## 2 Molecular Mechanisms of Antimicrobial Agents Recalcitrance in Clinically Important Biofilms
### Persister Cells  
Persisters are an antibiotic-tolerant differentiated phenotype of the wild type within a bacterial population [64]. Persister cells emerge stochastically, and their drug tolerance is non-inheritable. Therefore, persisters were presumed to be nongrowing, nondividing, metabolically inactive cells that arise in response to environmentally stressful conditions such as hypoxia, acidic pH, nutritional starvation, or antibiotic exposure being able to survive in this temporary dormant state until the stress is removed. After stress relieving, the persisters switch back to the actively growing state, thereby providing greater survival potential to the bacterial population. Whereas in a wild planktonic type _E. coli_ population, the frequency of persister formation was found to be only 1 in a million, in stationary cultures and biofilms, an about \(10^{4}\) fold increase of the persister subpopulation up to 1 in 100 was observed [65]. The molecular mechanism underlying the formation of persisters is largely unknown, and some authors even claimed that nongrowing cells in a bacterial population might not necessarily belong to the persister phenotype, while persisters might be metabolically active cells [66]. Nevertheless, some important insights into the mechanism of persister formation were gained through experimental studies ingeniously designed to overcome the inherent difficulties arising from the very low frequency of the persister phenotype. The main model underlying the genetic basis of persister formation involves the expression of toxin-antitoxin (TA) modules. The main role of TA systems is to regulate growth arrest and transition to the persistent state [60; 62; 63; 64; 65; 67]. TA systems consist of a pair of genes within an operon. One gene encodes a protein toxin, while the second gene encodes the cognate antitoxin, which might be either a protein or a non-coding RNA molecule [65]. The toxin component interferes with a vital cellular process determining growth arrest. However, under normal growth conditions, the toxin is inactivated through binding to its complementary antitoxin. However, under stress conditions, the antitoxin is degraded, thereby leaving the toxin to achieve its full toxic effects freely. There are several types of TA systems (I-VI), the best studied being the type II TA system in which both toxin and antitoxin are proteins [65]. [PERSON] and [PERSON] [68] were the first to demonstrate that bacterial persistence has a genetic basis through isolation of _E. coli_ K12 mutants with vastly increased levels of persisters, i.e., high persistence or hip mutants. Further research showed that the hip operon consists of two genes, hipA and hipB. The wild-type hipA gene encodes a 440-residue protein toxin, whereas the upstream hipB gene encodes a smaller 88-residue DNA-binding protein [66]. Since hip B and hip A are on the same hipA operon, they share the same promoter and are transcribed together. The hipB protein is a transcriptional repressor of the hipA operon and downregulates its own promoter by cooperatively binding to four operators upstream of hipBA [69; 70]. Hence, the expression of the hipB operon is autoregulated. However, at the same time, the hipB protein acts as the antitoxin counterpart of the hipA protein toxin. Under normal growing conditions, both genes are expressed and the hipB antitoxin binds to the hipA toxin, forming a tight dimer complex and thereby neutralising the deleterious effects, i.e., growth arrest, of the latter. However, during cellular stress periods, the stress-induced Lon protease degrades the less stable hipB antitoxin, thereby freeing the toxin from its antitoxin counterpart [59; 63; 71; 72; 73; 74]. In its unbound state, the hipA protein toxin acts as a kinase that phosphorylates and inhibits glutamyl-tRNA synthetase, resulting in an increased concentration of uncharged glutamyl tRNAs (tRNA\({}^{\text{Glu}}\)) [59; 74; 75]. In turn, this leads to the accumulation of stalled ribosomes.