Supporting Information. Chitosan Derivatives Active against Multi-Drug-Resistant. Bacteria and Pathogenic Fungi: In Vivo Evaluation as Topical

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1 Supporting Information Chitosan Derivatives Active against Multi-Drug-Resistant Bacteria and Pathogenic Fungi: In Vivo Evaluation as Topical Antimicrobials Jiaul Hoque, Utsarga Adhikary, Vikas Yadav, Sandip Samaddar, Mohini Mohan Konai, Relekar Gnaneshwar Prakash, Krishnamoorthy Paramanandham, Bibek R. Shome, Kaustuv Sanyal and Jayanta Haldar * Chemical Biology and Medicinal Chemistry Laboratory, New Chemistry Unit, Jawaharlal Nehru Centre for Advanced Scientific Research, Jakkur, Bengaluru , India jayanta@jnacsr.ac.in Molecular Mycology Laboratory, Molecular Biology and Genetics Unit, Jawaharlal Nehru Centre for Advanced Scientific Research, Jakkur, Bengaluru , India National Institute of Veterinary Epidemiology and Disease Informatics (NIVEDI) Ramagondanahalli, Yelahanka, Bengaluru , India

2 EXPERIMENTAL PROCEDURES Synthesis of Quaternized Chitosan Derivatives. Chitosan (2.5 g) was suspended in Millipore water (200 ml), and then acetic acid (1 ml, 0.5%, v/v) was added to the suspension. The chitosan-acetic acid mixture was stirred for about 12 h at room temperature to obtain a clear solution. GTMAC was then added to the polymer solution in three portions at nearly 2 h intervals. The GTMAC to chitosan mole ratio was varied from 4:1 to 8:1. The reaction was allowed to proceed at 55 C for 18 h after the final addition of GTMAC. The reaction mixture was then diluted with 300 ml water and the product was precipitated with excess of acetone. The chitosan derivatives were filtered through a sintered glass funnel and washed with acetone repeatedly and finally with acetone-ethanol mixture (1:1). The products were characterized by 1 H NMR, 13 C cross-polarization magic-angle spinning (CP-MAS) NMR and FT-IR spectroscopy (yield higher than 90%). The degrees of substitutions (DS) or degrees of quaternization (DQ) in the quaternary chitosan derivatives were determined by conductivity measurements. Determination of Degree of Substitution (DS). DS of the polymers was calculated by titrating the amount of chloride (Cl ) ions on the polymers with aqueous AgNO 3 solution. The moles of reacted GTMAC to the mole of repeating sugar unit of chitosan was defined as DS. HTCC polymers (0.025 g) were dissolved in Millipore water (50 ml) and then solution of AgNO 3 (0.02 M) was used to titrate the polymer solutions. The DS of HTCC polymers was calculated using the following Equation 1: C AgNO3 V AgNO3 = m HTCC DS)/ (DS M 3 ) + (1 DS DA) M 1 + (DA M 2 ) (1) In this equation, C AgNO3 is the concentration of AgNO 3, V AgNO3 is the volume of the AgNO 3 solution at the point of equivalence, m is the mass of HTCC used for titration, M 1 is the molecular weight of the glucosamine repeating unit, M 2 is the molecular weight of the N- acetyl glucosamine repeating unit, M 3 is the molecular weight of GTMAC substituted

3 repeating unit, i.e., quaternary unit, DA is the degree of acetylation of the native chitosan (0.15 or 0.21). Microorganisms and Culture Conditions. S. aureus, MRSA, A. baumannii, E. coli and K. pneumoniae were cultured in nutrient broth (2.0 g of yeast extract, 1.0 g of beef extract, 5.0 g of peptone, and 5.0 g of NaCl in 1000 ml of Millipore water). Brain heart infusion broth (12.5 g of calf brains infusion form, 10 g of peptone, 5.0 g beef heart infusion form, 2.0 g D- glucose, 2.5 g Na 2 HPO 4 and 5.0 g NaCl in 100 ml of Millipore water) was used as growth medium for VRE. Agar ( %) was used along with the above-mentioned growth medium to prepare the solid medium. Bacterial samples in 30% glycerol were stored at 80 ºC. These bacterial stock solutions (5 L) were added to 3 ml of the corresponding growth medium and were grown at 37 ºC for 6 h before antibacterial experiments. Clinical isolates (12 species including both Gram-positive and Gram-negative samples) were obtained from the Department of Neuromicrobiology, NIMHANS, Bangalore , India. Nutrient broth was used for all the clinical isolates to determine the antibacterial activity of the HTCC polymers. All the fungal strains were grown in YPD (1% yeast extract, 2% peptone and 2% dextrose) media at 30 C. Antibacterial Assay. Bacterial cultures, grown for 6 h, were diluted to give 10 5 CFU/mL in respective media. The HTCC polymers were dissolved in sterilized Millipore water at 4 mg/ml and were serially diluted (2-fold serial dilution). The polymer solutions (50 μl) were then added to the wells of a 96-well plate followed by the addition of 150 μl of bacterial suspension ( 10 5 CFU/mL). The plates were then kept at 37 C for 24 h in an incubator under constant shaking. Finally, the optical density (OD) of the suspension was recorded to evaluate the efficacy of the polymers in inhibiting bacterial growth. Each concentration was used in triplicate and the assay was repeated at least twice. The antibacterial activity of the polymers was expressed as minimum inhibitory concentration (MIC) where the OD value of the

4 polymer treated bacterial suspension was similar to that of control having no bacteria. The minimum concentration at which no bacteria survived (minimum bactericidal concentration, MBC) was determined by plating the suspension on solid agar that showed negligible turbidity in the MIC experiment. The agar plates were then incubated at 37 C for 24 h till the bacterial colonies developed. Concentration at which no bacterial colony was observed on agar plate was taken as the MBC of the respective polymer. Bactericidal Time-kill Assay. The rate of bactericidal action at which the polymers killed bacteria was evaluated by time kill kinetics. Briefly, S. aureus and E. coli were grown for 6 h as described in the microorganism and culture section. Bacterial suspension (150 L) were added to the solution of two most active polymers (HTCC 3 and HTCC 6) at two different concentrations (MIC and 6 MIC, 50 L) to give approximately CFU/mL S. aureus and CFU/mL of E. coli. The plate containing bacterial suspension was then incubated at 37 C. At different time intervals (0, 30, 60, 90, 120, 240 and 360 min), 10 µl of the aliquots from the bacterial suspension was withdrawn and serially diluted (10-fold serial dilution) in 0.9 % saline. 20 µl of the dilution was plated on solid agar plates and incubated at 37 C for 24 h. The bacterial colonies were counted and results were represented in logarithmic scale, i.e. log 10 (CFU/mL). A similar experiment was performed by using water (50 L) as control. Mechanism of Action. Cytoplasmic Membrane Depolarization Assay. Freshly grown (for about 6 h) bacteria were harvested (3500 rpm, 5 min), washed in 1:1 mixture of 5 mm glucose and 5 mm HEPES buffer (ph = 7.2) and resuspended in 1:1:1 mixture of 5 mm HEPES buffer, 5 mm glucose and 100 mm KCl. The suspension ( 10 8 CFU/mL, 150 L) was then added to the wells of a 96-well plate (black plate with clear bottom with lid). Then 3, 3 -dipropylthiadicarbocyanine iodide (DiSC 3 5) (8 M, 50 L) was mixed with the bacterial suspension and pre-incubated

5 for about 30 min for S. aureus and 40 min for E. coli (50 L of 800 M of EDTA was also mixed with E. coli suspension). After the incubation, fluorescence intensity of DiSC 3 5 was measured for the next 8 min at 2 min interval (excitation wavelength = 622 nm, emission wavelength = 670 nm). Bacterial suspensions were then transferred to another black wellplate containing 50 L of g/ml of HTCC polymers. The fluorescence intensity was then monitored immediately for another 12 min at every 2 min interval. A control experiment was performed by treating the pre-incubated bacterial suspension and dye solution only with sterile Millipore water (50 L). Intracellular K + ion Leakage Assay. Freshly grown were harvested similarly as described before, washed and resuspended in 1:1 mixture of 10 mm HEPES buffer and 0.5% glucose. Bacterial suspension ( 10 8 CFU/mL, 150 L) was then transferred into 96-well plate (black plate with clear bottom with lid). K + ion sensitive dye PBFI-AM (4 M, 50 L) was added to the wells containing bacterial suspension and pre-incubated for about 30 min for S. aureus and 40 min for E. coli. Fluorescence intensity was then monitored for next 8 min at 2 min interval (excitation wavelength = 346 nm, emission wavelength = 505 nm). The suspensions were transferred to another black well-plate containing HTCC polymer solution (50 L of g/ml) and fluorescence intensity was monitored for 12 min at every 2 min interval. A control experiment was performed by treating the pre-incubated bacterial suspension only with Millipore water (50 L). Outer Membrane Permeabilization Assay. Outer membrane permeabilization of Gramnegative bacteria was studied by the hydrophobic dye N-phenylnapthylamine (NPN). Freshly grown bacteria (E. coli) were harvested similarly as mentioned earlier, washed, and resuspended in 1:1 mixture of 5 mm glucose and 5 mm HEPES buffer. The suspension ( 10 8 CFU/mL, 150 L) was then transferred to a black 96-well plate. NPN dye (10 μm, 50 L) was added to the wells containing bacterial suspension and pre-incubated for about 40

6 min. After the incubation, fluorescence intensity was recorded for next 8 min at 2 min interval (excitation wavelength = 350 nm, emission wavelength = 420 nm). The bacterial suspensions were then transferred to another black plate containing 50 L of g/ml of polymers. Fluorescence intensity of the dye was monitored immediately for another 12 min at 2 min interval. A control experiment was made by treating the pre-incubated bacterial suspension only with Millipore water (50 L). Inner Membrane Permeabilization Assay. S. aureus and E. coli were grown and harvested, washed, and resuspended in 1:1 mixture of 5 mm glucose and 5 mm HEPES buffer. Bacterial suspension ( 10 8 CFU/mL, 150 L) was then added to the wells of a black 96-well plate. Then the membrane impermeable dye propidium iodide (PI) (10 μm, 50 L) was added to the wells containing bacterial suspension and equilibrated for about 30 min and 40 min for S. aureus and E. coli respectively. Then the fluorescence intensity was measured for next 8 min at 2 min interval (excitation wavelength = 535 nm, emission wavelength = 617 nm). The bacterial suspensions were then transferred to another black plate containing 50 L of g/ml of HTCC polymers and fluorescence intensity was recorded immediately for next 12 min at 2 min interval. A control experiment was made by treating the preincubated bacterial suspension only with Millipore water (50 L). Confocal Laser Scanning Microscopy (CLSM). Freshly grown bacteria ( 10 9 CFU/mL) were centrifuged (at rpm for 1 min) and the pellet was resuspended in 1X PBS to give 10 8 CFU/mL. The suspension (150 μl) was then added to the wells of 96-well plate followed by the addition of 50 L of 8000 g/ml of HTCC 3. The plate was then kept at 37 C for 2 h under constant shaking. After the treatment, bacteria were collected in an eppendorf tube, centrifuged and resuspended in PBS. The suspension (5 L) was then combined with a mixture containing 3.0 μm green fluorescent dye SYTO 9 and 15.0 μm red fluorescent dye PI. The mixture was incubated in the dark for 15 min. Finally, aliquot (5 μl) was placed on a

7 glass slide, covered with a cover slip, sealed, and examined under a confocal laser scanning microscope (Zeiss 510 Meta Confocal Microscope). Excitation wavelength for SYTO 9 was nm and for PI was nm. Emission wavelength was for SYTO 9 at nm and for PI at nm and was collected using a band-pass filter. Resistance Development Study. One of the most active polymers, HTCC 3, was used to evaluate the propensity of developing bacterial resistance towards HTCC polymers. First, MIC of the polymers was determined against both Gram-positive S. aureus and Gramnegative A. baumannii as described in the protocol for the antibacterial assay. Subsequently MIC values were determined repeatedly with the bacteria grown at the sub-mic (MIC/2) of the polymer. Two control antibiotics norfloxacin and colistin were chosen for S. aureus and for A. baumannii respectively. In the case of norfloxacin and colistin, the initial MIC values were determined against respective bacteria and serial passaging was initiated similarly by transferring bacterial suspension grown at the sub-mic level. After each MIC experiment, bacteria grown at the sub-mic of the test polymer/antibiotics were once again transferred and assayed for next MIC evaluation. The process was repeated for 14 passages for both the bacteria. The fold increase in MIC for test polymer and the control antibiotics was then plotted against the number of passages to evaluate the propensity of bacterial resistance development. Cytotoxicity Assay. Hemolytic Activity. Studies on human subjects such as human red blood cells (hrbc) and human embryo kidney cells (HEK 293) were performed according to the guidelines approved by Institutional Bio-Safety Committee (IBSC) at Jawaharlal Nehru Centre for Advanced Scientific Research (JNCASR). Blood was collected from a healthy donor in heparin containing vacutainer. RBCs were isolated by centrifugation at 3500 rpm for 5 min and resuspended in 1X PBS (5 vol%). RBC suspension (150 μl) was then added to the polymer

8 solutions of different concentrations in a 96-well plate (50 μl). Two control experiments were made, one with only water (50 μl) and the other with of 0.1 vol% solution of triton X- 100 (50 μl). The suspension was then kept at 37 C for 1 h. The plate was then centrifuged at 3500 rpm for 5 min. Supernatant (100 μl) from the wells was then transferred to another new 96-well plate and absorbance at 540 nm was measured. Percentage of hemolysis was calculated as (A A o )/(A total A o ) 100, where A is the absorbance for the polymer containing well, A o is the absorbance for the water containing well, and A total is the absorbance for triton-x containing well, all at 540 nm. To visualize the effect of cationic polymers, treated and non-treated RBC were also imaged by optical microscopy. Fluorescence Microscopy. Human embryo kidney cells (HEK 293) were cultured in DMEM media supplemented with 10% FBS and penicillin-streptomycin solution at 37 C under 5% CO 2-95% air atmosphere. First, cells were first seeded in 96 well plates at a concentration of 10 4 cells/well and were allowed to adhere to the wells of the plate overnight. The seeded cells were treated with HTCC 3 polymer at three different concentrations (at MIC or higher MIC values, e.g., 125, 250 and 500 g/ml). Media and triton-x (0.1 vol%) were used as negative and positive controls respectively. After the incubation, both treated and untreated cells were washed with 1X PBS and stained with the mixture of membrane permeable dye calcein AM (2 μm) and membrane impermeable dye propidium iodide (4.5 μm) for 15 min. Finally, the cells were washed and then imaged with a 10X objective in Leica DM2500 fluorescence microscope. Excitation and emission wavelengths for SYTO 9 were nm and nm, and for PI were nm and nm respectively. Antifungal activity Antifungal Assay. The fungi were grown overnight in YPD media and diluted in fresh media next day to get a cell concentration of 10 5 cells/ml. The polymers were dissolved in Millipore water, serially diluted and taken into the wells of 96-well plate as described before

9 in antibacterial assay section. Fungal cells (150 L) were then added to the polymer solutions and incubated at 30 C for 20 h. Minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) were determined for all the polymers in a similar way as described for the bacteria after recording the OD values of fungal suspension. Kinetics of Antifungal Activity. C. albicans cells were grown overnight in YPD and diluted to get a concentration of 10 5 cells/ml. Approximately, 150 µl of the cell suspension was added into wells of a 96-well plate containing polymer solutions at two different concentrations (MIC and 8 MIC, 50 L). Subsequently, 3 L of cells were spotted on YPD agar plates as the control (0 h) followed by spotting 2 h intervals till 8 h. The agar plates were then incubated at 30 C for about 24 h and visual growth of fungal colonies was monitored to determine the killing kinetics. Mechanism of Antifungal Action. To evaluate the nature of interaction of the cationic chitosan derivatives with the fungal cell membrane, live/dead assay was performed using green fluorescent dye SYTO 9 and red fluorescent dye PI. C. albicans cells were grown as described previously. Cells were taken (150 L, 10 6 cells/ml) into the wells of 96-well plate containing polymer solutions (50 L) to give two different concentrations (MIC and 8 MIC). After 6 h, cells were collected from the wells and centrifuged at 4000 rpm for 5 min. Finally, the cells were washed with 1 ml of 1X PBS and resuspended in 100 µl of the buffer. Then the cells were stained using 1:1 mixture of SYTO 9 and PI (3.0 µm SYTO 9 and 15.0 µm PI) and incubated at room temperature for 30 min in dark. Both treated and nontreated cells were imaged under fluorescence microscope (Olympus microscope, model BX51) using Olympus DP71 camera. SYTO 9 fluorescence was monitored using the green emission filter and PI fluorescence was determined in the red emission filter. The microscopy images were processed using Image Pro-Plus software, Image J and Adobe Photoshop.

10 In-vivo Toxicity (Acute Dermal Toxicity). The numbers of animals per groups, dosage of polymers etc. were used according to the Organisation for Economic Cooperation and Development (OECD) guidelines for the Testing of Chemicals (OECD 425). BALB/c mice (female, 6-8 weeks, g) were used for the toxicity studies. Mice were divided into control and test groups with 5 mice per group. Fur of the mice was shaved 24 h before the experiment. To the shaved region, the polymer solution (80 L) of different concentration was applied to give various dosages (50, 100 and 200 mg/kg). Adverse effect on the skin of mice was monitored along with mortality rate for 14 days post treatment. After 14 day, the skin section where the polymer was applied was taken, fixed in formalin for 24 h and imaged by haemotoxylin and eosin staining.

11 Table S1. Characterization of chitosan derivatives Sample MW of chitosan used (kda) GTMAC/glucosamine molar ratio Degree of substitution (DS) (%) Solubility in water HTCC :1 31 HTCC :1 48 HTCC :1 58 HTCC :1 29 HTCC :1 45 HTCC :1 54 Table S2: Bactericidal activity of chitosan derivatives MBC (µg/ml) Polymer Drug-sensitive bacteria Drug-resistant bacteria S. aureus E. coli P. aeruginosa MRSA VRE K. pneumoniae HTCC HTCC HTCC HTCC HTCC HTCC MBC = Minimum bactericidal concentration; MRSA = methicillin-resistant Staphylococcus aureus; VRE = vancomycin-resistant Enterococcus faecium

12 Figure S1. 1 H NMR spectrum of cationic chiitosan derivative (HTCC 3) in D 2 O performed at 80 C. Figure S2. 13 C CP-MAS spectrum of cationic chiitosan derivative (HTCC 3).

13 Figure S3. Conductivity of HTCC polymers (a) HTCC 1, (b) HTCC 2, (c) HTCC 3, (d) HTCC 4, (e) HTCC 5, and (f) HTCC 6, (concentration = 0.5 mg/ml) as a function of AgNO 3 volume added (concentration = 20 mm; temperature = 28 C).

14 Figure S4. Rate of the antibacterial action of the chitosan derivatives (HTCC 3 and HTCC 6) at two different concentrations: MIC and 6 MIC towards (a) S. aureus and (b) E. coli respectively. Stars represent 50 CFU/mL.

15 Figure S5. Mechanism of antibacterial action of quaternary chitosan derivatives. Outer membrane permeabilization of E. coli by HTCC polymers.

16 Figure S6. Minimum fungicidal concentration of the polymers: Fungal colonies obtained after plating 3 L of media on YPD agar plate from the MIC experiment at different concentration of polymers.

17 Figure S7. Fluorescence microscopy images of HEK 293 cells after treatment with HTCC 3 for 24 h and staining with calcein AM and propidium iodide (PI). (a-c) Non-treated cells (negative control); (d-f) cells treated with HTCC 3 at 250 μg/ml; (g-i) cells treated with HTCC 3 at 500 μg/ml; and (j-l) cells treated with 0.1% triton-x (positive control). Scale bar 100 m.