**5. Persister phenomena**

Bacterial biofilms include persisters, cells that neither grow nor die during exposure to bactericidal agents, thus exhibit multidrug tolerance (MDT) (Lewis, 2005; Cheng & Hardwick, 2007; Lewis, 2008). While measuring a dose-response of a *Pseudomonas aeruginosa*  biofilm to ofloxacin, Brooun et al. (2000) observed that a fraction of persister cells was not killed even by very high doses of the antibiotics. These cells appeared invulnerable in contrast to fairly sensitive *Pseudomonas aeruginosa* biofilm (Brooun, et al., 2000). Also in *Escherichia coli*, increasing concentration of ciprofloxacin or imipenem leaded to an initial 100- to 1000-fold reduce of live cells of a biofilm, remaining small population insensitive persisters to further increases in drug concentration (Ashby et al., 1994). These data suggest that most of the cells in the biofilm are as susceptible to bactericidal agents as planktonic bacteria. Only the persister fraction is responsible for survival of the whole sessile population (Ashby et al., 1994; Brooun et al., 2000). Also Spoering & Lewis (2001) noticed that stationary phase planktonic and sessile bacteria were tolerant to anitimicrobials at similar level and that resistance of stationary phase and biofilm bacteria was dependent on the persister fraction. In addition, the increased resistance to killing of biofilm is due to high level of persisters produced by stationary phase bacteria inside biofilm (Spoering & Lewis,

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

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.

Bacterial biofilms include persisters, cells that neither grow nor die during exposure to bactericidal agents, thus exhibit multidrug tolerance (MDT) (Lewis, 2005; Cheng & Hardwick, 2007; Lewis, 2008). While measuring a dose-response of a *Pseudomonas aeruginosa*  biofilm to ofloxacin, Brooun et al. (2000) observed that a fraction of persister cells was not killed even by very high doses of the antibiotics. These cells appeared invulnerable in contrast to fairly sensitive *Pseudomonas aeruginosa* biofilm (Brooun, et al., 2000). Also in *Escherichia coli*, increasing concentration of ciprofloxacin or imipenem leaded to an initial 100- to 1000-fold reduce of live cells of a biofilm, remaining small population insensitive persisters to further increases in drug concentration (Ashby et al., 1994). These data suggest that most of the cells in the biofilm are as susceptible to bactericidal agents as planktonic bacteria. Only the persister fraction is responsible for survival of the whole sessile population (Ashby et al., 1994; Brooun et al., 2000). Also Spoering & Lewis (2001) noticed that stationary phase planktonic and sessile bacteria were tolerant to anitimicrobials at similar level and that resistance of stationary phase and biofilm bacteria was dependent on the persister fraction. In addition, the increased resistance to killing of biofilm is due to high level of persisters produced by stationary phase bacteria inside biofilm (Spoering & Lewis,

only inhibits their growth (Lewis, 2000; Singh et al., 2006).

**5. Persister phenomena** 

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

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 metabolic activity.

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

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 targets, formatting the resistant, dormant persister cells.

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.

2007).

Mechanisms Determining Bacterial Biofilm Resistance to Antimicrobial Factors 221

*aeruginosa* biofilm with a wild-type structure: loosely packed biomass with a mushroom appearance with notable amount of extracellular polysaccharides and water channel traversing the entire the biological layer. Whereas, signal molecules-negative mutants of *Pseudomonas aeruginosa*, *Brukholderia cenocepacia* and *Aeromonas hydrophila* showed defects in the late stages of biofilm maturation and thus were unable to form biofilms with the wildtype architecture (Huber et al., 2001; Lynch et al., 2002; Steidla et al., 2002; Labbate et al.,

Because heterogonous architecture of biofilms and the synthesis of degradative enzymes deactivate biocides, it seems reasonable to speculate that biofilm antimicrobial agents resistance could also be influenced by *quorum sensing* system. Moreover, coordinated expression of *quorum sensing*-mediated phenotypes is crucial in cells migration to a more suitable environment/better nutrient supply and in adaptation to a new modes of growth, which may afford protection from deleterious environment (Whitehead et al., 2001; Abee et al., 2011). However, to date *quorum sensing* system as factor decreasing the biofilm susceptibility to antimicrobial agents has been studied in a limited number of strains. Davies et al. (1998) and Hassett et al. (1999) reported that exposure of *quorum sensing*-negative mutant biofilms to the antimicrobial agents SDS and hydrogen peroxide caused detachment and dispersion of surface-anchored bacteria. In addition, Hassett et al. (1999) have reported that cell-to-cell signaling mechanism in *Pseudomonas aeruginosa* controls the expression of the catalase and superoxide dismutase genes and mediates biofilms resistance to hydrogen peroxide. According Shih & Hoang (2002) *quorum sensing*-deficient mutant biofilms susceptibility to kanamycin correlated with thinner biofilm formation and lower EPS production. Above results provide evidences that biofilm respond directly or indirectly to

Interestingly, resent reports have also demonstrated chelating properties of cell-to-cell signals (Schertzer, et al. 2009). Such non-signaling features were stated for *Pseudomonas aeruginosa quorum sensing* molecules. Weinberg (2008) examined multiple meaning of *quorum sensing* system in mixed-species bacterial population. The author performed that *Pseudomonas aeruginosa* may kills competing bacteria in the growth environment by hijacking the bacteria's iron stores using 2-heptyl-3-hydroxy-4-quinolone signal. According to Weinberg (2008) *Pseudomonas* quinolone signal is a high affinity iron chelator. The ability of signal molecules to trap external positive-charged compounds is similar to antimicrobial action of glycocalyx matrix (Schertzer, et al, 2009). However, this role of cell-to-cell signal

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

molecules to biofilm resistance properties needs to be examined in more detail.

environmental stress via a *quorum sensing* system.

**7. General stress response** 

The work of Moyed & Bertrand (1983) supported this statement. Moyed & Bertrand (1983) discovered in *Escherichia coli* a toxin-antitoxin system (*hipAB* locus) that has a potential of both killing the cells and improving survival after exposure to lethal doses of antimicrobial factors. The inactivation of the toxin-antitoxin systems by insertional elements or by mutation, induced defects in PCD system in *Escherichia coli* and made the bacteria more susceptible to antimicrobial agents (Han et al., 2011).
