Innovative Strategies to Overcome Biofilm Resistance

We review the recent literature concerning the efficiency of antimicrobial photodynamic inactivation toward various microbial species in planktonic and biofilm cultures. The review is mainly focused on biofilm-growing microrganisms because this form of growth poses a threat to chronically infected or immunocompromised patients and is difficult to eradicate from medical devices. We discuss the biofilm formation process and mechanisms of its increased resistance to various antimicrobials. We present, based on data in the literature, strategies for overcoming the problem of biofilm resistance. Factors that have potential for use in increasing the efficiency of the killing of biofilm-forming bacteria include plant extracts, enzymes that disturb the biofilm structure, and other nonenzymatic molecules. We propose combining antimicrobial photodynamic therapy with various antimicrobial and antibiofilm approaches to obtain a synergistic effect to permit efficient microbial growth control at low photosensitizer doses.


Introduction
Photodynamic therapy dates to the time of the pharaohs and ancient Romans and Greeks, for whom the connection between the sun and health was obvious. Until the 19th century, heliotherapy was the only known form of phototherapy [1]. Heliotherapy was used in thermal stations to cure tuberculosis and to treat ulcers or other skin diseases [2]. e 20th century brought signi�cant developments in phototherapy, particularly in photodynamic therapy (PDT) directed against cancer as well as photodynamic inactivation (PDI) of microorganisms, also known as antimicrobial PDT (APDT). PDT has gained clinical acceptance, and many clinical trials are being conducted, while APDT is in its infancy. As antibiotic therapies become less effective because of increasing microbial resistance to antibiotics, alternative methods such as APDT for �ghting infectious diseases are urgently needed. Microbial bio�lms cause a large number of chronic infections that are not susceptible to traditional antibiotic treatment [3,4]. Bio�lm-forming microbes are held together by a self-produced matrix that consists of polysaccharides, proteins and extracellular DNA [5,6].

�. �io�lm� �tructure, �iolog�, and Treatment Problems
A microbial bio�lm is de�ned as a structured community of bacterial cells enclosed in a self-produced polymeric matrix that is adherent to an inert or living surface [4,7]. e matrix contains polysaccharides, proteins, and extracellular microbial DNA, and the bio�lm can consist of one or more microbial (bacterial or fungal) species [5,8]. e matrix is important because it provides structural stability and protection to the bio�lm against adverse environmental conditions, for example, host immunological system and antimicrobial agents [6,9]. Bio�lm-growing microorganisms cause chronic infections which share clinical characteristics, like persistent in�ammation and tissue damage [3]. A large number of chronic bacterial infections involve bacterial bio�lms, making these infections very difficult to be eradicated by conventional antibiotic therapy [4]. Bio�lm formation also causes a multitude of problems in the medical �eld, particularly in association with prosthetic devices such as indwelling catheters and endotracheal tubes [10]. Bio�lms can form on inanimate surface materials such as the inert surfaces of medical devices, catheters, and contact lenses or living tissues, as in endocardium, wounds, and the epithelium of the lungs, particularly in cystic �brosis patients [8,11,12]. Microbial antigens stimulate the production of antibodies, which cannot effectively kill bacteria within the bio�lm and may cause immune complex damage to surrounding tissues [13]. Regardless of the presence of excellent cellular and humoral immune reactions, host defense mechanisms are rarely able to resolve bio�lm infections [14]. e symptoms caused by the release of planktonic cells from the bio�lm can be treated by antibiotic therapy, but the bio�lm remains unaffected [15]. us, bio�lm infection symptoms are recurrent even aer several antibiotic therapy cycles, and the only effective means of eradicating the cause of the infection is the removal of the implanted device or the surgical removal of the bio�lm that has formed on live tissue [16]. Bio�lmgrowing bacteria differ from planktonic bacteria with respect to their genetic and biochemical properties. Bio�lm-forming bacteria coaggregate with each other and with multiple partners and form coordinated groups attached to an inert or living surface; they surround themselves with polymer matrix, communicate effectively via quorum sensing mechanisms, and express low metabolic activity limiting the impact of conventional antimicrobials acting against actively metabolizing cells [4,7,12].
�.�. �io�lm �ormation. Bio�lm formation can be divided into three main stages: early, intermediate, and mature [17]. During the early stage, planktonic cells swim along the surface oen using their �agella mode of movement or they can be transferred passively with the body �uids ( Figure 2). Next, the contact between microorganisms and a surface is made, resulting in the formation of a monolayer of cells [18][19][20]. At this stage, the bacteria are still susceptible to antibiotics, and perioperative antibiotic prophylaxis can be critical for successful treatment [6,9]. e importance of the �rst attachment step was con�rmed by experiments with surface attachment-defective (sad) mutant strains of Pseudomonas aeruginosa, which are unable to form bio�lms [21]. e next step involves irreversible binding to the surface, multiplication of the microorganisms, and the formation of microcolonies [6,9]. During this stage, the polymer matrix is produced around the microcolonies and generally consists of a mixture of polymeric compounds, primarily polysaccharides (the matrix contributes 50%-90% of the organic matter in bio�lms) [22,23]. Studies on Candida albicans have demonstrated that during the third stage (the maturation phase), the amount of extracellular material increases with incubation time until the yeast communities are completely encased within the material [17]. e matrix consists mainly of water, which can be bounded within the capsules of microbial cells or can exist as a solvent [24]. Apart from water and microbial cells, the bio�lm matrix is a very complex material. e bio�lm matrix consists of polymers secreted by microorganisms within the bio�lm, absorbed nutrients and metabolites, and cell lysis products; therefore, all major classes of macromolecules (proteins, polysaccharides, and nucleic acids) are present in addition to peptidoglycan, lipids, phospholipids, and other cell components [25][26][27]. e third step of bio�lm formation is the formation of a mature community with mushroom-shaped microcolonies [3]. During this stage, the bio�lm structure can be disrupted, and microbial cells can be liberated and transferred onto another location/surface, causing expansion of the infection [6,9]. Bio�lm formation is regulated at different stages through diverse mechanisms, among which the best studied is quorum sensing (QS) [28][29][30][31]. e QS mechanism involves the production, release, and detection of chemical signaling molecules, which permit communication between microbial cells. e QS process regulates gene expression in a celldensity-dependent manner; for bio�lm production, the genes involved in bio�lm formation and maturation are activated at a critical population density [32][33][34]. ere are three well-de�ned groups of signaling QS molecules in bacteria: oligopeptides, acyl homoserine lactones (AHLs), and autoinducer-2 (AI-2) [34]. Gram-positive bacteria predominately use oligopeptides as a communication molecule, and AHLs are speci�c for Gram-negative bacteria [35,36]. AI-2 is reported to be a universal signaling molecule that is used for both interspecies and intraspecies communication [34]. Boles and Horswill proposed that the Staphylococcus aureus agr quorum sensing system controls not only the switch between planktonic and bio�lm growth but also the mechanism of the dispersal of cells from an established bio�lm [37]. Moreover, results from our research group indicate that agr polymorphism could impact bio�lm formation and directly in�uence bacterial susceptibility to photoinactivation (data not shown).
�.�. �io�lm �esistance. Infections caused by bio�lm-forming bacteria are oen di�cult to treat. Bio�lm formation almost always leads to a large increase in resistance to antimicrobial agents (up to 1000-fold decrease in susceptibility) in comparison with planktonic cultures grown in conventional liquid media [4,7]. A few mechanisms of bio�lm resistance to antibiotics have been proposed. e �rst proposed mechanism involves the matrix, which represents a physical and chemical barrier to antibiotics. Ciofu et al. [38] demonstrated that the resistance of P. aeruginosa bio�lms to antimicrobial treatment is related to mucoidy. Mucoid bio�lms were up to 1000 times more resistant to tobramycin than nonmucoid bio�lms, in spite of similar planktonic MICs [38]. Anderl et al. demonstrated that cipro�oxacin and chloride ion could penetrate a wild-type Klebsiella pneumoniae bio�lm, while ampicillin could not [39]. By contrast, ampicillin rapidly penetrated a -lactamase-de�cient K. pneumoniae bio�lm. e authors assumed that the bio�lm matrix was not an inherent mechanical barrier to solute mobility and that ampicillin failed to penetrate the bio�lm because it was deactivated by the wild-type bio�lm at a faster rate than it could diffuse into the �lm [40]. Jefferson et al. suggested that even though the matrix may not inhibit the penetration of antibiotics, it may retard the rate of penetration enough to induce the expression of genes within the bio�lm that mediates resistance [41]. A second hypothesis to explain reduced bio�lm susceptibility to antibiotics concerns the metabolic state of microorganisms in a bio�lm. Some of the cells located deep inside the bio�lm structure experience nutrient limitation and therefore exist in a slow-growing or starved state [42]. Nutrient-depleted zones within the bio�lm can result in a stationary phase-like dormancy that may in�uence the general resistance of bio�lms to antibiotics. Walters et al. demonstrated that oxygen penetrated from 50 to 90 m into colony bio�lms formed by P. aeruginosa and that the antibiotic action is focused near the air-bio-�lm interface [43]. is study also showed that oxygen limitation has a role in antibiotic resistance [43]. Slow-growing or nongrowing cells are not very susceptible to many antimicrobial agents because the cells divide infrequently and antibiotics that are active against dividing cells (such as beta-lactams) are not effective. e third hypothesis involves genetic adaptation to different conditions. e mutation frequency of a bio�lm-growing microorganism is signi�cantly higher than that of its planktonic form; for P. aeruginosa, up to a 105-fold increase in mutability has been observed [44]. A recent study by Ma and Bryers demonstrated that donor populations in bio�lms (containing a plasmid with a kanamycin resistance gene) exposed to a sublethal dose of kanamycin exhibited an up to tenfold enhancement in the transfer efficiency of the plasmid [45]. At least some of the cells in a bio�lm are likely to adopt a distinct phenotype that is not a response to nutrient limitation but a biologically programmed response to growth on a surface [4,7]. Several genes are involved in bio�lm formation and some of the genes are exclusively expressed in bio�lm-growing microorganisms [46,47].
All published results indicate that a reduction in the efficiency of photodynamic treatment occurs when PDI is applied to bio�lm-related experimental models. us, it is necessary to identify factors that disrupt bio�lm structure or affect bio�lm formation.

Antimicrobial Photodynamic Therapy
Photodynamic therapy consists of three major components: light, a chemical molecule known as a photosensitizer, and oxygen. e photosensitizer (PS) can be excited by absorbing a certain amount of energy from the light. e excitation occurs when the wavelength range of the light overlaps with absorbance spectrum of the photosensitizer. Aer excitation, photosensitizers usually form a long-lived triplet-excited state, from which energy can be transferred to biomolecules or directly to molecular oxygen, depending on the reaction type ( Figure 1). Type I ( Figure 1) reactions involve electron transfer from the triplet state PS to a substrate, for example, unsaturated membrane phospholipids or aminolipids, leading to the production of lipid-derived radicals or hydroxyl radicals (HO • ) derived from water. ese radicals can combine or react with other biomolecules and oxygen to yield hydrogen peroxide, causing lipid peroxidation or leading to the production of reactive oxygen species that can cause cellular damage and cell death [48]. Type II (Figure 1 ground-state (triplet) molecular oxygen to produce excited singlet-state oxygen, which is a very reactive species with the ability to oxidize biomolecules in the cell such as proteins, nucleic acid, and lipids, causing cell damage and death [48]. Both mechanisms can operate in the cell simultaneously, but type II is generally considered the major APDT pathway [49]. ere are two major types of cellular damage: DNA damage and the destruction of cellular membranes and organelles. Because the cell is protected by DNA repair systems, DNA damage may not be the main cause of microbial cell death. A large portion of the microbicidal effect of APDT may be due to the disruption of proteins involved in transport and membrane structure and the leakage of cellular contents [49].
Recent studies have shown that the antimicrobial effect can be obtained with the use of photosensitizers belonging to different chemical groups. e most studied PSs are phenothiazine dyes (methylene blue (MB) and toluidine blue O (TBO)), porphyrin and its derivatives, fullerenes, and cyanines and its derivatives (Table 1). More studies have been conducted of forms of microbial growth other than planktonic growth (Table 2). e problem of several chronic microbial infections is now known to be inseparable from bio�lm formation by pathogens. us, in vitro studies have concentrated more on bio�lm models as well as in vivo models, particularly rat and mouse models of infected wounds ( Table 2). As we have discussed previously, when studying the use of photoinactivation in bio�lm-related models, considered as an important factor. Because APDT is related to the production of toxic radicals such as singlet oxygen, the quantity of toxic radicals that are generated should increase as the irradiation time increases [55]. However, our results show no such correlation; for photodynamic inactivation, the light dose is important, not the irradiation time. albicans) [60]. ey used fullerene derivatives and white light to illuminate the cells and were able to reduce the growth of all tested microorganisms by 3 to 5 log 10 units, depending on the microorganism and fullerene derivative. e most efficient was the BF2 derivative [60]. Park [62]. A signi�cant decrease in bio�lm density was observed, and the majority of the cells within the bio�lm were nonviable when 100 M TMP and 10 min of irradiation (mercury vapor lamp, 220-240 J/cm 2 ) were used. Moreover, the use of 225 M TMP and the same light dose resulted in almost complete disruption and clearance of the studied bio�lm [62]. e effect of ZnPc-mediated APDT on yeast bio�lms (C. albicans, non-albicans Candida species and non-Candida species) was studied by Junqueira et al. [63]. A gallium-aluminum-arsenide (GaAlAs) laser was used as the light source with the photosensitizer ZnPc at a concentration of 0.25 mg/mL. In all of the studied species, APDT caused reductions in CFU/mL values compared to the control group, but the levels of reduction ranged from 0.33 to 0.85 log 10 for the various fungal species. e Candida spp. that were most resistant to APDT were C. albicans, C. glabrata, C. norvegensis, C. krusei, and C. lusitaniae (reduction <0.5 log 10 ). e non-Candida pathogens T. mucoides and K. ohmeri were inactivated by APDT, with reductions of 0.85 log 10 and 0.84 log 10 , respectively [63]. Biel et al. demonstrated that MB-mediated APDT is highly effective in the photoeradication of multispecies bacterial bio�lms (multidrug-resistant P. aeruginosa and MRSA) [64,65]. A signi�cant decrease in CFU/mL (>6 log 10 units) was achieved when 300 g/mL MB and a light dose of 60 J/cm 2 (diode laser, 664 nm) were used. e reduction was >7 log 10 units when 500 g/mL MB and two light doses of 55 J/cm 2 separated by a 5-minute break were used [64,65]. Meire et al. observed a statistically signi�cant 1.9 log 10 reduction in the viable counts of E. faecalis bio�lms treated with 10 mg/mL MB and exposed to a so laser of an output power of 75 mW (660 nm) for 2 min [66]. For bio�lm-based cultures, much higher PS concentrations are required to obtain an APDT killing efficiency comparable to that observed for planktonic cultures. ese higher concentrations may be potentially toxic for eukaryotic cells. us, it is of great importance to propose a strategy to decrease the PS concentrations used in vivo to further facilitate the application of APDT for the treatment of infections in humans and animals. Light parameters such as total light dose, beside the PS concentration, play an important role in APDT efficacy. In general, photoinactivation of microbial cells is dependent on light dose delivered to the sample and its efficacy is increasing with increasing light dose, considering particular light source of speci�c power density. In fact, lower PS concentration can be substituted by higher light doses, thus giving good opportunity to improve selectivity of APDT in potential clinical applications. e complexity of biological effects of irradiation of microbial cells as well as molecular responses to a PS itself (light-independent effects) demand individual optimization protocols for each reaction.

Recent In Vivo Studies.
Because APDT is an alternative and promising method for treating patients, in vivo studies are being conducted. Park et al. performed experiments on bio�lms in an in vivo mouse model [61]. ey demonstrated that Ce6-PDT treatment signi�cantly reduced bio�lm formation by S. aureus when treated with 10 M Ce6 and 10 J/cm 2 of laser light. Because the S. aureus strain used in the study is bioluminescent, a bioluminescent in vivo imaging system (IVIS) was used. e group examined the effect of Ce6-mediated PDT on in vivo bacterial growth in a mouse model of skin infection with S. aureus, and the reduction in the intensity of bioluminescence was observed immediately aer PDT. Moreover, on the 5th day aer infection, the signal was almost undetectable in mice treated with Ce6-mediated PDT [61]. Dai et al. reported that a new MBmediated APDT effectively treated C. albicans skin abrasion infections in mice [67]. In that study, a combination of 400 M NMB and 78 J/cm 2 red light (Luma-Care lamp) was used to perform APDT 30 min aer fungal inoculation, which resulted in a signi�cant decrease in fungal luminescence (only few pixels corresponding to microbes could be observed immediately aer APDT). Moreover, no signi�cant reoccurrence of infection was observed at 24 h aer APDT [67]. In an in vivo study by Hashimoto et al., APDT with 10 M hypocrellin B with lanthanide ions (HB:La +3 ) and a light dose of 24 J/cm 2 (blue and red LED) reduced the number of P. aeruginosa in burn wounds, delaying bacteremia and decreasing bacterial levels in blood by 2-3 log 10 compared to an untreated group [68]. Moreover, mice survival was increased at 24 h [68]. Fullerene-mediated APDT against P. mirabilis and P. aeruginosa wound infection was investigated by Lu et al. [69]. For P. mirabilis infection, 1 mM fullerenes (B6) and illumination with white light yielded a reduction of 96% aer 180 J/cm 2 , which resulted in a highly signi�cant increase in mouse survival of 82% [69]. For P. aeruginosa, the treatment gave a maximum reduction of 95%, but there was no bene�cial effect on mouse survival (100% of the mice died within 3 days of infection) [69].
e infectious diseases that can be treated with APDT are mostly found in bio�lm form, emphasizing the importance of focusing on the bio�lm and its eradication, mass reduction, cell number reduction, and loss of viability. Photodynamic inactivation is a promising treatment option for eradication of microbial infections� however, as a bio�lm treatment strategy, it has to overcome the obstacle of exopolymer matrix constituting a physical barrier for the photosensitizers as well as light.
Bio�lm penetration by biocides or antibiotics is typically strongly hindered. To increase the efficiency of new treatment strategies against bacterial and fungal infections, factors that lead to bio�lm growth inhibition, bio�lm disruption, or bio�lm eradication are being sought. ese factors could include enzymes, sodium salts, metal nanoparticles, antibiotics, acids, chitosan derivatives, or plant extracts. All of these factors in�uence bio�lm structure via various mechanisms and with different efficiencies. (Japanese knotweed) extracts inhibited Propionibacterium acnes bio�lm formation by 64.8%, 98.5%, and 99.2%, respectively [70]. Moreover, active compounds within the extracts were identi�ed and tested against three P. acnes strains. e most effective compound was resveratrol from P. cuspidatum, which reduced bio�lm formation by 80% for each strain at a concentration of 0.32% (w/v). Icariin extracted from E. brevicornum reduced bio�lm formation by 40%-70% at concentrations of 0.01%-0.08% (w/v). e antibio�lm activity of salidroside (0.02%-0.25% concentration) extracted from R. crenulata was strain dependent and yielded a bio�lm reduction of 40% for P. acnes LMG 16711 and less than 20% for other tested strains.
Melia dubia (bead tree) bark extracts were examined by Ravichandiran et al.; at a concentration of 30 mg/mL, these extracts reduced E. coli bio�lm formation by 84% and inhibited virulence factors such as hemolysins by 20% [71]. Bacterial swarming regulated by quorum sensing mechanisms (QS) was inhibited by 75%, resulting in decreased bio�lm expansion [71]. Similar results were reported by Issac Abraham et al. concerning Capparis spinosa (caper bush) extract. At a concentration of 2 mg/mL, an inhibition of E. coli bio�lm formation by 73% was observed [72]. For the pathogens Serratia marcescens, P. aeruginosa, and P. mirabilis, bio�lm biomass was reduced by 79%, 75%, and 70%, respectively. Moreover, the mature bio�lm structure was disrupted for all of the studied pathogens. Furthermore, the addition of C. spinosa extract (100 g/mL) to a bacterial culture resulted in swimming and swarming inhibition [72]. For Lagerstroemia speciosa (giant crape myrtle) extract, 83% bio�lm inhibition was achieved at a concentration of 10 mg/ mL [73]. Moreover, the anti-QS activity of the L. speciosa extract affected tolerance to tobramycin and reduced the expression of virulence factors such as LasA protease, LasB elastase, and pyoverdin [73].
e inhibition of bio�lm formation is not the only antibio�lm strategy. Taganna et al. reported that a Terminalia catappa (bengal almond) extract at sub-MIC concentrations (500 g/mL and 1 mg/mL) stimulated bio�lm formation; P. aeruginosa bio�lm formation increased by 220% [74]. Despite increased bio�lm formation, the T. catappa extract disrupted bio�lm structure, and the administration of 1% SDS reduced the bio�lm by 46%. Moreover, anti-QS activity and a 50% reduction of LasA expression were observed when the T. catappa extract was applied [74].
Highly effective antibio�lm activity was observed for fresh Allium sativum extract (fresh garlic extract, FGE). Fourfold treatment of a P. aeruginosa bio�lm with FGE (24 hrs interval) resulted in bio�lm reduction by 6 log 10 units. Moreover, in vivo prophylactic treatment of a mouse model of kidney infection with FGE (35 mg/mL) for 14 days resulted in a 3 log 10 unit decrease in the bacterial load on the �h day aer infection compared to untreated animals. In addition, FGE protected renal tissue from bacterial adherence and resulted in a milder in�ammatory response and histopathological changes of infected tissues. Fresh garlic extract inhibited P. aeruginosa virulence factors such as pyoverdin, hemolysin, and phospholipase C. Moreover, killing efficacy and phagocytic uptake of bacteria by peritoneal macrophages were enhanced by garlic extract administration [75].
Next, Carneiro et al. [77] tested sub-MIC concentrations of casbane diterpene (CS) extracted from Croton nepetaefolius bark against two Gram-positive bacteria (S. aureus and S. epidermidis), �ve Gram-negative bacteria (Pseudomonas �uorescens, P. aeruginosa, Klebsiella oxytoca, K. pneumoniae, and E. coli), and three yeasts (Candida tropicalis, C. albicans, and C. glabrata). S. aureus and S. epidermidis bio�lms were signi�cantly disrupted when CS was applied (125 g/mL and 250 g/mL, resp.). Among Gram-negative bacteria, K. oxytoca bio�lms formation were not affected by CS, and K. pneumoniae bio�lms were reduced by 45%. Administration of CS at a concentration of 125 g/mL caused complete inhibition of P. �uorescens bio�lms (by 80%). However, lower concentrations of CS supported P. aeruginosa bio�lm formation. Similar results were obtained for E. coli. e authors explained the observed phenomena by the enhanced production of exopolysaccharides due to the stress induced by the presence of CS in the culture. Casbane diterpene activity against C. albicans and C. tropicalis was observed, reducing bio�lm formation by 50% (at concentrations of 62.5 g/mL and 15.6 g/mL, resp.) [77]. Candida bio�lm formation was inhibited more effectively by Boesenbergia pandurata (�ngerroot) oil [78]; bio�lms were reduced by 63% to 98% when sub-MIC volumes (from 4 L/mL to 32 L/mL) were used. Moreover, a signi�cant disruption of mature bio�lms was observed when similar volumes of the tested oil were applied [78].
ese data con�rm that plant extracts have anti-QS, antiseptic, and antivirulence factor properties and can easily inhibit bio�lm formation as well as disrupt the mature bio�lm structure. us, plant extracts in combination with other antimicrobial strategies such as antibiotics or photodynamic inactivation could provide an effective bactericidal tool for the treatment of various bacterial and yeast infections.

Bio�lm-Disrupting En�ymes.
Because the bio�lm matrix is composed of DNA, proteins, and extracellular polysaccharides, recent studies have indicated that the disruption of the bio�lm structure could be achieved via the degradation of individual bio�lm compounds by various enzymes.  BioMed Research International �aemophilus in�uen�ae, K. pneumoniae, E. coli, P. aeruginosa, S. aureus, and Streptococcus pyogenes. Using DNase I at a concentration of 10 g/mL, degradation of mature 24 h formed bio�lms by 53.85%, 52.83%, 50.24%, 53.61%, 51.64%, 47.65%, and 49.52%, respectively, was observed. Moreover, bacterial susceptibility to selected antibiotics increased in the presence of DNase I. Azithromycin, rifampin, levo�oxacin, ampicillin, and cefotaxime were more effective in the presence of DNase I (5 g/mL) [79].
Furthermore, Hall-Stoodley et al. [80] reported that DNase I induced bio�lm degradation by 66.7%-95% for six clinical isolates of Streptococcus pneumoniae, even though the bio�lms were grown for six days. e authors revealed that the average bio�lm thickness was reduced by 85%-97%, indicating that, within the bio�lm, areas composed of lower amounts of extracellular DNA in comparison to adherent cells exist [80].
Moreover, Eckhart et al. [81] investigated the use of DNase I and DNase 1L2 (20 g/mL) against S. aureus and P. aeruginosa bio�lms. Both enzymes revealed strong anti-bio�lm activity. A�er 7 hrs of incubation, P. aeruginosa bio-�lm formation was effectively reduced by DNase 1L2 treatment. However, eighteen hours of incubation in the presence of each enzyme resulted in weak inhibition of bio�lm formation. S. aureus bio�lm formation was signi�cantly reduced by both enzymes, independent of the incubation time [81].
Furthermore, the antibio�lm activity of deoxyribonuclease I (130 g/mL) in combination with selected antibiotics toward C. albicans bio�lms was estimated. A reduction of viable counts by 0.5 log 10 units was observed for bio�lmgrowing C. albicans incubated with DNase I. Treating C. albicans with amphotericin B alone (1 g/mL) resulted in a 1 log 10 unit reduction in cell viability, which increased to 3.5 log 10 units in combination with DNase I. At higher concentrations of amphotericin B (>2 g/mL) and DNase I, cell viability was reduced by 5 log 10 units. However, the fungicidal effectiveness of caspofungin and �uconazole decreased when combined with DNase I, indicating that the synergistic effect between the antibiotic and DNase I is dependent on the fungicidal agent used [82].

4.2.2.
Lysostaphin. �romising antibio�lm results were also obtained for lysostaphin. Lysostaphin is a natural staphylococcal endopeptidase that can penetrate bacterial bio�lms [83,84]. e antimicrobial properties of lysostaphin were analyzed by Walencka et al. [85], who reported the bio�lm inhibitory concentration (BIC) of the enzyme for 13 S. aureus and 12 S. epidermidis clinical strains. e BIC against 8 S. aureus strains was estimated to be between 4 and 32 g/ mL, and for the remaining 5 strains, the BIC value was higher than the maximum tested concentration (>64 g/mL). e majority of the studied S. epidermidis strains were more resistant to lysostaphin activity than were the S. aureus strains. Only 2 of the 12 S. epidermidis strains exhibited reduced bio�lm formation in the presence of 128 g/mL or 16 g/mL lysostaphin. For the remaining 10 strains, the BIC value was estimated to be greater than 254 g/mL. In addition, the combined use of lysostaphin with oxacillin increased the susceptibility of the bio�lm-growing bacteria to the antibiotic. However, no antibio�lm e�ciency was observed for hetero-vancomycin-intermediate S. aureus and methicillin-resistant S. epidermidis strains [85].
High antibio�lm effectiveness of lysostaphin toward S. aureus strains was con�rmed by Kokai-Kun et al. [86], who used a mouse model to determine the most effective treatment strategy for multiorgan bio�lm infection. S. aureus bio�lms, including methicillin-resistant S. aureus (MRSA), were completely eradicated in the presence of lysostaphin when animals were treated with the 15 mg/kg lysostaphin and 50 mg/kg of nafcillin, administered 3 times per day for four days. Moreover, lysostaphin (10 mg/kg) effectively protected indwelling catheters from bacterial infection [86].
In addition, Aguinaga et al. [87] reported that lysostaphin leads to signi�cantly increased antibiotic susceptibility, with strain-dependent activity. e minimal bio�lm eradication concentration (MBEC) for MRSA and MSSA strains was estimated for 10 antibiotics in combination with 20 g/mL lysostaphin. e highest synergistic effect was observed when lysostaphin was combined with doxycycline (MBEC decreased from 4 mg/mL to 0.5 mg/mL) or levo�oxacin (MBEC decreased from 2 mg/mL to <1.9 mg/mL) against MRSA and MSSA, respectively [87].

-Amylases.
Craigen et al. [88] analyzed the antibio-�lm activity of -amylases against strains of S. aureus and S. epidermidis. e tested enzymes effectively reduced formed bio�lm and decreased bio�lm formation in the case of S. aureus. However, no antibio�lm effect of the analyzed enzymes was observed for S. epidermidis. Time-course experiments for S. aureus showed that bio�lms were degraded by 79% within 5 min and by 89% within 30 min of incubation with -amylases. Amylase at doses of 10, 20, and 100 mg/mL reduced bio�lms by 72%, 89%, and 90%, respectively, and inhibited matrix formation by 82%. In fact, S. aureus clinical isolates exhibited strain-dependent responses to amylase, but the treatment was successful for each strain. In addition, the antibio�lm activities of amylases from different biological sources were evaluated. e most effective bio�lm reduction was reported for -amylase isolated from Bacillus subtilis. Although enzymes derived from human saliva and sweet potato had no effect against preformed bio�lms, all of the tested enzymes, regardless of origin, were highly effective in inhibiting bio�lm formation [88].

Lyase.
Bio�lms of two mucoid P. aeruginosa strains were treated with gentamycin (64 g/mL) in combination with alginate lyase (20 U/mL). e studied enzyme caused bio�lm matrix li�uefaction. Incubation of the bio�lm with lyase and gentamycin for 96 h resulted in the complete eradication of the bio�lm structure and living bacteria. A reduction of viable counts by 2-3 log 10 units was reported for both strains when the combined therapy was applied [89].

Silver Nanoparticles.
Silver is a nontoxic antimicrobial metal that can be used in medicine. Kalishwaralal et al. [91] analyzed the antibio�lm activity of silver nanoparticles (AgNPs) against P. aeruginosa and S. epidermidis strains. Nanoparticles were synthesized with Bacillus licheniformis and AgNO 3 . e mean diameter of the received particles was 50 nm. Incubation with AgNO 3 -containing nanoparticles (100 nM) inhibited the amount of bio�lm formed aer 24 h by 98%. Incubation with 50 nM AgNPs reduced exopolysaccharide content, indicating that bio�lm formation was inhibited, although bacterial viability was unaffected [91].
Next, Mohanty et al. [92] reported dose-dependent anti-bio�lm activity of AgNPs against S. aureus and P. aeruginosa. Silver nanoparticles were prepared in 1% soluble starch with an average particle size of 20 nm. Incubation of bio�lms (24 hr incubation) in the presence of 1 M or 2 M silver nanoparticles yielded greater than 50% or 85% inhibition of bio�lm formation, respectively. Prolonged (48 hr) treatment resulted in 65% and 88% reduction of bio�lm formation, respectively. A silver nanoparticle concentration of 0.1 M did not affect bio�lm growth. Moreover, no signi�cant cytotoxic effect was observed at any of the concentrations tested [92].
AgNP activity against C. albicans and C. glabrata bio�lm formation was also estimated. Addition of silver nanoparticles to cultures of Candida adherent cells at a concentration of 3.3 g/mL reduced the percentage of total biomass of adherent C. glabrata cells by >90%. Moreover, mature bio-�lms aer the treatment were signi�cantly disrupted (97%) by 54 g/mL AgNPs. C. albicans bio�lms exhibited increased resistance in comparison to C. glabrata with silver nanoparticle treatment, and an 85% reduction of adherent cell growth was observed at concentration >6.7 g/mL. No effect on mature bio�lms was reported [93].
Chitosan-based silver nanoparticles (CS-AgNPs) reduced P. aeruginosa 24 hrs-grown bio�lms by >65% at a concentration of 2 g/mL. S. aureus bio�lms formation were inhibited by 22% by the same concentration of CS-AgNPs. Treatment with higher dose (5 g/mL) reduced bio�lm formation by 65%. Scanning electron microscopy con�rmed the destruction of the P. aeruginosa cell membrane by 2 g/ mL CS-AgNPs. In addition, no cytotoxic effects toward macrophages were observed [94].

�ther Bio�lm-�isrupting Factors.
As bio�lm-related infections have become an increasingly prevalent problem in contemporary medicine, factors that disrupt bio�lm structure or exhibit antibio�lm activity have been the sub�ect of intense interest.
Arias-Moliz et al. [96] investigated lactic acid at concentrations of 2.5%-20% and demonstrated its antimicrobial activity toward E. faecalis and Enterococcus duran strains. Complete eradication of bio�lms was observed when 15% lactic acid was used for 1 min. In addition, 5% lactic acid reduced the viable cell count by 40.7%-100%. Simultaneous administration with 2% chlorhexidine slightly improved the killing efficacy of lactic acid, while administration with 0.2% cetrimide completely eliminated every tested strain independent of the lactic acid concentration used [96].
Recently, numerous antibio�lm researches were published. Considering the fact, that various compounds acting against Gram-positive bacteria, Gram-negative bacteria, or fungi were analyzed, and different stage of bio�lm growth (mature bio�lm eradication or inhibition of bio�lm formation) was assessed, it is difficult to reliably compare all the presented results. Some of the approaches seem, however, to be very promising. Among described plant extracts, fresh garlic showed the highest antibio�lm and antibacterial properties against P. aeruginosa. Also Japanese knotweed (P. cuspidatum) expresses good efficacy in the treatment of P. acnes bio�lm formation. What seems, however, to be the most interesting is the ability to search for synergistic effects between different approaches, exempli�ed by the action of bio�lm-disrupting enzymes in combination with antibiotics. S. aureus bio�lm was completely disrupted by lysostaphin with nafcillin and P. aeruginosa bio�lm by a combination of lyase with gentamycin, and DNaseI with amphotericin B effectively reduced C. albicans bio�lm. Chitosan and chitosan-based silver nanoparticles can easily disrupt mature bio�lm of P. aeruginosa and S. mutans and could provide penetration of bio�lm structures by antimicrobials. is data suggested that APDT, enzymes, plant extracts, and other compounds can be used in various combinations acting as good antibio�lm and antimicrobial agents. e presented innovative strategies may potentially strongly support classical treatments and cause an increase of their effectiveness.

Conclusions
In environments that include the human body, microbial cells form a well-organized structure termed a bio�lm. e development of strategies to combat bacteria growing in bio�lms is a challenging task; these bacteria are much more resistant to classical antimicrobial therapies and exchange genetic material more easily. us, under the pressure of a particular antibiotic, resistant clones are selected. Antimicrobial photodynamic therapy appears to be a very promising therapeutic option to effectively control the growth of microbial bio�lms. However, as with other antimicrobial therapies, APDT is generally less effective against microorganisms growing in bio�lms than against planktonic cells. Hence, there is a need to develop a therapeutic approach that would (i) increase the sensitivity of the microorganism to already established methods (e.g., antibiotic therapies) by violating the structure of the bio�lm or disturbing the communication between a population of microorganisms in the bio�lm or (ii) combine several modes of microbicidal action to achieve a synergistic effect. An example of the �rst approach is to use enzymes that affect the bio�lm, while the second approach could be achieved by combining APDT with antibiotics, plant extracts, or bio�lm-disrupting enzymes. Moreover, if we combine APDT with the use of enzymes that are speci�c for microbial structures; the selectivity of the approach will be increased as it potentially will permit the use of lower photosensitizer concentrations. One disadvantage of APDT is the limited amount of data based on animal models. However, the growing number of in vivo studies verifying APDT based on various photosensitizers is encouraging and will determine the direction of further research.