## 2 Molecular Mechanisms of Antimicrobial Agents Recalcitrance in Clinically Important Biofilms
### Cell-to-Cell Communication. Two-Component Systems, and Quorum Sensing  
In order to effectively monitor critical changes in the external environment and to regulate their internal physiology to adapt to such modifications, bacteria developed intricate signal transduction pathways. Bacteria are endowed with stimuli-responsive abilities due to a specialised signal transduction mechanism relying on the two-component systems (TCS). Moreover, since bacteria in the biofilm mode of life seem to respond as a community rather than just a simple collection of individual cells, cell-to-cell signaling appears as being essential for coordinated behaviour. As a further matter, bacterial cells are also engaged in active cross-talk with the host organism. Experimental evidence has been provided to support the involvement of some TCSs in the regulation of certain gene clusters, thereby promoting biofilm development [76; 77]. In general, a TCS consists of a sensor histidine kinase (HK) protein and a corresponding response regulator (RR) protein or domain [76]. Several different TCSs may be found in a bacterial cell, since each particular TCS is able to sense and respond to a specific environmental signal such as pH, nutrient levels, osmotic pressure, redox state, quorum-sensing proteins, and antibiotics [76; 78; 79]. Quorum sensing (QS) is a bacterial cell-to-cell communication mechanism relying on the production, release, and sensing by bacteria of signaling molecules called autoinducers [80; 81]. The amount of the autoinducers (AIs) produced is dependent on cell density. Once the concentration of an autoinducer overcomes a threshold concentration, the transcription of a target gene is activated. Thereby, gene expression is regulated in a cell density-dependent manner, allowing bacteria in a biofilm to gain a competitive advantage in surviving and spreading in natural environments. There are differences between the QS systems used by Gram-positive and Gram-negative bacteria regarding the regulatory components and molecular mechanisms. However, all QS systems share a series of common features [82]. First, bacterial response to the signaling molecules is triggered only when the local concentration of AIs overcomes a certain threshold necessary for detection that can be reached only at high cell densities. Second, all bacteria possess specific receptors located in the cytoplasm or in the plasma membrane specialised in the detection of the AI molecules. Third, AIs detection not only triggers the expression of specific target genes required for cooperative behavior, but also upregulates their own production through a positive feedback autoinduction loop. In Gram-negative bacteria, the signaling molecules are acyl-homoserine lactones (AHLs) produced by Luxl-type AHL synthases. AHL autoinducers are small free diffusible molecules that pass through the cell membrane and leave the cell. At high cell densities, the local concentration of the AI moleculesreaches the detection threshold, and the signal is recognised by the cognate LuxR-type receptors, which are cytoplasmic transcription factors. In the absence of the corresponding AIs, most LuxR proteins fail to fold properly and are degraded. However, by binding to the cognate AHL signaling autoinducers, LuxR receptors become stable, dimerise, and bind to DNA, resulting in QS-mediated gene regulation [77]. On the other hand, Gram-positive bacteria use oligopeptides as AIs (AIPs). AIPs produced by ribosomes undergo post-translational modifications before being exported out of the cell by a membrane-associated ATP-binding cassette (ABC) transporter [83]. Further detection and response to AIPs rely on a TCS signal transduction system. The detection of extracellular AIPs is achieved by a membrane-bound sensor kinase protein, which autophosphorylates at specific conserved histidine residues on HK. Next, the phosphoryl group is shuttled either intermolecularly to a conserved aspartate side chain in a cognate cytoplasmic response regulator protein or intramolecularly on a receiver domain.