## 7 Cellular Permeability, Proton Motive Force, and Persistence  
Carbon metabolism, particularly of sugars, has been demonstrated to have direct and indirect effects on antibiotic uptake and efflux in persistent and tolerant bacteria. Therefore, increasing efflux or decreasing membrane permeability may represent a complementary strategy to tolerate antibiotics by preventing their intracellular accumulation. For example, the uptake of aminoglycoside antibiotics has been demonstrated to be highly PMF-dependent [78; 79]. PMF is known to be significantly lower in metabolically quiescent persister cells, which significantly limits the uptake and effectiveness of this particular class of antibiotics [75; 77; 80]. As discussed previously, stimulating PMF through supplementation with TCA cycle metabolites has been shown to increase the uptake and efficacy of this class of antibiotics against persisters [75; 77]. PMF also has an indirect effect on antibiotic uptake through the action of efflux proteins. In total, there are four major families of efflux proteins found in prokaryotes that utilize PMF as an energy source: major facilitator (MF), multidrug and toxic efflux (MATE), resistance-modulation-division (RND), and small multidrug resistance (SMR) [79; 81; 82; 83]. _P. aeruginosa_ has been found to overexpression various efflux pumps that provide protection against multiple classes of antibiotics during aminoglycoside exposure or biofilm growth [84; 85; 86; 87]. Furthermore,this response appears to be dependent on dose and length of antibiotic exposure, suggesting that these are adaptive responses [84]. Conversely, inhibiting efflux pumps in _P. aeruginosa, E. coli_, and _M. tuberculosis_ have been found to sensitize those bacteria to various classes of antibiotics [88; 89]. Within _P. aeruginosa_ biofilms, the expression pattern of the MexAB-OrrM efflux pump was found to be highest at the substratum, where oxygen and nutrient availability is lowest [85]. Perhaps the most compelling evidence for the role of efflux in bacterial persistence can be found in a 2016 article by [PERSON] et al. [90]. In this work, _E. coli_ persister cells were observed to have reduced levels of cytoplasmic \(\beta\)-lactam accumulation due to enhanced expression and activity of the central efflux component TolC [90]. Eliminating the _ompF_ and _ompC_ channels (which allow for diffusion of \(\beta\)-lactams) did not significantly alter persister formation rates or change the intracellular antibiotic concentration relative to non-persister cells. However, knocking out or inhibiting TolC significantly attenuated persister formation and increased intracellular levels of antibiotics [90; 91]. It should be noted that these persister cells were confirmed to be metabolically dormant, suggesting that the persistence phenotype encompasses both passive (reduced metabolism) and active (efflux) responses to antibiotics. Exploiting the permeability of persister cells without modulating bacterial metabolism may present an alternative strategy to treating infections [92]. Early studies using daptomycin demonstrated that it was effective in a concentration-dependent manner against stationary phase and metabolically arrested MRSA [93]. Furthermore, daptomycin was found to be significantly more effective in these situations than \(\beta\)-lactams, which require active bacterial growth. Daptomycin increases cellular permeability by disrupting outer bacterial membranes, thus bypassing the requirement of active metabolism to be effective [93]. Modulating cellular permeability of bacteria may also increase the efficacy and expand the spectrum of activity of existing antibiotics [94; 95]. Supplementation with ionic silver has been shown to increase the membrane permeability of Gram-negative biofilm cells by stimulating production of hydroxyl radicals that disrupt disulfide bonds and result in misfolded membrane proteins [95].