# The Complex Relationship between Virulence and Antibiotic Resistance
### Drug Resistance in Biofilms  
Amid the torrent of environmental stresses, it is thought that the majority of bacteria, particularly in the presence of a foreign body or under prolonged exposure to sub-inhibitory antibiotic concentrations, reside in surface adherent biofilms [50; 51; 52]. Biofilm formation occurs through a series of events coordinated through cell-cell communications (i.e., quorum sensing), as mediated by excreted autoinducers (i.e., small molecules in gram-negative bacteria [53; 54] and peptides in gram-positive bacteria [55; 56; 57]). Signaling cascades initiate upon detection of a critical extra cellular concentration of autoinducer [4; 21] and culminate in adhesion, metabolic changes, production of a protective glycocalyx, up-regulation of virulence, and decreased antibiotic susceptibility among other factors [58; 59; 21]. Interrupting bacterial adhesion prior to glycocalyx formation may prove an antibiotic strategy with efficacy analogous to those therapies designed to kill planktonic bacteria [60; 61]. Importantly, once a surface adherent biofilm has been established therapies designed to kill planktonic bacteria are ineffective. The glycocalyx protects bacterial inhabitants from antibiotics, biocides, and other chemical or physical obstacles [62; 63] and is recognized a key element in the persistence of infections [64]. The secretion of an extracellular glycoprotein matrix provides a protected ecological niche for the proliferation and the propagation of antibiotic resistance through the exchange of genetic material [65; 66], allowing the accumulation of mutations and genetic elements that confer resistance over time [15; 29; 67]. These events have been thoroughly reviewed and are beyond the scope of this review [53; 68]. Evidence holds that the development of biofilm is a major pathway to development of resistance. Resistance conferred by a biofilm is likely not just a consequence of an encompassing glycocalyx but may also result from the underlying heterogeneous bacterial subpopulations. These sub-populations vary not only in their degree of antibiotic susceptibility but also the mechanism by which they achieve their states of antibiotic resistance [19; 63]. Certain sub-populations of biofilm bacteria may produce enzymes that degrade antibiotics while other sub-populations have up-regulated efflux pumps [53]. The overall ecology of the biofilm community also imposes endogenous oxidative stress on its members, which consequently drives biofilm bacteria to a hyper-mutability state [47; 69; 70]. In addition to being capable of quickly acquiring a multidrug resistant phenotype, this combination of resistance mechanisms has lead scientists to describe biofilms as having enhanced resistance for a vast array of antibiotic classes [53; 66]. Sub-populations can also be metabolically distinct, a fact that has critical implications for clinical treatment and the de novo development of resistance. Bacterial persisters are a metabolically senescent sub-population within a biofilm. Although several good reviews describe aspects of persister cells in biofilms [19], briefly exploring the impact of metabolic senescence is useful to frame a discussion of therapeutic development to circumvent this resistance [71; 72]. In general, the efficacy of most antibiotics is based on the metabolic activity of the bacteria (e.g., \(\beta\)-lactams that target cell wall synthesis in actively dividing _P. aeruginosa_[19; 53; 73] and quinolones that typically introduce nicks in actively replicating DNA [74]); therefore, non-growing or slow-growing cells are less susceptible to antibiotic effects, a fact that has been repeatedly demonstratedin vitro using bulk cultures forced into a prolonged stationary phase [75]. When cells are challenged with antibiotics during this stationary phase of slowed growth, they display a persistence phenotype, having increased survival when compared to cells in a logarithmic phase of growth under the same antibiotic pressure [19; 73; 76], giving rise to a property called--drug indifference [77].