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Functional biomaterials surfaces

Study on inhibitory activity of chitosan-based materials against biofilm producing Pseudomonas aeruginosa strains

Journal of Biomaterials Applications 0(0) 1–10 ! The Author(s) 2015 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav DOI: 10.1177/0885328215578781 jba.sagepub.com

Agnieszka Machul1, Diana Mikołajczyk1, Anna Regiel-Futyra2, Piotr B Heczko1, Magdalena Strus1, Manuel Arruebo3,4, Graz_ yna Stochel2 and Agnieszka Kyzioł2

Abstract Six antibiotic-resistant Pseudomonas aeruginosa strains, isolated from chronic diabetic foot infections, were chosen for studying the influence of different chitosan-based materials: chitosan solution and chitosan submicroparticles in both planktonic and 24 h-old biofilm-forming models. Chitosan solution occurred to be more effective in the reduction of bacterial populations than chitosan submicroparticles for both planktonic and biofilm-related Pseudomonas cells. It seems that the antimicrobial activity of the tested chitosan preparations depends on the individual bacterial strain susceptibility probably related to differences in the phenotypes and natural antioxidant abilities of Pseudomonas aeruginosa strains. Keywords Chitosan, antibacterial, Pseudomonas aeruginosa, biofilm, planktonic bacteria form

Introduction Pseudomonas aeruginosa (PAR) is a genus of aerobic Gram-negative bacteria regarded as important human pathogen. The mucoid strains of PAR forming biofilm are responsible for a wide range of chronic infections such as pneumonia in cystic fibrosis patients or diabetic foot infections. Moreover, PAR may form biofilm and colonize the surfaces of medical devices and implants, e.g. catheters and prostheses.1–4 Bacterial biofilms play a crucial role in the pathogenesis of several serious infections. In detail, bacteria attach to the living and nonliving surfaces and form biofilm composed of polysaccharides, proteins and other components. This complex structure hinders antibiotic activity, facilitates bacteria colonization, subsequent acquisition of ecological niches and finally allows it to invade host organism or to survive in adverse environmental conditions.2,5 Chitosan (CS) is a cationic polymer obtained from renewable resources (i.e. exoskeletons of marine crustaceans) upon a severe deacetylation of chitin in alkaline media.6,7 It has attracted considerable interest due to its unique combination of properties mainly excellent

biocompatibility, enzymatic biodegradability, ability to metal complexation, nontoxicity and antibacterial activity.8,9 Although CS’s antimicrobial activity has been previously well demonstrated and documented, the antibacterial activity of CS has never had its mechanism completely elucidated.10–16 Three possible action modes have been proposed. The first hypothesis postulates that the responsibility for the bacterial membrane structure loosening and consequent leakage of cell components is caused by the electrostatic interactions between CS’s polycationic molecules and negatively charged cell membrane components.17 CS is also 1 Jagiellonian University Medical College, Department of Microbiology, 31121 Krako´w, Czysta, Poland 2 Jagiellonian University, Faculty of Chemistry, Krako´w, Ingardena, Poland 3 Department of Chemical Engineering, Nanoscience Institute of Aragon (INA), Zaragoza, Spain 4 Networking Research Center on Bioengineering, Biomaterials and Nanomedicine, CIBER-BBN, Zaragoza, Spain

Corresponding author: Agnieszka Kyzioł, Jagiellonian University Medical College, Czysta 18, Krako´w 31-121, Poland. Email: [email protected]

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viewed as a cell penetrating molecule which is able to interact with DNA. This results in the inhibition of DNA transcription and protein synthesis. In contrast, other group of authors propose that CS chelates metal ions which leads to the production of toxins, the inhibition of enzymes activity and finally to the cell cycle arrest.11–13 A general accepted idea is that the antimicrobial activity of CS strongly depends on several factors. The most important are the studied strain of the tested bacteria and its growth phase, CS molecular weight and deacetylation degree, CS concentration, form (solution, colloid), pH, temperature, medium components, etc.18,19 Thus, since CS’s antibacterial effect has been tested under varied conditions, it remains problematic to compare its activity among results obtained by different authors. CS antimicrobial properties have been proven against planktonic bacterial cell growth,17,20–25 however, little is known about its effect on already established or mature biofilms. Orgaz et al.20 have demonstrated the significant inhibitory effect of CS with medium molecular weight and its enzymatically hydrolyzed products against mature biofilms of pathogenic bacterial strains associated with foodborne diseases and food spoilage. CS is also considered as a perfect candidate for fungalbased biofilm prevention and treatment, mainly on central venous catheters and other medical devices.17,21,22,25 To the best of our knowledge, only one paper has been primarily focused on the CS’s ability to interrupt PAR biofilms. CS gels at low concentrations (0.13 wt%) occurred to be effective in disrupting the biofilm integrity and causing cell death.24 We present here two different forms of CS-based materials: CS solution and CS submicroparticles tested for their antibacterial activity against six antibiotic-resistant PAR strains. An important objective of our studies was to determine whether different forms of CS materials maintain their antibacterial activity on biofilm forming Pseudomonas bacteria in comparison to planktonic forms of the same strains. Since CS-based materials attract considerable interest in various biomedical applications, a detailed study on their antibacterial activity on multiple strains isolated from clinical cases is required.

Materials and methods Chemicals CS with medium average molecular weight (Mw 1278  8 kDa; from chitin of crab shells) was purchased from Sigma-Aldrich and used as received. Glacial acetic acid (99.8 wt%; Sigma-Aldrich) was used as solvent. Sodium tripolyphosphate (TPP, tech., 85 wt%) was also obtained from Sigma-Aldrich. Sodium

cacodylatetrihydrate, sucrose, glutaraldehyde water solution (50 wt%), poly-L-lysine and anhydrous methanol (Sigma-Aldrich) were used for fixing and bacteria dehydration before scanning electron microscope (SEM) visualization. All solutions were prepared in deionized water (Milli-Q).

Preparation procedure Preparation of CS solutions. CS (medium average molecular weight) stock solution (1% (w/v)) was prepared as follows: CS flakes were dissolved in 0.1 M acetic acid aqueous solution using a heating plate and magnetic stirring. Heating (65 C) and mixing were maintained until a clear solution was obtained (around 12 h) indicating the complete CS dissolution. Before the antibacterial tests, the pH of the CS solution was adjusted to 5.5. Preparation of chitosan submicroparticles. Chitosan submicroparticles (CS_SMPs) were prepared by following the modified method of Calvo et al.26 First, CS (1 mg/ml, pH ¼ 4.1) and sodium TPP (2 and 4 mg/ml, pH ¼ 9.0) solutions were prepared. TPP solution was added drop wisely (volume ratio of TPP:CS was 1:1) to the CS solution and stirred at 750 rpm for 30 min. The resulting suspensions (CS_2TPP, CS_4TPP for 2 and 4 mg/ ml TPP, respectively) were treated with ultrasounds for 15 min (620 W, 50 Hz). Obtained particles were washed with water in triplicate and centrifuged (3  4000 rpm/ 10 min at 21 C, Thermo Scientific, Heraeus, Fresco 17). Samples were stored at 4 C. Part of the obtained CS_SMPs was freeze-dried.

Materials characterization Determination of the size and surface charge of submicroparticles. Particle size distribution with polydispersity index and zeta potential measurements was performed using a Zetasizer NanoZS (Malvern Instruments, UK) by dynamic light scattering technique (Backscatter optics, 25 C). Measurements were carried out using diluted submicroparticle-based suspensions in deionized water at pH ¼ 7.0. The particle size and zeta potential were determined in triplicates, and average values were calculated. Bacterial cell wall disruption visualization by SEM. Bacterial cell wall disruption, upon incubation with CS and CS_SMPs, was visualized by SEM (TESCAN, Vega 3). Coverslips were placed in a 12-well plate and treated with poly-L-lysine to enhance further bacterial cell adhesion. Glass slides were immersed in bacterial suspensions incubated with CS forms (control, CS 1 (w/v)%, CS_2TPP) for 0, 8, and 24 h. Then, 1 ml of fixing buffer (sodium cacodylate in 2.5 wt%

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glutaraldehyde and 0.1 M sucrose) was added and the slides were incubated for 1.5 h at 37 C. A dehydration procedure was applied in a gradient of methanol. Dried samples were sputtered with a 20 nm-gold layer (Quorum, Q150R S) to facilitate SEM visualization. Bacterial cultures. All tests were performed on six PAR strains: PAR 5, PAR 18, PAR 20, PAR 40, PAR 50 and PAR 54. The strains were randomly selected from a larger collection of PAR from clinical samples sent to Chair of Microbiology, Jagiellonian University Medical College in Krakow. The samples for microbiological analyses were taken from wounds of patients with chronic diabetic foot infections. They were plated on the following media: McConkey agar (Oxoid, UK) for Enterobacteriaceae, Columbia blood agar (Oxoid) with 5% sheep blood for Streptococci, BBL Enterococcosel agar (BD, Franklin Lakes, USA) for Enterococci and Pseudomonas agar (Oxoid) for Pseudomonas. Phenotypic identification of PAR isolates from McConkey agar was conducted with the commercial identification system API20NE (BioMerieux, Marcy l’Etoile, France). The strains were kept frozen at 70 C on glass beads in Trypticase-Soy Broth with 10% glycerol. Strains were previously characterized by different efficacy in biofilm production and chemical structure of the biofilm matrix. Strains were propagated in 10 ml of Trypticase-Soy Broth (TSB, Difco) at 37 C for 24 h under aerobic conditions. Then the cultures were centrifuged (2000 rpm; 10 min) and washed with 10 ml of saline buffer. Initial suspensions of bacteria (1  108 CFU/ml (colony forming units)) were prepared by serial dilutions in saline using the MacFarland’s scale and by counting bacterial colonies by plating them on McConkey Agar (Oxoid) using decimal dilutions.

Inhibitory activity of the tested materials on planktonic form of PAR Determination of the inhibitory properties of the CS-based materials against planktonic Pseudomonas cells. The tested materials CS solution and CS_SMPs were added to 96-well plates with Pseudomonas cultures. In detail, 270 ml of tested materials were added to 30 ml of fresh Pseudomonas (culture PAR prepared as described in Bacterial culture section) to obtain a final concentration of CS solution: 0.5% and 1.0% (w/v) and CS_SMPs: 0.9 mg/ml. The mixtures were incubated at 37 C under aerobic conditions. Numbers of viable bacteria were counted at 0 time and after 8 and 24 h of incubation. Then decimal dilutions of the bacterial suspensions were made in saline, plated on McConkey Agar (Oxoid), and incubated at 37 C for 24 h. Numbers of viable bacteria expressed as CFU/ml were determined by counting the colonies.

Inhibitory activity of the tested materials on PAR in biofilm Determination of the inhibitory properties of the CS-based materials against 24 h-old Pseudomonas cells in biofilm. At the beginning, to a 96-well plate were added 170 ml of fresh TSB (Oxoid) and 30 ml of 24 h-old Pseudomonas culture (prepared as above) to obtain a final bacterial concentration of 1  107 CFU per well. Then the bacteria were adhered to the bottom of the plates by centrifugation (2000 rpm; 10 min). The plates were further incubated for 24 h to reach a mature biofilm formation.27 After that, about 170 ml of culture was gently removed from each well, and 270 ml of the tested materials were added to obtain the final concentration of CS solution: 0.5 and 1.0 (w/v)% and CS_SMPs: 0.9 mg/ml. Following steps were performed as mentioned before.

Statistical analysis Statistical analyses were performed using STATISTICA, version 10 software. Statistical significance values of the groups’ means for six strains of PAR were evaluated using Student’s t-test. The statistical analyses performance were considered significant when P < 0.05. Since significant differences among the tested strains for their susceptibility to CS and CS_SMPs were obtained, activities of these two CS forms and control against PAR were calculated for mean values for six tested strains.

Results and discussion The antibacterial activity of CS against various strains of planktonic bacteria is well documented,10–16 whereas its inhibitory effect on biofilm-growing bacteria as well as on complex biofilm structures alone has not been previously considered. Here, two different CS forms were tested in order to study the impact of CS form on PAR cells embedded in biofilm matrix. The inhibitory activities of the tested CS-based materials against the bacteria entrapped in biofilm were compared with the one obtained for planktonic Pseudomonas cultures.

Materials characterization Based on our previous research regarding antibacterial activity of CS-based materials,28 CS of medium average molecular weight was chosen for this study. Additionally, due to our initial antibacterial test against PAR, CS with low and high molecular weights were rejected for further investigation mainly because of their unsuitable properties as solution viscosity or solubility at higher pH. CS solution and CS_SMPs

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were prepared in order to see differences in the antimicrobial activity of the polysaccharide, depending on the material form.

Determination of size and surface charge of CS_SMPs CS_SMPs were formed by ion gelation technique with polyanionic sodium triphosphate (TPP). Two different concentrations of a cross-linking agent were applied to provide various cross-linking degrees. Particle size and surface charge are critical variables determining the fate of therapeutically administrated particles. Mean particles size, polydispersity and zeta potentials measured in water (pH ¼ 7) are summarized in Table 1. The particle size was measured by dynamic light scattering revealing the hydrodynamic diameter of the submicroparticles prepared by ionotropic gelation technique. It is possible that cationic CS can interact with the negatively charged sodium TPP, forming inter- and intra-molecular cross-linkages, yielding smaller ionically cross-linked submicroparticles with lower surface charge. However, in our experimental system, the size of the submicroparticles increased with the concentration of TPP from 249  72 up to 342  73 nm for 2 and 4 mg/ml of TPP, respectively. This change may be explained by the reduced stability of the particle-

Table 1. Mean particle size and zeta potential of chitosan submicroparticles. Sample

Size (nm)

Polydispersity

Zeta potential (mV)

CS_2TPP CS_4TPP

249  72 342  73

0.3 0.6

15  7 14  5

(a)

based colloids with the increase of the cross-linking agent concentrations promoting agglomeration. Jonassen et al.29 have demonstrated that TPP in excess increases the inter-particle linkage over time, leading to aggregate formation which reduces the particle stability. Zeta potential is the surface charge, which influences the particle stability by the electrostatic repulsion between individual particles, and it was ca. þ15 mV for our studied CS_SMPs.

Submicroparticle surface morphology The shape and surface morphology of the CS-based materials were observed by SEM (Figure 1). Micrographs show large aggregates of CS_SMPs. Most of the CS_SMPs were found to be spherical. No significant differences in particles size and shape were observed after the freeze-drying process.

Inhibitory activity of the tested materials on PAR cultures in planktonic and 24 h-old biofilm models CS solution inhibitory activity. The inhibitory properties of the CS solution against tested Pseudomonas strains in planktonic and biofilm-associated form (24 h-old biofilm) were determined in vitro. First, influence of CS solutions (0.5 (w/v)% and 1 (w/v)%) on the number of viable PAR planktonic populations has been studied, and results are presented in Figure 2. The higher concentration of CS was more effective in both incubation times and resulted in a total reduction of the viable cells of all tested strains in 24 h. Noteworthy, only two strains (PAR 50 and PAR 54) were not fully inhibited by 1% (w/v) of CS when incubated for 8 h (Figure 2(c)). In addition, there were significant statistical differences between the averages for the six strains obtained for the studied groups

(b)

Figure 1. SEM images of CS_SMPs in suspension (a) and after freeze-drying (b). CS_SMPs: chitosan submicroparticles.

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p < 0.05

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p < 0.05

p < 0.05

109 108

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107 106 5

10

Culture control CS 0.5% (w/v) CS 1% (w/v)

p < 0.05

104 103 102 101

*

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PAR 5 PAR 18 PAR 20 PAR 40 PAR 50 PAR 54

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106 105 104 103 102 101

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**** 8h

(c) 108

PAR 5 PAR 18 PAR 20 PAR 40 PAR 50 PAR 54

107 106

CFU / mL

24 h

105 104 103 102 101 100 0h

****

******

8h

24 h

Figure 2. Influence of CS solutions on numbers of viable Pseudomonas aeruginosa planktonic populations: the average for the six strains (a), CS 0.5% (w/v) (b) and CS 1% (w/v) (c). Results depict CFU/ml for six Pseudomonas aeruginosa strains: PAR 5, PAR 18, PAR 20, PAR 40, PAR 50 and PAR 54 measured at three time intervals (0, 8 and 24 h). Detection limit was 100 CFU/ml, and differences among the tested strains were within this limit. * represents a total bactericidal effect. CS: chitosan; PAR: Pseudomonas aeruginosa.

(CS 0.5% (w/v) and 1% (w/v)) after 8 h of incubation (Figure 2(a)). Statistical significance was not observed between these groups for the other times: 0 and 24 h. However, we observed statistical differences between control and the other groups after 0, 8 and 24 h of incubation. As indicated in Materials and methods section, differences between CS, CS_SMPs and control

were calculated for mean values for six tested strains of PAR. Although there are many conflicting reports on chitosan antimicrobial activities, these data confirm the general opinion given in the literature that CSs show inhibitory properties against PAR. For example, as stressed by Fernandes et al.,30 PAR showed to be the most resistant microorganism out of six different Gram-negative and Gram-positive species to three CSs with different molecular weight and two chitooligosaccharide mixtures. It is known that antimicrobial activity of different CSs is highly changeable and dependent on their acetylation degree and molecular weight.31 However, it seems that the situation with anti-PAR inhibitory properties of CS is even more complicated in the light of the data obtained by us, indicating that anti-Pseudomonas activity of the CSs preparation is strongly strain-dependent. This fact has not yet been shown in the literature since the vast majority of the papers on antibacterial properties of different CSs have been based on testing individual, collection derived, strains of PAR but not on clinical isolates. Second, inhibitory activities of CS solutions against 24 h-old PAR biofilm-associated cells regarded as mature biofilm were investigated. The results are presented in Figure 3. Both tested CS concentrations showed a total reduction of bacterial growth in 24 þ 24 h for strains PAR 18, PAR 20 and PAR 40 (Figure 3). Again, 1% (w/v) of CS was generally more effective in its inhibitory activity against all tested strains than 0.5% (w/v), which caused the bacteria growth inhibition in 24 þ 8 h only for one strain PAR 18 (Figure 3(b)). Statistical difference was observed only between culture control and CS 1% (w/v) at the beginning of experiment (24 h-old biofilm PAR culture). After 24 þ 8 and 24 þ 24 h of incubation, statistical differences were observed between culture control and CS 0.5% (w/v) and CS 1% (w/v). Moreover, there were significant statistical differences between the averages for the six strains obtained for the studied groups (CS 0.5% (w/v) and 1% (w/v)) in case of both 24 þ 8 and 24 þ 24 h of incubation time (Figure 3(a)). Importantly, CS in 0.5% (w/v) solution had almost no influence on the growth of strains PAR 50 and PAR 54 in both 24 þ 8 and 24 þ 24 h of incubation time (Figure 3(b)). However, a higher (1% (w/v)) CS concentration showed a noticeable reduction in the growth of these strains (Figure 3(c)). In case of strain PAR 50, this concentration of CS caused a 5-log reduction in 24 þ 8 h, which was reversed in 24 þ 24 h, and a 3-log increase of the bacteria numbers was observed. Such effect was not observed for strain PAR 54, and 6 logarithms growth reduction was found only in 24 þ 24 h of

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(a)

p < 0.05

p < 0.05

p < 0.05

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Culture control CS 0.5% (w/v) CS 1% (w/v)

p < 0.05 Culture control CS_2TPP CS_4TPP

106

107

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CFU / mL

107 105 104 103

106 105 104 103

102

102

1

10

0

101

10

24 h

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100

(b) 109

0h PAR 5 PAR 18 PAR 20 PAR 40 PAR 50 PAR 54

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PAR 5 PAR 18 PAR 20 PAR 40 PAR 50 PAR 54

108 107 106

104

CFU / mL

CFU / mL

p < 0.05

108

108

103 102 101

105 104 103

***

*

100 24 h

24 + 8 h

102

24 + 24 h

101

(c) 109 PAR 5 PAR 18 PAR 20 PAR 40 PAR 50 PAR 54

108 107 106 105

0h

103

106

**

100 24 h

24 + 8 h

**** 24 + 24 h

Figure 3. Influence of CS solution on numbers of 24 h-old biofilm-associated viable Pseudomonas aeruginosa cells (mature biofilm): the average for the six strains (a), CS 0.5% (w/v) (b) and CS 1% (w/v) (c). Results depict CFU/ml for six Pseudomonas aeruginosa strains: PAR 5, PAR 18, PAR 20, PAR 40, PAR 50 and PAR 54 measured at two time intervals (24, 24 þ 8 and 24 þ 24 h). Detection limit was 100 CFU/ml, and differences among the tested strains were within this limit. *represents a total bactericidal effect. CS: chitosan; PAR: Pseudomonas aeruginosa.

incubation with a CS solution (1 (w/v)%). Our results indicate differences among PAR strains in their susceptibility to CS, which may explain controversies on its antibacterial activity present in the literature.32 Moreover, partial and reversible growth inhibition exerted by CS against strain PAR 50 may indicate inactivation of the CS by its degradation by some PAR strains during prolonged incubation. Such a degradation caused by chitinase of Bacillus cereus was recently described by Liang et al.33

24 h PAR 5 PAR 18 PAR 20 PAR 40 PAR 50 PAR 54

108 107

101

* 8h

(c) 109

104 102

*

100

CFU / mL

CFU / mL

p < 0.05 109

109

105 104 103 102 101

*

100 0h

8h

* 24 h

Figure 4. Influence of CS_SMPs (0.9 mg/ml) on number of viable Pseudomonas aeruginosa planktonic populations: the average for the six strains (a), CS_2TPP (b) and CS_4TPP (c). Results depict CFU/ml for six Pseudomonas aeruginosa strains: PAR 5, PAR 18, PAR 20, PAR 40, PAR 50 and PAR 54 measured at different time intervals (0, 8 and 24 h). Detection limit was 100 CFU/ml, and differences among the tested strains were within this limit. *represents a total bactericidal effect. CS: chitosan; CS_SMPs: chitosan submicroparticles; PAR: Pseudomonas aeruginosa.

CS_SMPs inhibitory activity. Then, inhibitory activity of CS_SMPs at a final concentration of 0.9 mg/ml was tested in vitro against PAR in planktonic and 24 hold biofilm forms. Results are presented in Figures 4 and 5 for planktonic and biofilm-related bacteria, respectively. CS:TPP weight ratios of 1:2 and 1:4 resulting in particles size of 249  72 and 342  73 nm for

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(b)109

PAR 5 PAR 18 PAR 20 PAR 40 PAR 50 PAR 54

108

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107 106 105 104 103 102 101 100 24 h

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24 + 8 h

24 + 24 h

109

PAR 5 PAR 18 PAR 20 PAR 40 PAR 50 PAR 54

108

CFU / mL

107 106 105 104 103 102 101 100 24 h

24 + 8 h

24 + 24 h

Figure 5. Influence of CS_SMPs (0.9 mg/ml) on number of viable cells of Pseudomonas aeruginosa in the 24 h-old biofilm (mature biofilm): the average for the six strains (a), CS_2TPP (b) and CS_4TPP (c). Results depict CFU/ml for six Pseudomonas aeruginosa strains: PAR 5, PAR 18, PAR 20, PAR 40, PAR 50 and PAR 54 measured at different time intervals (24, 24 þ 8 and 24 þ 24 h). Detection limit was 100 CFU/ml, and differences among the tested strains were within this limit. CS: chitosan; CS_SMPs: chitosan submicroparticles; PAR: Pseudomonas aeruginosa.

CS_2TPP (TPP: 2 mg/ml) and CS_4TPP (TPP: 4 mg/ ml), respectively were investigated. In general, CS_SMPs turned out to be less effective when compared with CS solutions against planktonic form of PAR. A total reduction of the bacterial growth was observed only for two strains, i.e. PAR 18 for both CS_SMPs (CS_2TPP and CS_4TPP) and PAR 40 for CS_SMPs CS_4TPP after 24 h of incubation with the

tested materials (Figure 4(c)). In addition, there were no statistically significant differences between averages for six strains of PAR for the two tested groups (CS 0.5% (w/v) and 1% (w/v)) (Figure 4(a)). Statistical difference was observed after 8 and 24 h of incubation between culture control and (CS 0.5% (w/v) and 1% (w/v)). Again, striking differences were observed in individual susceptibility of the tested PAR to the CS_SMPs. The bacterial cells present in 24 h-old biofilm seemed to be more resistant, as compared to their planktonic form (Figure 5). Comparing observed antibacterial effect of CS solutions and CS_SMPs for both planktonic and 24 h-old biofilm forms, it can be concluded that the CS solution with positively charged functional groups was more effective than CS_SMPs. It can be explained by privileged reaction of those groups with biofilm matrix and negatively charged outer components of the bacterial membrane. It is in agreement with results published by other authors claiming that inhibitory activity depends mainly on positive charges of CS available in the initial solution. In case of submicroparticles, these charges are partially neutralized by the cross-linker resulting in a weaker biological activity.13,34 Also, diffusion constraints probably hinder submicron particles to permeate through the extracellular polymeric matrix that constitutes the biofilm whereas CS solutions rapidly diffuse through this exopolysaccharide reaching the embedded bacteria and enhancing the antimicrobial action. The antibacterial activity of CSs is strongly dependent on their molecular weight and deacetylation degree.18,19,31 In our case, we chose medium molecular weight CS-based on our previous evaluation of its antimicrobial action.28 It should be also stressed here that on the contrary to other studies on antimicrobial properties of the CSs, our studies were done on several, selected, clinically relevant, antibiotic-resistant and biofilm producing strains of PAR. Moreover, some of the tested strains appeared to be much more resistant than others to activity of the tested CS preparations. It is very difficult to provide any explanations in this respect, but it can be only speculated that some clinically important PAR strains may enzymatically decompose CS thus interfering with its antibacterial activity. It is known that some PAR strains, mostly isolated from soils, possess strong chitinase activity,35 which property makes them important bioactive waste degrading microorganisms, able even to utilize shellfish shells.36 Quite recently, the presence of both chitinase and chitin-binding proteins was identified from the complete genome sequence of PAR strain YL84, a quorum-sensing strain isolated from compost.37

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CS

CS_2TPP

Control

CS

CS_2TPP

t0

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t0

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Figure 6. SEM micrographs representing the morphology of the bacteria cell wall upon contact with CS and CS_SMPs (CS_2TPP) for strains PAR 5 (a) and PAR 54 (b). Red arrows indicate disrupted cells; green arrows – undamaged ones. CS: chitosan; CS_SMPs: chitosan submicroparticles.

Bacterial cell wall disruption SEM visualization. The bacterial morphology changes were studied by SEM after 24 þ 24 h of treatment with the tested materials: CS solution and CS_SMPs (CS_2TPP) incubated with 24 h-old PAR culture forming biofilm. Selected images are presented in Figure 6 (red arrows indicate disrupted cells while green arrows – undamaged ones). SEM examination of two selected strains (PAR 5 and PAR 54) was applied to correlate the results of the tests on inhibitory activities with the visual effect of biofilm destruction and bacteria cell wall damage. Red arrows show bacteria with disruptions in cell membranes, while green ones indicate viable and intact

bacteria cells. Clearly, no changes in the shape of the cells and in their conditions upon the incubation time can be observed for the control samples. The morphological effect for bacteria incubated with different CS forms is evidenced by the change in the shape of the cells, which finally may lead to the cell wall disruption. To conclude, bacteria growing in planktonic form generally seemed to be more susceptible to CS-based materials than bacteria growing surrounded by a mature biofilm. The observed differences in the antibacterial activity of the tested materials could be explained by the fact that bacteria living within the biofilm are phenotypically different from their planktonic forms.

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Moreover, bacteria in biofilms exhibit an increased antimicrobial resistance compared with planktonic forms. First of all, the reduction of the susceptibility to antimicrobial agents is carried out by the bacteria mainly by gene activation and surface changes as well as by specific molecular-target production (intrinsic resistance). Second, the complicated structure of the biofilm matrix delays or impedes the diffusion of many bactericidal agents, e.g. antibiotics into deeper layers (extrinsic resistance).38 Finally, the exposed surface per volume ratio is higher when the planktonic bacteria is exposed to the antimicrobial compared to the surface exposed when forming a biofilm. In the later form, bacteria attach to the solid surface and agglomerate one to another in a scaffold of self-produced extracellular polymeric substances minimizing their exposed area to the antimicrobial.

Conclusions The influence of different forms of CS-based materials: CS solution and CS_SMPs on viable PAR populations in mature biofilms (after 24 h) was evaluated in vitro. Importantly, six investigated PAR strains with different efficacies in their biofilm production and chemical structure of the biofilm matrix were isolated from suppurated wounds of six patients with chronic diabetic foot infections. CS solution showed higher antibacterial action than CS_SMPs for both planktonic and 24 h-old biofilm PAR populations. To the best of our knowledge, there are no reports on CS antibacterial activity exerted against multiple PAR strains isolated from clinical cases and on differences in their susceptibility to this activity. Thus, our results indicate the importance of conducting studies on antimicrobial activity of different biomaterials on multiple strains of the potentially pathogenic bacteria before making conclusions on their antimicrobial spectrum and on their usefulness in biomedical applications.\ Declaration of conflicting interests None declared.

Funding This work was supported by the grant N N401 547040 (2012–2014) and statutory grant K/ZDS/002861 (2012). The research was carried out with the equipment purchased through the financial support of the European Regional Development Fund in the framework of the Polish Innovation Economy Operational Program (contract no. POIG.02.01.00-12-023/08). Financial support from the EU in the form of the ERC Consolidator Grant program (ERC-2013-CoG-614715, NANOHEDONISM) is gratefully acknowledged.

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Study on inhibitory activity of chitosan-based materials against biofilm producing Pseudomonas aeruginosa strains.

Six antibiotic-resistant Pseudomonas aeruginosa strains, isolated from chronic diabetic foot infections, were chosen for studying the influence of dif...
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