phase of some TB drugs, due to hydroxyl radical-mediated genome-wide random mutagenesis [38; 39; 40]. In this view, drug combinations should contain complementary drugs preferentially distributing in lesions in which their most vulnerable target population resides [17].  
In the event of caseous granulomas expansion, the necrotic centers fuse with the airway structures of bronchi to form pulmonary cavities in which are found both extracellular bacilli from liquefied caseum and intracellular bacilli derived from the lysis of infected macrophages of the cavity walls. In contact with the atmospheric oxygen, these bacilli rapidly proliferate in the lumen of cavities, and later appear in the sputum of TB patients [17]. Due to high bacterial load in pulmonary cavities, genetically resistant bacilli with chromosomal mutations may be generated, playing an important role in the development of resistance [16]. Noticeably, in comparison with paired sputum isolates, additional resistances were found in Mtb isolates recovered from surgically resected cavities of the same patient [41]. A single founder Mtb strain underwent genetic mutations during treatment, leading to the acquisition of additional drug resistance in different sections of the lung of the same patient, preferentially in the cavity wall [42]. In keeping with this observation, drug-specific gradients in the walls of human pulmonary cavities were reported to be associated with the development of acquired resistance in patients with MDR-TB, due to the low level of some drugs in the cavities centers, where there is a high number of replicating bacilli [43]. In the latter study, spatial heterogeneity of drug concentrations across the pulmonary cavity resulted in the development of mutations in the Mtb genes _gyrA_ (FQ resistance) and _gydB_ (aminoglycosoxide resistance), consistent with evolution from MDR- to XDR-TB after about five months of therapy [43]. Overall, these observations indicate that acquired Mtb resistance may be related to the formation of drug-penetration gradients in TB lesions generating suboptimal drug concentrations in non-vascularized caseous granulomas and in liquefied caseum in the cavity centers [16; 17; 43].