**4. Metabolic and growth rate heterogeneity**

Differences in nutrients and oxygen availability within biofilm affect in differences in growth rate and metabolic activity of bacteria. Wentland et al. (1996) and Xu et al. (1998) used fluorescent probes and reporter genes to visualized patterns of bacterial growth and cells metabolic activities in biofilm. Different concentrations of the key metabolic substrates and products within biofilm proved that surface-bound communities contain cells at all phases of bacterial growth and cells at the different activity levels (Stewart, 2002). This leads to microbial population heterogeneity. The problem occurs both in single-species and mixed-species bacterial biofilms (Xu et al., 2000). Better access to nutrients and oxygen in the periphery region of biofilm promotes metabolic activity of cells. In this part of biological layer the bacteria are able to proliferate. In contrast, in the deeper part of biofilm the metabolic potential of bacteria is limited by the worse diffusion process of nutrients (Senior, 2004). Chapman et al. (1993), Wentland et al. (1996) and Xu et al. (1998) identified slow-

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* 

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

Differences in nutrients and oxygen availability within biofilm affect in differences in growth rate and metabolic activity of bacteria. Wentland et al. (1996) and Xu et al. (1998) used fluorescent probes and reporter genes to visualized patterns of bacterial growth and cells metabolic activities in biofilm. Different concentrations of the key metabolic substrates and products within biofilm proved that surface-bound communities contain cells at all phases of bacterial growth and cells at the different activity levels (Stewart, 2002). This leads to microbial population heterogeneity. The problem occurs both in single-species and mixed-species bacterial biofilms (Xu et al., 2000). Better access to nutrients and oxygen in the periphery region of biofilm promotes metabolic activity of cells. In this part of biological layer the bacteria are able to proliferate. In contrast, in the deeper part of biofilm the metabolic potential of bacteria is limited by the worse diffusion process of nutrients (Senior, 2004). Chapman et al. (1993), Wentland et al. (1996) and Xu et al. (1998) identified slow-

biofilm (Dunne et al., 1993; Boles & Horswill, 2011).

bacteria are susceptible to antimicrobials agents.

**4. Metabolic and growth rate heterogeneity** 

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 arrested (Sternberg et al., 1999).

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 cell wall.

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

Mechanisms Determining Bacterial Biofilm Resistance to Antimicrobial Factors 219

2001). It is also important to emphasize that persisters are not simply non-growing cells in stationary culture. Keren et al. (2004b) noticed that fluoroquinolones and mitomycin C eliminated the bulk of *Escherichia coli* biofilm and left 1-10% intact persisters. From a medical perspective, the presence of persisters in biofilm is problematic. In planktonic population, a fraction of persisters that survive antibiotic action, is eliminate by the immune system (Hoyle et al., 1990; del Pozo & Patel, 2007). Biofilm persisters are protected from the immune system by glycocalyx matrix. In sessile bacterial population persisters are responsible for biofilm regrowth when the antibiotics concentration decrease or when the treatment is

The formation of persisters is dependent on the bacteria growth state (Lewis, 2007). Keren et al. (2004b) performed a test for measuring a rate of persisters after adding spent stationary medium to early log cells of *Escherichia coli* and *Pseudomonas aeruginosa*. Authors noticed that spent medium did not increase presisters of examined bacteria. In addition, persisters are rapidly lost if a stationary population is diluted (Keren et al., 2004b). The work of Keren et al. (2004b) demonstrated that formation of persisters dependent on the level of bacterial

Falla & Chopra (1998) suggested that presisters are not mutant, but rather dormant variant of the wild type cells. Keren et al (2004a) observed that repeated reinoculation maintaining the cells in an log phase affects to a complete loss of persisters in *Escherichia coli* population. The work of Keren et al. (2004a) suggest that persisters are not formed in response to bactericidal agents exposure. According to Lewis (2005) persisters representing specialized survival cells whose formation is controlled by the growth stage of the bacterial culture. Moreover persisters are the cells with forfeiting rapid propagation system which ensures

The tolerance of persisters to antibiotics works, not by preventing bactericidal binding, but by interfering with the lethal action of the compounds. Lewis (2007) postulated that persisters produce multidrug resistance protein (MDR protein) that shut down the antibiotic targets. It is worth point out that bactericidal properties of antibiotics occur by corrupting the target function of cells, rather than by inhibiting it. For instant, erythromycin blocks protein synthesis (Menninger & Otto, 1982). Streptomycin leads translational misreading, that produces truncated toxic peptides, causing the cell death. Shutting down the ribosome in a persister cells would produce tolerance to bactericidal aminoglycosides (Kornder, 2002; Lewis, 2005). According to Lewis (2005) persister protein can shut down most of antibiotics

The phenomenon of tolerance of persisters to antimicrobial agents has also been linked with programmed cell death (PCD) system (Webb et al., 2003; Lewis, 2005; Lewis, 2007). Lewis (2000) suggests that actions of antimicrobial compounds are not responsible for cell death, but that they lead to cell damage that indirectly trigger PCD. The most common observation of PCD in bacterial biofilm is autolysis of cells. Autolysis is a self-digestion of the cell wall by peptidoglycan hydrolases termed autolysin (Shockman et al., 1996). Both production and hydrolysis of peptidoglycan are essential for creating the cell wall, therefore some autolysisns are the part of normal bacteria growth activity in biofilm (Lewis, 2000). Because a bactericidal compound that diffuses throughout biofilm would not able to eliminate whole sessile population, Lewis (2005) proposed that persisters have a defective PCD mechanism.

survival of cells in presence of lethal doses of antimicrobial factors (Lewis, 2005).

targets, formatting the resistant, dormant persister cells.

discontinued (Hoyle et al., 1990; Lewis, 2000).

metabolic activity.

bacteria to tobramycin. In this experiment the susceptibility of majority of *Pseudomonas aeruginosa* cells within biofilms were not much different from what is stated for planktonic bacteria. The greater parts of *Pseudomonas aeruginosa* biofilm were killed by clinically achievable range of antibiotics concentrations (about 5µg/mg) (Brooun et al., 2000). Brooun et al. (2000) also reported that after biofilm maturation, further increase in the antibiotic concentration had no effect on killing of *Pseudomonas aeruginosa* biofilm. The results of Tanaka et al. (1999) and Brooun et al. (2000) reinforced the idea that under the particular circumstances metabolic and growth rate heterogeneity may only contribute to increasing tolerant of bacterial biofilms to antimicrobials agents. Brooun et al. (2000) also stated that only a small fractions of bacteria are responsible for the very high level of resistance of *Pseudomonas aeruginosa* biofilms. According to Lewis (2000) the greater number of bacteria in biofilms are usually not more resistance to killing than free-floating cells and die more rapidly after treatment with a lethal dose of antibiotics. Under particular circumstances bacteria in non-growing zones of biofilms are preserved by the presence of biocides that only inhibits their growth (Lewis, 2000; Singh et al., 2006).

In biofilms metabolic activities of bacteria are controlled by oxygen availability. Biofilms of *Pseudomonas aeruginosa* grow in a gaseous environment of pure oxygen were killed by ciprofloxacin and tobramycin antibiotics (Walters et al., 2003). In contrast, Tresse et al. (1995) reported that reduction of oxygen availability enhanced of antibiotic resistance of agarentrapped *Escherichia coli*. Also Hill et al. (2005) observed that anaerobically biofilm-grown isolates of *Pseudomonas aeruginosa* were significantly less susceptible for meropenem, tobramycin and ciprofloxacin treatments. According to Yoon et al. (2002) under strict anaerobic conditions, bacteria form robust biofilm, and that specific gene products were essential to develop such anaerobic biofilms. Metabolic and phenotypic changes under anaerobic conditions lead to increased levels of biocide resistance of bacterial biofilms. Sauer et al. (2002) based on analysis of protein patterns of *Pseudomonas aeruginosa* mature biofilm, demonstrated that a large part of biological layer is exposure to oxygen limitation.
