Inhibition of mixed fungal and bacterial biofilms on silicone by carboxymethyl chitosan

Accepted Manuscript Title: Inhibition of Mixed Fungal and Bacterial Biofilms on Silicone by Carboxymethyl Chitosan Author: Yulong Tan Matthias Leonhard Doris Moser Su Ma Berit Schneider-Stickler PII: DOI: Reference:

S0927-7765(16)30638-5 http://dx.doi.org/doi:10.1016/j.colsurfb.2016.08.061 COLSUB 8131

To appear in:

Colloids and Surfaces B: Biointerfaces

Received date: Revised date: Accepted date:

24-4-2016 1-8-2016 30-8-2016

Please cite this article as: Yulong Tan, Matthias Leonhard, Doris Moser, Su Ma, Berit Schneider-Stickler, Inhibition of Mixed Fungal and Bacterial Biofilms on Silicone by Carboxymethyl Chitosan, Colloids and Surfaces B: Biointerfaces http://dx.doi.org/10.1016/j.colsurfb.2016.08.061 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Inhibition of Mixed Fungal and Bacterial Biofilms on Silicone by Carboxymethyl Chitosan

Yulong Tan1*, Matthias Leonhard1, Doris Moser2, Su Ma3, Berit Schneider-Stickler1

1

Department of Otorhinolaryngology and Head and Neck Surgery, Medical

University of Vienna, Vienna, Austria. 2

Department of Cranio-Maxillofacial and Oral Surgery, Medical University of

Vienna, Vienna, Austria. 3

Food Biotechnology Laboratory, Department of Food Sciences and

Technology, BOKU-University of Natural Resources and Life Sciences, 1190 Vienna, Austria.

*Corresponding author: Yulong Tan Email address: [email protected] Tel.: +43-1-40400 33170 Fax.: +43-1-40400 42840

Number of words: 2423

Number of tables/figures: 8

Matthias Leonhard Email address: [email protected] Doris Moser Email address: [email protected] Su Ma Email address: [email protected] Berit Schneider-Stickler Email address: [email protected]

Graphical abstract

Highlights 

Carboxymethyl chitosan inhibited mixed biofilm of fungi and bacteria.



Carboxymethyl chitosan decreased metabolic activity and inhibited cells adhesion.



Carboxymethyl chitosan blocked further development of biofilms at different stages.

Abstract Mixed biofilms with fungi and bacteria are the leading cause for the failure of medical silicone devices, such as voice prostheses in laryngectomy. In this study, we determined the effect of carboxymethyl chitosan (CM-chitosan) on mixed biofilm formation of fungi and bacteria on silicone which is widely used for construction of medical devices. Mixed biofilm formations were inhibited 72.87% by CM-chitosan. Furthermore, CM-chitosan significantly decreased the

metabolic

activity

of

the

biofilms

(2-methoxy-4-nitro-5-sulfo-phenyl)-2H-tetrazolium-5

using

2,

3-bis

carboxanilide

(XTT)

reduction assay. The examination using confocal laser scanning microscopy and scanning electron microscope confirmed that CM-chitosan inhibited the mixed biofilm and damaged the cells. Effects of CM-chitosan on different stages of biofilms were also evaluated. CM-chitosan inhibited the adhesion of fungi and bacteria with an efficiency of >90%. It prevented biofilm formation at efficiencies of 69.86%, 50.88% and 46.58% when CM-chitosan was added at 90 min, 12h and 24h after biofilm initiation, respectively. Moreover,

CM-chitosan inhibited Candida yeast–to-hyphal transition. CM-chitosan was not only able to inhibit the metabolic activity of biofilms, but also active upon the establishment and development of biofilm. Therefore, CM-chitosan may serve as a possible antibiofilm agent to limit biofilm formation on voice prostheses. Keywords: mixed fungal-bacterial biofilm; carboxymethyl chitosan 1. Introduction Silicone is widely used as medical material due to its biocompatibility and mechanical and moulding properties [1-3]. However, silicone has the propensity to become rapidly colonized by microorganisms that form a biofilm on the surfaces of the medical devices, which increases the risk of infection and limits the devices life time. Voice prostheses are mainly made of silicone rubber [4-6]. The insertion of voice prosthesis in a surgically created tracheoesophageal fistula is a standard method for voice rehabilitation after total laryngectomy [7, 8]. However, the biofilm on the surface of the prosthesis limits it life time to an average in situ life time of around 3 months [9-11]. These biofilms comprise a mixture of fungi and bacteria. In mixed fungal and bacterial biofilms, a range of different interactions can occur, such as enhanced surface colonization, increased resistance to antimicrobials compared with single species biofilms. The fungi most routinely identified are species of Candida, in which Candida albicans and Candida tropicalis are

regarded as the main fungal species [9, 12]. Next to Candida species, several bacterial members of the commensal oral and skin flora of the host have been detected, including Staphylococcus, Streptococcus species, Lactobacilli and Rothia dentocariosa [13-15]. Chitosan is low toxicity and good biocompatibility, which is widely used in biomedical and pharmaceutical fields. Several works have reported its antimicrobial activity [16-20]. The antibiofilm activities of chitosan and some of its derivatives on fungi and bacteria also have been evaluated [21-23]. However, little information is known about the effect of chitosan and its derivatives on mixed species biofilm. In our previous study [23, 24], carboxymethyl chitosan (CM-chitosan), a chitosan derivative, could inhibit bacterial biofilm formation. In this study, the antibiofilm effect of CM-chitosan on mixed species biofilm formation of fungi and bacteria on in vitro silicone should be determined. 2. Materials and methods 2.1. Strains and media The strains used in this study were C. albicans, C. tropicalis, Lactobacillus gasseri, Streptococcus salivarius, R. dentocariosa, and Staphylococcus epidermidis. All strains were clinical isolates from voice prostheses of laryngectomized patients in routine follow up examinations. Dysfunctional voice prostheses due to biofilm formation were explanted from the tracheoesophageal fistulas and processed within 24 h. The prostheses were

vortexed in 5 ml PBS for 3 min, the microbial specimen were isolated and analyzed using standard microbiology methods, stored in -80°C and thawed before use. Yeast peptone dextrose (YPD) medium (1% yeast extract, 2% peptone, 2% dextrose [Sigma-Aldrich, Austria]) was used for culturing Candida species while Tryptic Soy Broth (TSB) was utilized for bacteria. TSB was previously determined to be an optimal medium for supporting both candidal and bacterial biofilm growth [25, 26]. Medical

grade

silicone

was

purchased

from

Websinger,

Austria.

CM-chitosan (molecular weight= 30 kDa, deacetylation degree= 90%, viscosity= 16 MPa s and O-carboxymethylation degree = 91.9%) was obtained from G.T.C. Bio Corporation (Qingdao, China). 2.2. Growth of mixed biofilms on silicone plates Mixed biofilm formation on silicone plates was performed as described previously [25] with minor modifications. In brief, medical grade silicone plates were sterilized and placed into wells of a 96-well microplate, and then air dried in a biological safety cabinet. Overnight cultures of fungi and bacteria were diluted to OD600 0.01 with TSB media. Equal volumes of these cell suspensions were mixed, and 200 μl of this mixed cell suspension was added to the wells of 96-well microplate. After the adhesion phase, nonadherent cells were removed by rinsing with PBS. The plates were incubated at 37°C for 48 h without shaking. 2.3. Effect of CM-chitosan on biofilm

Biofilms were grown as described above, using medical silicone plates as the substrate. 200 μl of the mixed cell suspension containing CM-chitosan at different concentrations (0, 1.25 and 2.5 mg/ml) were added to the wells. Afterwards, biofilms were formed as described above. 2.4. Crystal violet assay In order to quantify biofilm biomass during polymicrobial growth, biofilms were grown as described above and processed for crystal violet staining as previously described [25], with some modifications. Briefly, mixed biofilm formation formed on silicone plates in 96-well microplate and wells were extensively washed three times with PBS. Biofilms were stained with 200 μl of 0.1% (w/v) crystal violet solution for 30 min, washed and incubated in 200 μl of 30% (v/v) acetic acid for 15 min to extract the crystal violet retained by the cells. The extract was used to determine the amount of biofilm by measuring its A 590 with a microtiter plate reader. 2.5. Measurement of biofilm metabolic activity The

metabolic

activity

of

biofilms

was

calculated

using

a

2,

3-bis(2-methoxy-4-nitro-5-sulfo-phenyl)-2H-tetrazolium-5 carboxanilide (XTT) reduction assay as described previously [21] with minor modifications. Mixed biofilm formation formed on silicone plates in 96-well microplate and wells were washed once in PBS. Silicone plates were incubated at 37°C for 2 h with 150 μl XTT working reagent (XTT 180 mg/l; AppliChem, Darmstadt, Germany); conversion of the XTT substrate to a soluble colored formazan

product correlates with cell viability. The resulting absorbance was read at 490 nm. 2.6. Confocal laser scanning microscopy (CLSM) Images of mixed biofilm with or without CM-chitosan treatment were observed by CLSM. For CLSM observation, mixed biofilm developed on silicone platelets were rinsed with PBS and stained with a Live/Dead ® BacLight™ Bacterial Viability and Counting Kit (L34856, Invitrogen) for 30 min at room temperature in the dark. This kit contains two nucleic acid-specific dyes: Syto9 is membrane-permeable, will stain all cells and can be detected by green

fluorescence,

and

propidium

iodide

(PI)

which

is

membrane-impermeable, stains cells with damaged membranes and gives red fluorescence. 2.7. Adhesion assays Initial adhesion was performed as described previously [27] with minor modifications. In brief, 200 μl of the mixed cell suspension was added to each well of a 96-well microplate and incubated at 37°C for 90 min. The 96-well microplate was added 200 μl of PBS and discarded slowly for five times to remove unattached cells, and then vigorously pipetting up and down. After serial dilutions, viable counts were assessed on agar plates. Results were given as inhibition percentages using the following formula by Pierce et al. [27]: % I = 100 − (CFUsample/ CFUcontrol) × 100

2.8. Cell-cell interaction assays Cell-cell interaction was determined as described previously [28] with minor modifications. After inoculation, CM-chitosan was added at different times (90 min, 12h and 24h) to different concentrations. After being incubated at 37°C for an additional 48 h, biofilms were rinsed, stained with crystal violet and quantified as described above. 2.9 Effect of CM-chitosan on Candida yeast–to-hyphal transition Candida yeast–to-hyphal transition was performed as described previously [29]. Fungal cells were inoculated into YPD plus 10% fetal bovine serum (FBS) at a final OD600 0.1 with or without CM-chitosan (2.5 mg/ml). After 4 h, hyphal morphology was assessed microscopically. 2.10. Scanning electron microscope (SEM) Silicone plates with adhesion of cells or mixed biofilms were fixed in 3 vol.% glutaraldehyde solution overnight at 4°C, and then subjected to serial dehydration with 70%, 80%, 96%, 100% ethanol for 20 min each. The substrates were chemically-dried with Hexamethyldisilazane (Sigma-Aldrich, St. Louis, US), coated with gold, and then examined with SEM (JSM 6310, Jeol Ltd, Akishima, Tokyo, Japan) at an acceleration voltage of 15 kV. 3. Results 3.1. Inhibition of CM-chitosan on mixed biofilm In order to examine the ability of CM-chitosan to inhibit mixed species biofilm formation on medical grade silicone surface, mixed species biofilms were

grown attached to the surfaces of medical grade silicone with or without CM-chitosan. The crystal violet assay was used to measure the mixed species biofilms formation grown on silicone plates. Quantization of biofilm remaining attached to the plates revealed that CM-chitosan treatment resulted in a 72.87% reduction in the amount of surface-associated biofilm with the concentration of CM-chitosan 2.5 mg/ml (Fig. 1). The metabolic activity of biofilm was measured with XTT assay. As shown in Fig. 2, a significant difference was observed between CM-chitosan-treated group and the control group. The metabolic activity of mixed species biofilms was reduced 70% after treatment with 2.5 mg/mL CM-chitosan. 3.2. Live/Dead assay by CLSM To visualize mixed biofilm, we used the Live/Dead assay in which viable microbe stain with the membrane permeable Syto9 dye (green) but exclude PI (red). The CLSM images of the biofilm changes with CM-chitosan treatment are displayed in Fig. 3. The results showed that thick green clusters formed on silicone surfaces in control group (Fig. 3A), which indicated the presence of microbial foci, characteristic of biofilm formation. With CM-chitosan treatment, the area occupied by those live (green) cells was drastically reduced and the fluorescence is very low (Fig. 3B), indicating that CM-chitosan resisted microbial colonization. 3.3. Analysis of Biofilm Architecture by SEM

SEM examination was used to correlate the crystal violet and XTT assay results with the visual effects on biofilm formation structure (Fig. 4). From the SEM images, it can be seen that the mature biofilm formed on the silicone surface consisting of densely packed Candida species and bacteria, embedded within a matrix of exopolysaccharides with a high degree of co-adhesion (Fig. 4A). In contrast, on platelets treated by CM-chitosan the biofilm was less dense than on the controls, and demonstrated few layers of cells, profuse cellular debris, together with degrading and morphologically altered yeast cells (Fig. 4B). 3.4. Cell-surface initial interaction assay Fig. 5 summarizes the effects of CM-chitosan on initial adhesion of the mixed species of fungi and bacteria to medical grade silicone. After incubation in 90 min, CM-chitosan reduced the adhesion of Candida and bacteria by 92.58% and 90.23%, respectively. Effect of CM-chitosan on adhesion is also visualized by SEM micrographs (Fig. 6) and showed disruption in number of cells and alteration in structural design of cells. In contrast, the silicone surface in control group is colonized by bacterial and fungal cells (Fig. 6A). 3.5. Cell-cell interaction assay CM-chitosan was also added to biofilms already developed at different stages. The concentration of CM-chitosan 2.5 mg/ml prevented biofilm

formation at efficiencies of 69.86%, 50.88% and 46.58% when added at 90 min, 12h and 24h after biofilm initiation, respectively (Fig. 7). 3.6 Effect of CM-chitosan on Candida yeast–to-hyphal transition We also examined the effect of CM-chitosan on hyphal growth. Fig. 8 shows micrographs of C. albicans cultures after treatment with or without CM-chitosan. Microscopic observation of the CM-chitosan-treated fungal cells revealed an absence of hyphae (Fig. 8B), whereas the control (untreated) culture had an abundant component of hyphal elements (Fig. 8A). CM-chitosan showed marked inhibition of hyphal development of C. albicans. However, we did not observe the hyphal development of C. tropicalis in this work. 4. Discussion Silicone is widely in use for implantable biomedical devices, such as voice prostheses, with its excellent biocompatibility and biodurability [1-3]. However, voice prostheses based on silicone are associated with a high likelihood of nosocomial infections because of biofilm formation on the silicone surface [8]. Numerous efforts have been undertaken to inhibit or control the biofilm formation on silicone-based voice prostheses, such as adjusting the properties of the voice prostheses material or surface modification [30-33]. Chitosan and its derivatives were also used to prevent the biofilm on the silicone by coating or modification [34-36]. Here, the results showed that CM-chitosan can effectively reduce the metabolic activity of cells, which may be attributed to the

ability of cationic chitosan to disrupt negatively charged cell membranes as microbes settle on the surface [37, 38]. The pictures of SEM and CLSM also confirmed the results. For the CLSM observation, CM-chitosan treatment did not bring noticeable changes in the dead cell number, because dead cells could not adhere to the surfaces when co-cultured with CM-chitosan. Similar findings have also been reported [39]. The interaction between the negative charge of the bacterial and fungal cellular membrane and the cationic charge of chitosan also may interfere with surface colonization or adhesion and cell-cell interactions during biofilm formation [40]. In this study, CM-chitosan inhibits the adhesion of Candida and bacteria to the surface and blocks the further development of biofilms at different stages. Planktonic cells are able to attach on the surfaces and form biofilm through a process that includes several steps. The initial stage of biofilm formation is adhesion stage, which constitutes the beneficial contact between planktonic microorganisms and a conditioned surface. After adherence to the surface, the microorganisms begin to produce intercellular connections and polymeric matrix, and then multiply embedded within the exopolysaccharide matrix [41-43]. By reducing the adhesion and the process of biofilm formation, CM-chitosan minimizes biofilm formation. The net positive charge of the microbial surfaces may keep fungal and bacterial cells as planktonic lifestyle, which is easier to clear for dynamic flow and the innate immune system [44-46].

It is well known that C. albicans is a polymorphic fungus which can exist as yeast cells, pseudohyphae, or hyphae [47, 48]. Hyphae play an important role in Candida biofilm maturation [49]. Yeast cells first adhere to the surface, followed by proliferation of yeast cells across the substrate surface and the beginning of hyphal development [50]. The hyphae of Candida are able to penetrate the substrate they are in contact with, such as medical silicone, which compromises the function of the device [11, 51]. In mixed fungal and bacterial biofilms, bacteria can form the dense biofilm on Candida hyphae, leading to macroscopic deposits on the surfaces [52]. It has been reported that chitosan can inhibit hyphal growth and spore germination [53-55]. Here, our results show CM-chitosan can suppress C. albicans hyphal growth. The inhibition of yeast-to-hyphal transition is also involved in, at least in part, charge interaction between the microbes and the chitosan [54, 55]. Chitosan-coated surfaces that have antibiofilm properties against fungi and bacteria have been evaluated in vitro, recently [34, 56, 57]. Moreover, several in-vivo studies also have proved non-toxicity and good biocompatibility of CM-chitosan [58, 59]. Our data demonstrated that CM-chitosan exhibits strong antibiofilm activity against mixed fungal and bacterial biofilms and is not toxic to human immortalized keratinocytes in vitro. The inhibited microbial composition is similar to oropharyngeal biofilms on dysfunctional voice prostheses. Therefore, the findings of this study suggest that CM-chitosan might offer a flexible and biocompatible platform for designing novel antibiofilm

coatings to protect medical devices from mixed biofilms-associated infection and for example prolong in vivo device lifetimes of voice prostheses in laryngectomized patients. 4. Conclusion The findings of this study suggest that CM-chitosan can effectively inhibit the mixed biofilm of fungi and bacteria on silicone in vitro. Although further studies in-vivo are needed to explore, the antibiofilm activity of CM-chitosan on silicone promises it to be a simple and practical agent for biofilm control on voice prostheses.

Conflict of interest The authors claim no conflict of interest. Submission declaration The work described above has not been published previously or under consideration for publication elsewhere. Author contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

References [1] R. Bayston, L.E. Fisher, K. Weber, An antimicrobial modified silicone peritoneal catheter with activity against both Gram positive and Gram negative bacteria, Biomaterials, 30 (2009) 3167-3173. [2] L. Rodrigues, I.M. Banat, J. Teixeira, R. Oliveira, Strategies for the prevention of microbial biofilm formation on silicone rubber voice prostheses, J Biomed Mater Res B Appl Biomater, 81 (2007) 358-370. [3] N. Venkatesan, S. Shroff, K. Jayachandran, M. Doble, Polymers as Ureteral Stents, J Endourol, 24 (2010) 191-198. [4] A.J. Timmermans, H.J. Harmsen, C. Bus-Spoor, K.J. Buijssen, C. van As-Brooks, M.C. de Goffau, R.H. Tonk, M.W. van den Brekel, F.J. Hilgers, B.F. van der Laan, Biofilm formation on the Provox ActiValve: Composition and ingrowth analyzed by Illumina paired-end RNA sequencing, fluorescence in situ hybridization, and confocal laser scanning microscopy, Head Neck, (2015). [5] H.C. van der Mei, K.J. Buijssen, B.F. van der Laan, E. Ovchinnikova, G.I. Geertsema-Doornbusch, J. Atema-Smit, B. van de Belt-Gritter, H.J. Busscher, Voice prosthetic biofilm formation and Candida morphogenic

conversions in absence and presence of different bacterial strains and species on silicone-rubber, PLoS One, 9 (2014) e104508. [6] L. Rodrigues, I.M. Banat, J. Teixeira, R. Oliveira, Strategies for the prevention of microbial biofilm formation on silicone rubber voice prostheses, J Biomed Mater Res B Appl Biomater, 81 (2007) 358-370. [7] A.Y. Attieh, J. Searl, N.H. Shahaltough, M.M. Wreikat, D.S. Lundy, Voice restoration following total laryngectomy by tracheoesophageal prosthesis: effect on patients' quality of life and voice handicap in Jordan, Health Qual Life Outcomes, 6 (2008) 26. [8] M.J. Talpaert, A. Balfour, S. Stevens, M. Baker, F.A. Muhlschlegel, C.W. Gourlay, Candida biofilm formation on voice prostheses, J Med Microbiol, 64 (2015) 199-208. [9] J.J. Oosterhof, K.J. Buijssen, H.J. Busscher, B.F. van der Laan, H.C. van der Mei, Effects of quaternary ammonium silane coatings on mixed fungal and bacterial biofilms on tracheoesophageal shunt prostheses, Appl Environ Microbiol, 72 (2006) 3673-3677. [10] F.J. HILGERS, A.J. BALM, Long‐ term results of vocal rehabilitation after total laryngectomy with the low‐ resistance, indwelling ProvoxTM Voice prosthesis system, Clin Otolaryngol Allied Sci, 18 (1993) 517-523. [11] H.F. Mahieu, H.K.F. Vansaene, H.J. Rosingh, H.K. Schutte, Candida Vegetations on Silicone Voice Prostheses, Arch Otolaryngol, 112 (1986) 321-325.

[12] T.G.M. Bauters, M. Moerman, H. Vermeersch, H.J. Nelis, Colonization of voice prostheses by albicans and non-albicans Candida species, Laryngoscope, 112 (2002) 708-712. [13] G.J. Elving, H.C. van der Mei, R. van Weissenbruch, H.J. Busscher, F.W. Albers, Comparison of the microbial composition of voice prosthesis biofilms from patients requiring frequent versus infrequent replacement, Ann Oto, Rhinol Laryn, 111 (2002) 200-203. [14] K.J.D.A. Buijssen, H.J.M. Harmsen, H.C. van der Mei, H.J. Busscher, B.F.A.M. van der Laan, Lactobacilli: Important in biofilm formation on voice prostheses, Otolaryng Head Neck, 137 (2007) 505-507. [15] K.J. Buijssen, B.F. van der Laan, H.C. van der Mei, J. Atema–Smit, P. van den Huijssen, H.J. Busscher, H.J. Harmsen, Composition and architecture of biofilms on used voice prostheses, Head neck, 34 (2012) 863-871. [16] A. Peña, N.S. Sánchez, M. Calahorra, Effects of chitosan on Candida albicans: conditions for its antifungal activity, BioMed Res Int, 2013 (2013). [17] L. Yien, N.M. Zin, A. Sarwar, H. Katas, Antifungal activity of chitosan nanoparticles and correlation with their physical properties, Int J Mater, 2012 (2012). [18] A.H. Doulabi, H. Mirzadeh, M. Imani, N. Samadi, Chitosan/polyethylene glycol fumarate blend film: Physical and antibacterial properties, Carbohyd Polym, 92 (2013) 48-56.

[19] T. Wang, X.-K. Zhu, X.-T. Xue, D.-Y. Wu, Hydrogel sheets of chitosan, honey and gelatin as burn wound dressings, Carbohyd Polym, 88 (2012) 75-83. [20] E. Costa, S. Silva, F. Tavaria, M. Pintado, Antimicrobial and antibiofilm activity of chitosan on the oral pathogen Candida albicans, Pathogens, 3 (2014) 908-919. [21] Y. Pu, A. Liu, Y. Zheng, B. Ye, In vitro damage of Candida albicans biofilms by chitosan, Exp Ther Med, 8 (2014) 929-934. [22] A. Silva-Dias, A. Palmeira-de-Oliveira, I.M. Miranda, J. Branco, L. Cobrado, M. Monteiro-Soares, J.A. Queiroz, C. Pina-Vaz, A.G. Rodrigues, Anti-biofilm activity of low-molecular weight chitosan hydrogel against Candida species, Med Microbiol Immun, 203 (2014) 25-33. [23] Y. Tan, F. Han, S. Ma, W. Yu, Carboxymethyl chitosan prevents formation of broad-spectrum biofilm, Carbohyd Polym, 84 (2011) 1365-1370. [24] Y. Tan, S. Ma, C. Liu, W. Yu, F. Han, Enhancing the stability and antibiofilm activity of DspB by immobilization on carboxymethyl chitosan nanoparticles, Microbiol Res, 178 (2015) 35-41. [25] B. Adam, G.S. Baillie, L.J. Douglas, Mixed species biofilms of Candida albicans and Staphylococcus epidermidis, J Med Microbiol, 51 (2002) 344-349.

[26] R.M. Shanks, J.L. Sargent, R.M. Martinez, M.L. Graber, G.A. O'Toole, Catheter lock solutions influence staphylococcal biofilm formation on abiotic surfaces, Nephrol Dial Transplant, 21 (2006) 2247-2255. [27] C.G. Pierce, P. Uppuluri, A.R. Tristan, F.L. Wormley, E. Mowat, G. Ramage, J.L. Lopez-Ribot, A simple and reproducible 96-well plate-based method for the formation of fungal biofilms and its application to antifungal susceptibility testing, Nat protoc, 3 (2008) 1494-1500. [28] J. Valle, S. Da Re, N. Henry, T. Fontaine, D. Balestrino, P. Latour-Lambert, J.-M. Ghigo, Broad-spectrum biofilm inhibition by a secreted bacterial polysaccharide, P Natl Acad Sci USA, 103 (2006) 12558-12563. [29] M.R. Cruz, C.E. Graham, B.C. Gagliano, M.C. Lorenz, D.A. Garsin, Enterococcus faecalis inhibits hyphal morphogenesis and virulence of Candida albicans, Infect Immun, 81 (2013) 189-200. [30] D.J. Balazs, K. Triandafillu, P. Wood, Y. Chevolot, C. van Delden, H. Harms, C. Hollenstein, H.J. Mathieu, Inhibition of bacterial adhesion on PVC endotracheal tubes by RF-oxygen glow discharge, sodium hydroxide and silver nitrate treatments, Biomaterials, 25 (2004) 2139-2151. [31] F. Abbasi, H. Mirzadeh, A.A. Katbab, Bulk and surface modification of silicone rubber for biomedical applications, Polym Int, 51 (2002) 882-888. [32] A. Ramakrishnan, R. Dhamodharan, J. Ruhe, Controlled growth of PMMA brushes on silicon surfaces at room temperature, Macromol Rapid Comm, 23 (2002) 612-616.

[33] L. Rodrigues, H. Van der Mei, J.A. Teixeira, R. Oliveira, Biosurfactant from Lactococcus lactis 53 inhibits microbial adhesion on silicone rubber, Appl Microbiol Biot, 66 (2004) 306-311. [34] R. Wang, K.G. Neoh, Z. Shi, E.T. Kang, P.A. Tambyah, E. Chiong, Inhibition of Escherichia coli and Proteus mirabilis adhesion and biofilm formation on medical grade silicone surface, Biotechnol Bioeng, 109 (2012) 336-345. [35] K. Mai-ngam, Comblike poly (ethylene oxide)/hydrophobic C 6 branched chitosan surfactant polymers as anti-infection surface modifying agents, Colloid Surface B, 49 (2006) 117-125. [36] X. Lu, W. Cui, Y. Huang, Y. Zhao, Z. Wang, Surface modification on silicon with chitosan and biological research, Biomed Mater, 4 (2009) 044103. [37] E.I. Rabea, M.E.-T. Badawy, C.V. Stevens, G. Smagghe, W. Steurbaut, Chitosan as antimicrobial agent: applications and mode of action, Biomacromolecules, 4 (2003) 1457-1465. [38] L.R. Martinez, M.R. Mihu, M. Tar, R.J. Cordero, G. Han, A.J. Friedman, J.M. Friedman, J.D. Nosanchuk, Demonstration of antibiofilm and antifungal efficacy of chitosan against candidal biofilms, using an in vivo central venous catheter model, J Infect Dis, 201 (2010) 1436-1440. [39] V. Antoci, C.S. Adams, J. Parvizi, H.M. Davidson, R.J. Composto, T.A. Freeman, E. Wickstrom, P. Ducheyne, D. Jungkind, I.M. Shapiro, The

inhibition

of

Staphylococcus

epidermidis

biofilm

formation

by

vancomycin-modified titanium alloy and implications for the treatment of periprosthetic infection, Biomaterials, 29 (2008) 4684-4690. [40] Y. Miyake, T. Tsunoda, S. Minagi, Y. Akagawa, H. Tsuru, H. Suginaka, Antifungal drugs effect adherence of Candida albicans to acrylic surfaces by changing the zeta-potential of fungal cells, FEMS Microbiol Lett, 69 (1990) 211-214. [41] B. Prakash, B. Veeregowda, G. Krishnappa, Biofilms: a survival strategy of bacteria, Curr Sci India, 85 (2003) 1299-1307. [42] H.-C. Flemming, T.R. Neu, D.J. Wozniak, The EPS matrix: the “house of biofilm cells”, J Bacteriol, 189 (2007) 7945-7947. [43] R. Bachmann, R. Edyvean, AFM study of the colonisation of stainless steel by Aquabacterium commune, Int Biodeter Biodegr, 58 (2006) 112-118. [44] T. Savard, C. Beaulieu, I. Boucher, C.P. Champagne, Antimicrobial action of hydrolyzed chitosan against spoilage yeasts and lactic acid bacteria of fermented vegetables, J Food Protect, 65 (2002) 828-833. [45] C. de la Fuente-Núñez, F. Reffuveille, L. Fernández, R.E. Hancock, Bacterial biofilm development as a multicellular adaptation: antibiotic resistance and new therapeutic strategies, Curr Opin Microbiol, 16 (2013) 580-589.

[46] H.J. Busscher, E. Engels, R.J. Dijkstra, H.C. van der Mei, Influence of a chitosan on oral bacterial adhesion and growth in vitro, Eur J Oral Sci, 116 (2008) 493-495. [47] S.W. Martin, J.B. Konopka, Lipid raft polarization contributes to hyphal growth in Candida albicans, Eukaryot Cell, 3 (2004) 675-684. [48] M. Chevalier, E. Medioni, I. Precheur, Inhibition of Candida albicans yeast-hyphal transition and biofilm formation by Solidago virgaurea water extracts, J Med Microbiol, 61 (2012) 1016-1022. [49] J.R. Blankenship, A.P. Mitchell, How to build a biofilm: a fungal perspective, Curr Opin Microbiol, 9 (2006) 588-594. [50] J.S. Finkel, A.P. Mitchell, Genetic control of Candida albicans biofilm development, Nat Rev Microbiol, 9 (2011) 109-118. [51] A. Brand, Hyphal growth in human fungal pathogens and its role in virulence, Int J Microbiol, 2012 (2012) 517529. [52] M.E. Shirtliff, B.M. Peters, M.A. Jabra-Rizk, Cross-kingdom interactions: Candida albicans and bacteria, FEMS Microbiol Lett, 299 (2009) 1-8. [53] M. Plascencia‐ Jatomea, G. Viniegra, R. Olayo, M.M. Castillo‐ Ortega, K. Shirai, Effect of chitosan and temperature on spore germination of Aspergillus niger, Macromolecular Bioscience, 3 (2003) 582-586. [54] J. Palma-Guerrero, H.B. Jansson, J. Salinas, L.V. Lopez-Llorca, Effect of chitosan on hyphal growth and spore germination of plant pathogenic and biocontrol fungi, J Appl Microbiol, 104 (2008) 541-553.

[55] J. Xu, X. Zhao, X. Han, Y. Du, Antifungal activity of oligochitosan against Phytophthora capsici and other plant pathogenic fungi in vitro, Pesticide Biochemistry and Physiology, 87 (2007) 220-228. [56] R.P. Carlson, R. Taffs, W.M. Davison, P.S. Stewart, Anti-biofilm properties of chitosan-coated surfaces, J Biomater Sci Polym Ed, 19 (2008) 1035-1046. [57] S. Bratskaya, D. Marinin, F. Simon, A. Synytska, S. Zschoche, H.J. Busscher, D. Jager, H.C. van der Mei, Adhesion and viability of two enterococcal

strains

on

covalently

grafted

chitosan

and

chitosan/kappa-carrageenan multilayers, Biomacromolecules, 8 (2007) 2960-2968. [58] W. Dong, B. Han, Y. Feng, F. Song, J. Chang, H. Jiang, Y. Tang, W. Liu, Pharmacokinetics and biodegradation mechanisms of a versatile carboxymethyl derivative of chitosan in rats: in vivo and in vitro evaluation, Biomacromolecules, 11 (2010) 1527-1533. [59] L.Q. Yang, Y.Q. Lan, H. Guo, L.Z. Cheng, J.Z. Fan, X. Cai, L.M. Zhang, R.F. Chen, H.S. Zhou, Ophthalmic drug-loaded N,O-carboxymethyl chitosan hydrogels: synthesis, in vitro and in vivo evaluation, Acta Pharmacol Sin, 31 (2010) 1625-1634.

Figure legends

Figure 1 Inhibition effect of CM-chitosan on mixed species biofilm formation. Mixed cells were co-incubated with various concentrations (0, 1.25 and 2.5 mg/ml) of CM-chitosan for 48 h; and their biofilm formation was compared to that without CM-chitosan. The results shown represent the means and standard deviations (error bars) of three independent experiments, *p<0.05 for comparison between the untreated and CM-chitosan-treated groups.

Figure 2 Inhibition effect of CM-chitosan on the metabolic activity of mixed species biofilm. Mixed cells were co-incubated with various concentrations (0, 1.25 and 2.5 mg/ml) of CM-chitosan for 48 h; and their biofilm metabolic activity was compared to that without CM-chitosan. Values obtained are given as the percentage of biofilm metabolic activity. The results shown represent the means and standard deviations (error bars) of three independent

experiments,

*p<0.05

for

CM-chitosan-treated groups.

comparison

between

the

untreated

and

Figure 3 CLSM images of mixed species biofilm formations on medical grade silicone surface with media supplemented without (A) or with (B) CM-chitosan. Biofilms were stained with the Live/Dead® BacLight™ Bacterial Viability and Counting Kit. CLSM reconstructions show the three-dimensional staining pattern for live cells (SYTO-9, green) and dead cells (propidium iodide, red). Magnification, ×20.

Figure 4 SEM images of mixed species biofilm formations on medical grade silicone surface with media supplemented without (A) or with (B) CM-chitosan.

Figure 5 Inhibition of cell-surface initial interaction by CM-chitosan. Mixed cells were co-incubated with various concentrations (0, 1.25 and 2.5 mg/ml) of

CM-chitosan for 90 min; and viable counts of Candida and bacteria on agar plates was compared to that without CM-chitosan. Values obtained given as the percentage of adhesion inhibition. The results shown represent the means and standard deviations (error bars) of three independent experiments, *p<0.05 for comparison between the untreated and CM-chitosan-treated groups.

Figure 6 SEM images of mixed species adhesion on medical grade silicone surface with media supplemented without (A) or with (B) CM-chitosan.

Figure 7 Biofilm formations of mixed species by addition of the CM-chitosan at different times. After inoculation, CM-chitosan was added at different times (90 min, 12h or 24 h) to different concentrations (0, 1.25 and 2.5 mg/ml) and co-incubated for an additional 48 h. The biofilm production was compared to that without CM-chitosan. (A) CM-chitosan was added at 90 min. (B) CM-chitosan was added at 12 h. (C) CM-chitosan was added at 24 h. Values obtained are given as the percentage of biofilm formation. The results shown represent the means and standard deviations (error bars) of three independent experiments,

*p<0.05

for

CM-chitosan-treated groups.

comparison

between

the

untreated

and

Figure 8 Effect of CM-chitosan on hyphal growth of C. albicans. C. albicans was grown in YPD medium containing 10% FBS in the absence (A) or presence (B) of CM-chitosan. Samples were withdrawn after incubation at 37 °C for 4 h, and photographed.