**8. Efflux pumps**

Efflux pumps can affect both intrinsic and acquired resistance to antimicrobial agents by applying the energy to limit the cytoplasmic compound concentration to subtoxic level (Nikaido, 1992; Hogan & Kolter, 2002; Liaw, et al., 2010). Efflux system was first described as a mechanism of negatively impact to tetracycline susceptibility in *Escherichia coli* population. It was the plasmid-encoded single component Tet protein export of tetracycline throughout the cytoplasmic membrane (Ball et al., 1980).

A set of efflux systems facilitates bacteria to survive in extreme environments. Bacterial efflux pumps are involved in the multidrug resistance (MDR) phenotype combined with other more specific resistance systems including target mutation and enzymatic

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

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.,

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.

Efflux pumps can affect both intrinsic and acquired resistance to antimicrobial agents by applying the energy to limit the cytoplasmic compound concentration to subtoxic level (Nikaido, 1992; Hogan & Kolter, 2002; Liaw, et al., 2010). Efflux system was first described as a mechanism of negatively impact to tetracycline susceptibility in *Escherichia coli* population. It was the plasmid-encoded single component Tet protein export of tetracycline throughout

A set of efflux systems facilitates bacteria to survive in extreme environments. Bacterial efflux pumps are involved in the multidrug resistance (MDR) phenotype combined with other more specific resistance systems including target mutation and enzymatic

an early general stress response (Brown & Barker, 1999).

1991; Lange & Hengge-Aronis, 1991; McCann et al., 1991).

**8. Efflux pumps** 

the cytoplasmic membrane (Ball et al., 1980).

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

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; Breidenstein, et al., 2011).

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 (Whiteley et al., 2001).

Mechanisms Determining Bacterial Biofilm Resistance to Antimicrobial Factors 225

Upon the extreme conditions setting the general stress response mechanism by surface-

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Numerous of works have focused on the identification of genes that could contribute efflux system-mediate resistance of bacterial biofilms. Maira-Litran et al. (2000) examined the systems of *mar* and *acrAB* that confer on *Escherichia coli* biofilm the multidrug resistance phenotype. The *mar* operon is a regulator controlling the expression of various genes in *Escherichia coli* cells constituting the *mar* regulon. Upregulation of *mar* in planktonic bacteria effects a resistance phenotype to antimicrobial agents such as antibiotics (penicillins, cephalosporins, rifampicin, nalidixic acid and fluoroquinolones), oxidative stress agents and organic solvents (Alekshun & Levy, 1997). *mar* can be induced by sub-lethal doses of commonly used therapeutics such as tetracycline, chloramphenicol, salicylate and paracetamol (Cohen et al., 1993; Seoane & Levy, 1995). The *acrAB* efflux pump is upregulated in *mar* mutants and determined the multidrug resistant phenotype of *mar* mutant isolates (Ma et al., 1995; Ma et al., 1996). According to Maira-Litran et al. (2000) the constitutive expression of *acrAB* efflux pump effects lower susceptibility of *Escherichia coli*  biofilm to sub-lethal doses of ciprofloxacin. In addition, the expression of *mar* and its target genes is related to stationary phase of bacteria growth. Authors observed the highest level of *mar* expression within the depth of *Escherichia coli* biofilm, where the metabolic activity of examined bacteria were the most suppressed (Maira-Litran et al., 2000).

Brooun et al. (2000) and De Kievit et al. (2001) examined the expression of the genes associated with efflux pumps (MexAB-OprM and MexCD-OprJ) in developing biofilms of *Pseudomonas aeruginosa*. Brooun et al. (2000) underscored the importance of these pumps in the resistance to ofloxacin. Authors demonstrated that at low concentration of ofloxacin *Pseudomonas aeruginosa* mature biofilm with lacking MexAB-OprM was less resistant to antibiotic than mature biofilm that overexpressed the pump (Brooun et al. 2000). De Kievit et al. (2001) found that expression of the genes that encode MexAB-OprM and MexCD-OprJ, are decreased over time during biofilm maturation. In addition, authors, using the overexpressing and efflux pumps mutants of *Pseudomonas aeruginosa* revealed that none of efflux pumps analyzed plays a significant role at decreasing susceptibility of *Pseudomonas aeruginosa* biofilm to antibiotics (De Kievit, et al., 2001). Therefore to assess the true function of efflux pump in bacterial biofilm resistance to antimicrobial agents, further experiments of additional not yet characterized loci with homology to efflux system are needed.
