## 4 Drug Resistance Mechanisms in TB
### Intrinsic Drug-Resistance of Mtb
#### 4.3.2 Drug Efflux  
Efflux pumps (EPs) are transmembrane proteins that provide resistance by expelling the drugs from the interior to the exterior of the cell. Five EP families are known, organized on the basis of energetic and structural characteristics: the ATP-binding cassette (ABC) superfamily, the major facilitator superfamily (MFS), the multidrug and toxic compound extrusion (MATE) family, the small multidrug resistance (SMR) family and the resistance nodulation division (RND) superfamily [18; 19; 46; 57]. The ATP-energized ABC members are primary transporters, while the others are secondary transporters energized by proton gradients (MFS, SMR, RND) or sodium gradients (MATE). The EP of Mtb belongs to the ABC (representing 2.5% of the entire Mtb genome), MFS and RND superfamilies, and to the SMR family.  
Following exposure of Mtb to sub-inhibitory concentration of INH and EMB, EP genes are overexpressed resulting in the development of low-level resistance for a prolonged period of time. After several weeks, a high level of acquired resistance develops, caused by chromosomal mutations in the genes encoding the target proteins [58; 59]. These observations indicate that inappropriate TBtreatment may generate pressure by sub-inhibitory drug concentrations that increase drug efflux, allowing a subsequent selection of mutants with high-level resistance [46; 57].  
Several EPs are known to be associated with resistance. For instance, Mtb exposure to INH induces the overexpression of \(MmpL7\) and \(mmR\) EP genes [60; 61]. Furthermore, several EPs are involved in resistance to several drugs. Thus, the EP Tap mediates low-level resistance to tetracycline (TC) and aminoglycos, whereas EPs encoded by the _Rv0194_ gene is associated with resistance to \(\beta\)-lactams, SM, TC, CF and vancomycin. Mutations in the _Rv0678_ gene caused an up-regulation of the transport protein, MmpL5, which caused EP-mediated cross-resistance to both BDQ and CFZ [60; 62; 63]. This is a potentially dangerous evolution of Mtb against antibiotics particularly in recent times, since BDQ and CFZ have just been included in the new WHO treatment guidelines of MDR/XDR-TB [8; 11].  
A strategy used to inhibit efflux-mediated drug resistance is efflux inhibition by non-antibiotic molecules that block the EP or inhibit the EP energy sources [57; 64]. The most studied inhibitors are vergamill (VP), thioridazine (TZ), reserpine, piperine, protonophores [57; 64], to be used in combination with anti-TB drugs in order to decrease or abolish the drug resistance caused by EP activity. Verapamil, an FDA-approved calcium channel blocker, decreased the MICs of BDQ, CFZ and other drugs [57]. This synergism was confirmed in various studies, but it was found that the effect of VP was not due to intra-mycobacterial drug accumulation, but on the disruption of membrane functions [65]. In Mtb-infected mice, VP increased the bioavailability and efficacy of BQ but not CFZ [66]. EP inhibitors are not presently used for the treatment of human TB, with the exception of TZ, which was administered in compassionate therapy for some XDR-TB cases [67].