**7. General stress response**

A general stress response is characterized by numerous changes in bacteria physiology and morphology that increasing cellular stress resistance (Hengge-Aronis, 1999; Lee et al., 2009). The formation of cell envelope and synthesis of thin aggregative fimbriae in *Escherichia coli*  and *Salmonella enteritis* serovar *Typhimurium* are both under control of general stress response. These features affect cell to cell contact (Atlung & Brøndsted, 1994; Römling et al., 1998). Moreover, the study of Hengge-Aronis et al. (1993) performed that under extreme conditions, the general stress response functions as a factor preventing cellular damage rather than repaired it. This mechanism induced by many different stresses including nutrients deprivation (which results in stationary phase of bacteria growth cycle), high or

Mechanisms Determining Bacterial Biofilm Resistance to Antimicrobial Factors 223

modification of antimicrobial agents (Zgurskaya & Nikaido, 2000; Davin-Regli et al., 2008; Bolla et al., 2011). The mechanism of efflux pumps in *Escherichia coli*, *Enterobacter aerogenes* and *Klebsiella pneumoniae* may also serve down regulation of porin production that slow down the penetration of hydrophilic solutes, and decrease the transmembrane diffusion of lipophilic solutes (Nikaido & Vaara, 1985; Plésiat & Nikaido, 1992; Li & Nikaido, 2004; Pagés et al., 2008). However, under particular circumstances, the outer membrane barrier cannot be the whole explanation of the bacteria resistance to inhibitors (Nikaido 1996). In fact, the equilibration across the outer membrane is reached very quickly, in the part of the surfaceto-volume ratio that is very large to compare with bacterial cell size. Thus, the periplasmic concentration of many antibiotics may achieve 50% of their external value (Nikaido, 1989). In the literature, numerous plasmid and chromosome-encoded efflux systems, both agentor class-specific and multidrug have been performed in a various of microorganisms where they are the major determinant in the intrinsic resistance of the bacteria to action of dyes, detergents and different classes of antibiotic including β-lactams (Nikaido, 1989; Nikaido, 1994; Markham & Neyfakh, 2001; Butaye et al., 2003). Bacterial efflux pumps compose of five classes of systems including: the major facilitator superfamily (MF), the ATP-binding cassette family (ABC), the resistance-nodulation-division family (RND), the small multidrug resistance family (SMR), and the multidrug and toxic compound extrusion family (MATE) (Putman et al., 2000; Kumar & Schweizer, 2005; Poole & Lomovskaya, 2006). To drive antimicrobial agents efflux, the ABC family system hydrolyses ATP, whereas the MF family, the RND family and the MATE family function as secondary transporters, catalysing drug-

The RND family transporters are most commonly found in bacteria cells (Poole, 2001). In gram-negative bacteria this system operates as a part of a tripartite mechanism that includes: a membrane fusion protein that is associated with the cytoplasmic membrane, a transporter protein that export substrates throughout the inner membrane, and an outer membrane factor (OMF) that enables the passage of the substrate throughout the outer membrane (Poole, 2005). The RND family transporters are the first line of bacterial defense that can promote the acquisition of additional resistance mechanisms such as target mutations or drug modification (Davin-Regli et al., 2008; Li & Nikaido, 2009). Pagés et al. (2008) and Pagés et al. (2010) performed that the expression of RND efflux pumps is an important prerequisite for the selection of fluoroquinolone resistant strains carrying the target mutation. According to Stover et al. (2000), *Pseudomonas aeruginosa* encode 12 efflux systems of the class of the RND family. However, to date only MexAB-OprM, MexCD-OprJ, MexEF-OprN, MexGHI-OmpD, MexJK and MexXY have been detailed characterized (Poole & Srikumar, 2001; Chuanchuen et al., 2002; Blair & Piddock, 2009;

Molecular analysis of efflux pumps assesses the role of this mechanism in biofilm resistance to antimicrobial agents. Exposure the bacterial biofilms to insufficient dose of antibiotics, such as tetracycline and chloramphenicol, and to xenobiotics, such as salicylate and chlorinated phenols, induces the expression of multi-drug resistance operons and efflux pumps (Levy, 1992; Ma et al., 1993). Also DNA microarray analysis of mature *Pseudomonas aeruginosa* PA01 biofilm demonstrated that none of genes encoding the RND efflux system were induced in sessile bacterial population grown in antibiotic-free environments

ion antiport (H+ or Na+) (Poole, 2005).

Breidenstein, et al., 2011).

(Whiteley et al., 2001).

low temperature, high osmolarity and acidic pH (Lange & Hengge-Aronis, 1991; Lee et al., 1995; Xu et al., 2001). Some evidences suggest also that biofilm development process leads to an early general stress response (Brown & Barker, 1999).

Exposure of *Escherichia coli* to adverse environments can induce RpoS, a sigma subunit of RNA polymerase, that acts as a central regulator. In *Escherichia coli* above 50 sigma factorcontrolled genes determine stress tolerance of cells, whereas others mediate the physiological rearrangement or redirect the metabolism of bacteria upon stress condition (Hengge-Aronis, 1999; Whiteley et al., 2000). Analysis of the molecular reactions in dense population of *Escherichia coli* revealed the influence of sigma factor-controlled genes on production of trehalose (Liu et al., 2000). Trehalose is the stress protectant in bacteria. In *Escherichia coli*, this molecule acts as osmoprotectant and is essential for bacteria desiccation tolerance (Strøm & Kaasen, 1993; Welsh & Herbert, 1999). Trehalose also plays an important role in thermotolerane of *Escherichia coli* (Hengge-Aronis et al., 1991). *rpoS* mutants that devoided of the typical features associated with the general stress response were unable to accumulate trehalose and they died off rapidly in stationary phase (Hengge-Aronis et al., 1991; Lange & Hengge-Aronis, 1991; McCann et al., 1991).

In bacterial populations, RpoS-controlled promoter regions include multiple binding sites for additional regulators such as cAMP-CRP or the histone-like proteins H-NS, leucineresponsive regulatory protein (Lrp), integration host factor (IHF) and FIS (Barth et al., 1995; Marschall et al., 1998). These regulators determining RpoS specificity (Marschall, et al. 1998).

As focused in literature, the general stress response acts both as a rapid emergency response and as a long-term mechanism, that enables the cell adaptation to nutrient deprivation and other environmental stresses that cause changes in cellular metabolism (Gentry et al., 1993; Hengge-Aronis, 1999). Activation of the general stress response in the cells, immobilized in biofilm matrix, may results in increasing resistance to biocides action (Drenkard, 2003). However, this mechanism needs to be examined in more detail. Drenkard (2003) demonstrated that the general stress response maintain cell viability upon stationary phase when nutrients availability is limited. It is highly probable that environments within biofilm would promote the expression of the RpoS. This process affecting the physiological changes that mediate protection of biofilms to environmental stresses (Drenkard, 2003). Adams & McLean (1999) observed that *Escherichia coli* that lack *rpoS* are unable to form biofilm of wild type architecture. The study of Cochran et al. (2000) demonstrate that thin biofilms formed by *Pseudomonas aeruginosa* mutants of *rpoS* are susceptible to hydrogen peroxide.
