**6.** *Quorum sensing*

A mechanism which cannot be overlooked when discussing bacterial biofilm resistance to antimicrobial factors is *quorum sensing*. Within biofilm, bacteria are able to sense an increase of the cell population density and respond to it by the induction of particular set of genes (Whitehead et al., 2001; Shirtliff, et al., 2002; González & Keshavan, 2006; Turovskiy et al., 2007). *Quorum sensing* termed also cell-to-cell signaling system, includes in gram-negative bacteria the production and secretion of an acyl homoserine lactones (AHL), which diffuse through the cell wall, from the cell to the medium (Eberl, 1999; Williams et al., 2007). *Quorum sensing* mechanism in gram-positive bacteria typically use secreted peptides as signal compounds and a two-component regulatory system (composed of a membranebound histidine kinase receptor and an intracellular response regulator) to detect the peptide and trigger the required changes in gene expression (Kleerebezem et al., 1997; Suntharalingam & Cvitkovitch, 2005). A third examined form of *quorum sensing* mechanism employes a family of related molecules termed autoinducers-2. This system was found in both gram-negative and gram-positive bacteria (Platt & Fuqua, 2010).

According to Whitehead et al. (2001) and González & Keshavan (2006) several important biofilm features are likely to affect signal molecules production. The number of active cells in the biological layer, which is influenced by the bacteria growth and the synthesis of both of glycocalyx matrix and degradative enzymes, may affects signal molecules production (Chopp, 2003; Mentag et al., 2003; Newton & Fray, 2004; Sakuragi & Kolter, 2007). Moré et al. (1996), Schaefer et al. (1996) and Parsek et al. (1999) observed that metabolic activity of gram-negative bacteria will likely affect the availability of cellular substrates pools for signal molecules production, *S*-adenosylmethionine and acyl-carrier protein, thereby increasing signal molecules production. For gram-negative bacteria, *S*-adenosylmethionine is the amino acid substrate necessary for the synthesis of *quorum sensing* signal compounds, whereas acyl carried protein is the donor of fatty acid chain in the biosynthesis of signal molecules of *Vibrio fischerii* (Eberhard et al., 1991).

*Quorum sensing* mechanism controls also the biofilm maturation process (Davies et al., 1998; Costerton, 1999; Watnick & Kolter, 2000). The work of Kjelleberg & Molin (2002) and Williams et al. (2007) demonstrated that the diffusion process of signal molecules within biofilm is unlimited. Inside biological layers there are shorter-distance migration of signal molecules and therefore the contact between the cells and reaction to signal molecules by the cells is more probable (Whitehead et al., 2001). The role of signal molecules-mediated *quorum sensing* in biofilm formation has been examined for *Brukholderia cenocepacia*, *Aeromonas hydrophila*, *Pseudomonas aeruginosa*, *Pseudomonas putida* and *Serratia marcescens* (Davies et al., 1998; Huber et al., 2001; Lynch et al., 2002; Steidla et al., 2002; Labbate et al., 2007). Davies et al. (1998) demonstrated that cell-to-cell signal *N*-(3-oxododecanoyl)-Lhomoserine lactone (3-oxo-C12-HSL) is needed for the development of *Pseudomonas* 

The work of Moyed & Bertrand (1983) supported this statement. Moyed & Bertrand (1983) discovered in *Escherichia coli* a toxin-antitoxin system (*hipAB* locus) that has a potential of both killing the cells and improving survival after exposure to lethal doses of antimicrobial factors. The inactivation of the toxin-antitoxin systems by insertional elements or by mutation, induced defects in PCD system in *Escherichia coli* and made the bacteria more

A mechanism which cannot be overlooked when discussing bacterial biofilm resistance to antimicrobial factors is *quorum sensing*. Within biofilm, bacteria are able to sense an increase of the cell population density and respond to it by the induction of particular set of genes (Whitehead et al., 2001; Shirtliff, et al., 2002; González & Keshavan, 2006; Turovskiy et al., 2007). *Quorum sensing* termed also cell-to-cell signaling system, includes in gram-negative bacteria the production and secretion of an acyl homoserine lactones (AHL), which diffuse through the cell wall, from the cell to the medium (Eberl, 1999; Williams et al., 2007). *Quorum sensing* mechanism in gram-positive bacteria typically use secreted peptides as signal compounds and a two-component regulatory system (composed of a membranebound histidine kinase receptor and an intracellular response regulator) to detect the peptide and trigger the required changes in gene expression (Kleerebezem et al., 1997; Suntharalingam & Cvitkovitch, 2005). A third examined form of *quorum sensing* mechanism employes a family of related molecules termed autoinducers-2. This system was found in

According to Whitehead et al. (2001) and González & Keshavan (2006) several important biofilm features are likely to affect signal molecules production. The number of active cells in the biological layer, which is influenced by the bacteria growth and the synthesis of both of glycocalyx matrix and degradative enzymes, may affects signal molecules production (Chopp, 2003; Mentag et al., 2003; Newton & Fray, 2004; Sakuragi & Kolter, 2007). Moré et al. (1996), Schaefer et al. (1996) and Parsek et al. (1999) observed that metabolic activity of gram-negative bacteria will likely affect the availability of cellular substrates pools for signal molecules production, *S*-adenosylmethionine and acyl-carrier protein, thereby increasing signal molecules production. For gram-negative bacteria, *S*-adenosylmethionine is the amino acid substrate necessary for the synthesis of *quorum sensing* signal compounds, whereas acyl carried protein is the donor of fatty acid chain in the biosynthesis of signal

*Quorum sensing* mechanism controls also the biofilm maturation process (Davies et al., 1998; Costerton, 1999; Watnick & Kolter, 2000). The work of Kjelleberg & Molin (2002) and Williams et al. (2007) demonstrated that the diffusion process of signal molecules within biofilm is unlimited. Inside biological layers there are shorter-distance migration of signal molecules and therefore the contact between the cells and reaction to signal molecules by the cells is more probable (Whitehead et al., 2001). The role of signal molecules-mediated *quorum sensing* in biofilm formation has been examined for *Brukholderia cenocepacia*, *Aeromonas hydrophila*, *Pseudomonas aeruginosa*, *Pseudomonas putida* and *Serratia marcescens* (Davies et al., 1998; Huber et al., 2001; Lynch et al., 2002; Steidla et al., 2002; Labbate et al., 2007). Davies et al. (1998) demonstrated that cell-to-cell signal *N*-(3-oxododecanoyl)-Lhomoserine lactone (3-oxo-C12-HSL) is needed for the development of *Pseudomonas* 

susceptible to antimicrobial agents (Han et al., 2011).

both gram-negative and gram-positive bacteria (Platt & Fuqua, 2010).

molecules of *Vibrio fischerii* (Eberhard et al., 1991).

**6.** *Quorum sensing*

*aeruginosa* biofilm with a wild-type structure: loosely packed biomass with a mushroom appearance with notable amount of extracellular polysaccharides and water channel traversing the entire the biological layer. Whereas, signal molecules-negative mutants of *Pseudomonas aeruginosa*, *Brukholderia cenocepacia* and *Aeromonas hydrophila* showed defects in the late stages of biofilm maturation and thus were unable to form biofilms with the wildtype architecture (Huber et al., 2001; Lynch et al., 2002; Steidla et al., 2002; Labbate et al., 2007).

Because heterogonous architecture of biofilms and the synthesis of degradative enzymes deactivate biocides, it seems reasonable to speculate that biofilm antimicrobial agents resistance could also be influenced by *quorum sensing* system. Moreover, coordinated expression of *quorum sensing*-mediated phenotypes is crucial in cells migration to a more suitable environment/better nutrient supply and in adaptation to a new modes of growth, which may afford protection from deleterious environment (Whitehead et al., 2001; Abee et al., 2011). However, to date *quorum sensing* system as factor decreasing the biofilm susceptibility to antimicrobial agents has been studied in a limited number of strains. Davies et al. (1998) and Hassett et al. (1999) reported that exposure of *quorum sensing*-negative mutant biofilms to the antimicrobial agents SDS and hydrogen peroxide caused detachment and dispersion of surface-anchored bacteria. In addition, Hassett et al. (1999) have reported that cell-to-cell signaling mechanism in *Pseudomonas aeruginosa* controls the expression of the catalase and superoxide dismutase genes and mediates biofilms resistance to hydrogen peroxide. According Shih & Hoang (2002) *quorum sensing*-deficient mutant biofilms susceptibility to kanamycin correlated with thinner biofilm formation and lower EPS production. Above results provide evidences that biofilm respond directly or indirectly to environmental stress via a *quorum sensing* system.

Interestingly, resent reports have also demonstrated chelating properties of cell-to-cell signals (Schertzer, et al. 2009). Such non-signaling features were stated for *Pseudomonas aeruginosa quorum sensing* molecules. Weinberg (2008) examined multiple meaning of *quorum sensing* system in mixed-species bacterial population. The author performed that *Pseudomonas aeruginosa* may kills competing bacteria in the growth environment by hijacking the bacteria's iron stores using 2-heptyl-3-hydroxy-4-quinolone signal. According to Weinberg (2008) *Pseudomonas* quinolone signal is a high affinity iron chelator. The ability of signal molecules to trap external positive-charged compounds is similar to antimicrobial action of glycocalyx matrix (Schertzer, et al, 2009). However, this role of cell-to-cell signal molecules to biofilm resistance properties needs to be examined in more detail.
