## 4 Growth, Metabolism, and ATP Production  
The formation of biofilms and persister cells represent two interrelated yet phenotypically distinct strategies utilized by bacteria to tolerate antibiotic treatment. Despite their differences, however, growth rate and the underlying metabolic state are crucial determinants of the antibiotic tolerance displayed by both biofilm and persister cells. As a complex ecological environment, biofilms exhibit heterogeneity in their population structure and metabolic activity. Cells proximal to the center of the biofilm can exhibit marked dormancy relative to cells in the periphery [49]. As a result, antibiotic efficacy is highest at the air interface, where metabolic activity is highest [49]. Additionally, the growth rate of biofilm cells has been shown to be a major determinant of antibiotic susceptibility in both _Pseudomonas aeruginosa_ and _E. coli_[50; 51]. Similar dynamics have also been observed in non-biofilm persister cells. Tolerance and persistence are closely associated with the rate and phase of bacterial growth [52; 53; 54; 11; 55]. For example, slow growth rates have been shown to permit stable tolerant phenotypes in _E. coli_[56], and both _P. aeruginosa_ and _S. aureus_ display an increase in persister formation in mid-exponential and stationary phase while remaining unchanged in early exponential phase [14]. Conversely, maintaining bacterial cultures in early exponential phase has been found to completely eliminate persisters [7; 14]. Furthermore, _E. coli_ has demonstrated an ability to modulate its lag time to match the duration of antibiotic exposure when subjected to repeated treatments [57]. Therefore, modulation of growth rate appears to be an adaptive and transient response to antibiotic exposure. A major contributing factor to variations in growth rate is nutrient availability, with nutrient limitation having long been known to induce persistence. Glucose deprivation has been shown to increase the formation of persisters and increase biofilm tolerance to fluoroquinolone and \(\beta\)-lactam treatment [58; 59]. Conversely, stationary phase _E. coli_ cells can be sensitized to ciprofloxacin by supplementing oxygen and carbon sources [55]. Furthermore, _E. coli_ grown in minimal media with limited glucose availability have higher expression of the efflux pump _acrB_, suggesting that sugar metabolism may have wide-ranging effects that include drug efflux [60]. It appears that amino acid deprivation is prerequisite for tolerance; however, deprivation of glucose in addition to amino acids produces bacteria that are highly tolerant of \(\beta\)-lactams, fluoroquinolones, and aminoglycos [56]. Long-term starvation of _Mycobacterium tuberculosis_ reduces susceptibility to rifampicin, isoniazid, and metronidazolezole and induces shifts in the expression of central metabolic pathways such as amino acid biosynthesis, energy metabolism, and lipid biosynthesis [53]. Most notably, starvation down-regulates the expression of many glycolysis and TCA cycle enzymes. Additionally, the NADH dehydrogenase operon and most of the ATP synthase complex, both of which contribute to the production of ATP, are dramatically down-regulated [53]. Conversely, starvation induced a significant up-regulation of the fumarate reductase gene _frdA_, which is a component of a complex which serves as an anaerobic electron transport chain in similar bacteria [53]. These changes allow _M. tuberculosis_ to enter into a tolerant state by decreasing growth rate while maintaining viability [53]. Taken together, these findings further demonstrate that phenotypic plasticity in bacteria is critical to surviving antibiotic exposure events. Bacteria may also reduce their metabolic flux and enter a persistent state through the utilization of the glyoxylate shunt. The glyoxylate shunt is a variant of the TCA cycle that enables net carbon assimilation by bypassing steps that generate carbon dioxide [61; 62; 63]. In _M.