**1. Introduction**

212 Antimicrobial Agents

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In most natural environments, the process of bacterial surface association is prevailing cells lifestyle. The tendency of bacteria to colonize solid materials is advantageous from an ecological standpoint. This mechanism allows bacteria the colonization of a nutritionally favorable new niche and encouraging symbiotic relationships between the cells. Sessile mode of growth provides also some level of protection from external stresses (Costerton et al., 1995; Dunne, 2002; Russell, 2002). Anchored bacteria are being linked to common human diseases ranging from tooth decay and paradontose to nosocomial infections and both biliary tract and kidney infections (Costerton et al., 1999; Potera, 1999). According to Russell (1999) and Wood et al. (2011) 80% of bacterial chronic inflammatory and infectious human diseases involve biofilm. In industrial environments surface-bound bacteria are the potential source of contamination of processed material that in consequence may lead to spoilage or transmission of pathogens (Bower et al., 1996; Gunduz & Tuncel, 2006; Myszka & Czaczyk, 2011).

Attached bacteria to organic or inorganic surfaces form thin layer called biofilm or biological layer. Biofilms consist of a single microbial species or multiple microbial species (O'Toole et al., 2000). However mixed-species biological layers predominate in most environments, single-species biofilms occur in a variety of infections and on the abiotic surface exploited in medicine and industry practice (Adal & Farr, 1996; Donlan, 2002). Despite of difference of ecosystems in which biofilms can develop, in each case the component microbial cells reach homeostasis and are optimally organized to convert all available nutrients to usefulness products for cells (O'Toole et al., 2000; Sutherland, 2001; Myszka & Czaczyk, 2009).

Biofilm-associated bacteria perform chemically diverse biocide-resistance phenotype (Mah & O'Toole, 2001; White & McDermott, 2001). It has been estimated that biofilms can tolerate antimicrobial agents (disinfectants, antibiotics, surphactants) at concentrations of 10-1000 times that needed to inactivate genetically equivalent planktonic bacteria (Jefferson, 2004). It is worth point out that almost all clinically and industrially approved antimicrobial agents are being less active against sessile bacteria. So far selection of antimicrobial agents for industry and medical properties based on their activity against planktonic bacteria (estimation the indexes of the minimal inhibitory concentration (MIC) and the minimal bactericidal concentration (MBC) for different antimicrobial agents).

Mechanisms Determining Bacterial Biofilm Resistance to Antimicrobial Factors 215

of the drug degrading enzyme β-lactamase. Ampicillin was able to penetrate biological layer formed by a β-lactamase–deficient mutant without difficulty. In contrast, ciprofloxacin diffused through *Klebsiella pneumoniae* biofilm without delay. Differences in the effect of penetration of both ciprofloxacin and ampicillin through *Klebsiella pneumoniae* wild-type and β-lactamase-deficient mutant biofilms, suggesting that biofilm resistance is multifactorial

Costerton et al. (1978) termed glycocalyx as the integral part of the biofilms of Gram-positive and Gram-negative bacteria. Glycocalyx known as either as slime or capsule may provide the forces responsible for cohesion and adhesion to the solid surfaces (Flemming, 1995; Mayer et al., 1999). This is performed by the weak interaction such as electrostatic interactions, hydrogen bonds and van der Waals forces (Flemming, 1995; Dunne, 2002). During biofilm maturation process, slime cementing and immobilizing the cells (Sutherland, 2001). Glycocalyx in biofilm structure varies in its thickness from 0.2 to 1.0µm (Flemming et al., 1992; Flemming & Wingender, 2001; Branda et al., 2005). Its composition is remarkably flexible and is control by the nature of the biofilm growth environment (Brown & Williams, 1985; Costerton, 1988; Anwar et al., 1990). The fibrous polysaccharides and globular glycoproteins components of the capsule are influenced by the condition applied upon cultivation. Brown & Williams (1985) and Costerton (1988) demonstrated that for the bacterial biofilm it is pivotal importance to maintain plasticity in the composition of its envelope to respond to changes in the growth environment. Such mechanisms enable the pathogenic bacteria surviving an extremely hostile environment when they enter the host

Recent reports suggest that slimes are responsible for the microbial biofilm resistance (Drenkard, 2003; Leid et al., 2005). Glycocalyx may cause alterations in the gaining access of antibacterial molecules to its targets located inside the cells (Anwar et al., 1990; Beech et al., 2005). According to Lewis (2001) glycocalyx matrix provides effective resistance for biofilm bacteria against large molecules such as antimicrobial proteins and their components. This physiological barrier is also effective against smaller peptides-defensins and their analogs (Lewis, 2001). Studies from a number of laboratories have concluded that the glycocalyx acting as a barrier, trapping antibacterial molecules from external environment and isolating the enclose cells from fluctuations in the surrounding environments (Gilbert et al., 1990; Flemming, 1995). The slime changes the charge and the free energy on bacterial surfaces, thereby limiting biocides transport (Hogt et al., 1986). Molecules binding capacity based on estimated number of available carboxyl and hydroxyl groups. The diffusion barrier's role of glycocalyx may also vary according to its soluble state (Siegrist & Gujer, 1985; Hoyle et al., 1992). Glycocalyx have been shown to accumulate antibacterial molecules up to 25% of their weight (Jang et al., 1990; Drenkard, 2003). Extracellular alginate, a slime produced by *Pseudomonas aeruginosa* has been studied for its ability to trap antimicrobial agents. This ability appears to be related to anionic nature of the exopolymer. Cationic substances can thus be retained within the matrix and prevented from acting upon the biofilm bacteria. Alginate has also been shown to bind positively-charged biocides and inhibit their activity (Suci et al., 1994). Also Hentzer et al. (2001) observed that alginate overproduction affects *Pseudomonas aeruginosa* biofilms resistance to antibiotic tobramycin treatment. On the other

(Anderl et al., 2000).

(Anwar et al., 1990).

**3. Glycocalyx** 

The problem of high resistance of biofilm to antimicrobials has not been dissolved yet. In the United States annual cost of eradication of biofilms in hospital conditions exceeded \$ 1 billion per year (Costerton et al., 1995; Archibald & Gaynes, 1997; Potera, 1999). Recent study demonstrated that biofilm resistance has a multifactorial character (Izano et al., 2009; Simões et al., 2009). Analysis of all described data can enable control of detrimental biofilms.

#### **2. Structure of biofilm**

Tolker-Nielsen & Molin (2000) stated that biofilms communities in natural environments have unique architecture although some structural features can be considered universal. Application of scanning confocal laser microscopy performed that biofilms formed on solid surfaces and exposed to a continuous flow of nutrients, are highly hydrated layers composed of microcolonies embedded in an organic polymer matrix of microbial origins (Lawrence et al., 1991; Gilbert et al. 2002a; Czaczyk & Myszka, 2007). Microcolonies are separated by water channels that allow the fluids to flow throughout the biofilm, making the distribution of nutrients and oxygen easer (Lindsay & von Holy, 2006; Shafahi & Vafai, 2009). Moreover, the water channels between the microcolonies provide a means of removing metabolic end products (Davey et al., 2003; Lindsay & von Holy, 2006). This system of nutrients and metabolic end products distribution functions only in periphery regions of biofilms. The cells within biofilms are more tightly packed and have worse access to nutrients and oxygens. Differences in nutrients and oxygen availability within the biofilm structure affect in differences in metabolic activity among the cells. In addition, the cells within biofilms secrete signal molecules that control formation of microcolonies of complicated architecture and diverse function (Parsek & Greenberg, 2005). Structural heterogeneity of biofilm provides an effective barrier that limit penetration of antimicrobial agents throughout the biological layer (Nobile & Mitchell, 2007; Roeder et al., 2010). Kinetic diffusion of antimicrobial compound of relative molecular weight of 100kDa through mature biofilm might be reduced to 60-80% as compared with its action against planktonic cells (DeBeer et al., 1994; Stewart, 1996). Moreover, suspended cells are directly exposure to toxic compounds. Biofilm-associated bacteria are much less permeable to the biocides. DeBeer et al. (1994) observed this phenomenon investigating the rate of penetration of chlorine into *Pseudomonas aeruginosa/Klebsiella pneumoniae* biofilm matrix. Also Suci et al. (1994) noticed transport limitation of ciprofloxacin through *Pseudomonas aeruginosa* biofilm. In this study, during the 21-min exposure, the presence of the antibiotic in periphery region of tested biofilm reached only 20% of ciprofloxacin concentration in the bulk medium (Suci et al., 1994). Gilbert et al. (1989) used perfused biofilm fermentors, in combine with continuous culture and observed that much of resistance of Gram-positive and Gramnegative biofilms was associated with the presence of nutrient-starved microocolonies.

Darouiche et al. (1994) noticed that although the presence of vancomycin in a *Staphylococcus epidermidis* biofilm exceeded bactericidal concentration, it was not sufficient to kill surfacebound bacteria. These authors support the notion that vancomycin resistance of *Staphylococcus epidermidis* biofilm, was not due to limited diffusion of the compound through biological layer, but rather to a reduction in the antimicrobial effect of the drug (Darouiche et al., 1994). Anderl et al. (2000) observed similar effect during investigation of rate of penetration of ampicillin and ciprofloxacin through *Klebsiella pneumoniae* biofilm. In this work, the inability of transport of ampicillin through biofilm was affected by the production of the drug degrading enzyme β-lactamase. Ampicillin was able to penetrate biological layer formed by a β-lactamase–deficient mutant without difficulty. In contrast, ciprofloxacin diffused through *Klebsiella pneumoniae* biofilm without delay. Differences in the effect of penetration of both ciprofloxacin and ampicillin through *Klebsiella pneumoniae* wild-type and β-lactamase-deficient mutant biofilms, suggesting that biofilm resistance is multifactorial (Anderl et al., 2000).
