**3. Glycocalyx**

214 Antimicrobial Agents

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.

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

**2. Structure of biofilm** 

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 (Anwar et al., 1990).

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

Mechanisms Determining Bacterial Biofilm Resistance to Antimicrobial Factors 217

growing or stationary-phase cells inside biofilm matrix. It was characterized by the decreased level of RNA (tRNA and rRNA) synthesis and accumulation of a guanine nucleotide-guanosine 3',5'-bis-pyro-phosphate (ppGpp). The authors demonstrated these effects in *in vitro* experiments by changing a conditions of biofilm maturation process from a nutrient-rich to a minimal ones (Chapman et al., 1993; Wentland et al., 1996; Xu et al. 1998). Similar information concerning the metabolic and growth rate heterogeneity of cells within biofilms has come from studies of cellular enzyme synthesis (Poulsen et al., 1993; Wimpenny et al., 2000). Mitchison (1969) performed that level of enzyme synthesis is influenced by a series of sequenced changes in the particular stage of the bacteria growth cycle. For instant in periphery sphere of bacterial communities where cells are able to proliferate, part of the cellular enzymes are continuously active, and part of them only double at specific point to allow equality in the daughter cells (Mitchison, 1969). Mitchison (1969) also demonstrated that during division stage cellular enzymes may be proportional to cell mass. In slow-growing or stationary-phase bacteria, cellular enzymes synthesis is

Because most of biocides killing metabolically active bacteria, it has been proposed that bacteria at the dormant growth phase in the deeper region of biofilm are less susceptible to antimicrobial agents (Evans, et al., 1989; Toumanen, et al., 1989; Lewis, 2001; Stewart, 2002; Gilbert et al., 2002b; Bulter et al., 2010). These effects were observed in amino acid-starved communities where the cells were able to produce ppGpp (Pissbaro et al., 1990). Evans et al. (1989), Toumanen et al. (1989) and Duguid et al. (1992) investigated growth-rate-related effects upon laboratory conditions for biofilms of *Pseudomonas aeruginosa*, *Escherichia coli* and *Staphyloccocus epidermidis*. The authors stated that the sensitivities of biofilms cells to penicillin, tobramycin and ciprofloxacin increased with the increasing growth rate of examined bacteria. These results suggest that the dormant phase of bacteria biofilm protects the cells from antimicrobial action of antibiotics (Evans et al., 1989; Toumanen et al., 1989; Duguid et al., 1992). The slow growth rate plays also an important role in mediating resistance of *Pseudomonas aeruginosa* biofilm to β-lactams (Tanaka et al., 1999; Alvarez-Ortega et al., 2010). According to Betzner et al. (1990) *Escherichia coli* at the dormant growth phase, activates the RelA-dependent synthesis of ppGpp that limits anabolic processes in cells. The presence of ppGpp suppressed the activity of a major *Escherichia coli* autolysin, SLT that makes the bacteria in non-growing zones of biofilm more tolerant to antibiotic treatment (Betzner et al., 1990). In addition, a mutation in *relA*, a gene coding ppGpp synthase, did not effect the growth rate. The population of *relA* mutants was more sensitive to killing by antibiotics. Rodionov and Ishiguro (1995) stated that ppGpp inhibits peptidoglycans production, that would explain the reduced levels of activity of the bacteria cell wall inhibitors. From a practical standpoint, it would be interesting to examine whether *relA* mutants become also eliminating by other antimicrobial agents that do not target the

In contrast, the Tanaka et al. (1999) researchers also demonstrated that growth rate heterogeneity in *Pseudomonas aeruginosa* biofilm did not limited bactericidal action of fluoroquinolones (Tanaka et al., 1999). In addition, Brooun et al. (2000) observed that *Pseudomonas aeruginosa* in non-growing zones of biofilms are resistant only to part of commercially available antibiotics. For instant, slow growth rate increased resistance of *Pseudomonas aeruginosa* to tetracycline, but did not influence on the resistance of examined

arrested (Sternberg et al., 1999).

cell wall.

hand, Dunne et al. (1993) and Yasuda et al. (1994) noticed that rifampicin, vancomycin, cefotiam and ofloxacin penetrated *Staphylococcus epidermidis* biofilms that formed on the dialysis membrane upon long-term exposure to antibiotics. These results support the notion that limitation of diffusion by glycocalyx matrix cannot always define resistance to antibacterial compounds. Transport limitations of biocides by glycocalyx depends on the present of the adsorption sites in the matrix (Carlson & Silverstein, 1998). After long-term exposure to antibiotics, saturating all possible binding sites in the glycocalyx matrix by the drugs enabled delivering and killing *Staphylococcus epidermidis* and *Staphylococcus aureus*  biofilm (Dunne et al., 1993; Boles & Horswill, 2011).

In addition, adsorption sites within glycocalyx may also serve to anchor exoenzymes from external environments. Such immobilized enzymes are capable to impede the penetration and action of susceptible drugs (Hoyle et al., 1990). Giwercman et al. (1991) found that βlactamases may accumulate in the glycocalyx of *Pseudomonas aeruginosa* giving the whole biofilm population the potential for decreased β-lactam susceptibility. In addition, in mixedspecies biological layers, the synthesis of neutralizing enzymes by one member of the community may confer protection for whole tested sessile populations (Stewart et al., 2000). Exoenzymes trapped within the biofilm matrix, may not only protect the sessile population from the antimicrobial activity of particular agents but also serve as a source of substrates scavaging the metabolites of biocides degradation and elimination (Morton et al., 1998).

Another form of biocides quenching by glycocalyx matrix has been demonstrated by Characklis (1989). The author found that chlorine react with extracellular polysaccharides in the mature biofilms and that this results in disruption of the structure of biological layer. The effect of this process may cause problems especially in industry practice by release of biofilm fragments of pathogenic microorganisms into water phase (Characklis, 1989). On the other hand, under particular circumstances, released biofilm fragments are more sensitive to biocides treatment. Gaylarde and Videla (1994) reported that eradication of biofilm from The North Sea pipelines by biocides caused initially increasing of the sulphate reducing bacterial count in the liquid from 2x102CFU/ml to 3.1x103CFU/ml. Interestingly, 2 hours later, the amount of the sulphate reducing bacterial amount fell to the value of 5,0x101CFU/ml. The study of Gaylarde and Videla (1994) indicated that liberated sessile bacteria are susceptible to antimicrobials agents.
