Fungistatic Activity of Modified Chitosans against Saprolegnia

Dec 5, 2000 - Chitosan derivatives were claimed as antimicrobials for fish and shellfish against infection from Vibrio anguillarum, Edwardsiella tarda...
0 downloads 9 Views 382KB Size
Download PDF
Biomacromolecules 2001, 2, 165-169

165

Fungistatic Activity of Modified Chitosans against Saprolegnia parasitica Riccardo A. A. Muzzarelli,*,† Corrado Muzzarelli,† Renato Tarsi,‡ Michele Miliani,† Francesca Gabbanelli,† and Massimo Cartolari† Centre for Innovative Biomaterials, and Institute of Microbiology, Faculty of Medicine, University of Ancona, Via Ranieri 67, IT-60100 Ancona, Italy Received September 11, 2000; Revised Manuscript Received October 16, 2000

Five chemically modified chitosans were tested for their antifungal activities against Saprolegnia parasitica by the radial growth assay in chitosan-bearing agar, and the fungal growth assay in chitosan-bearing broth. Results indicated that methylpyrrolidinone chitosan, N-carboxymethyl chitosan and N-phosphonomethyl chitosan exerted effective fungistatic action against S. parasitica: in fact the radial growth was nil for 50 h at 20 °C, and the fungus was precipitated when the Bacto YM broth contained one of these chitosans. Electron microscopy observations (TEM and SEM) provided evidence of ultrastructural alterations, damaged fungal structures, uptake of modified chitosans, and hyphal distortion and retraction. Introduction Members of the genus Saprolegnia are responsible for infections of fish and eggs in aquaculture facilities. They multiply on injured, stressed, or infected fish. Saprolegnia parasitica appears as a circular or crescent-shaped cottonlike mycelium around the caudal and anal fins and the head and spreads by radial extension.1 Prevention and treatment of saprolegniasis is therefore outstandingly important. Chitin and chitosan show antimicrobial activity and have been studied in terms of bacteriostatic/bactericide agents, to control growth of algae and to inhibit viral multiplication.2-5 The fungicidal effect of chitosan has been studied,6 in particular for agricultural applications: for instance the soilborn phytopathogenic fungi Fusarium solani and Colletotrichum lindemuthianum were inhibited by chitosan and N-carboxymethyl.7-9 Fusarium acuminatum, Cylindrocladium floridanum, and other pathogens of interest in forest nurseries were inhibited by chitosan in vitro.10 Similarly, Aspergillus flaVus was completely inhibited in field growing corn and peanut.11,12 The effects of chitosan on growth inhibition of fungi such as Botrytis cinerea in tomato and strawberries was correlated with reduction of aflatoxin, elicitation of phytoalexin and phenolic precursors, enhanced production of chitinases, and other factors relevant to the plant defenses; direct contact of A. flaVus with chitosan was reported to produce weakening and swelling of the hyphae. The fungistatic properties of chitosan against Rhizopus stolonifer were related to its ability to induce morphological changes in the cell wall. After seed treatment of wheat, peas and lentils during a 5 year trial, plant yield increased 2030%, and the potential use of chitosan in postharvest preservation of fruits and vegetables was proposed.13,14 Chitosan pyrithione based personal care products active * Corresponding author. Fax +39 071 2204683. † Centre for Innovative Biomaterials, University of Ancona. ‡ Institute of Microbiology, University of Ancona.

against a number of fungi including Candida albicans were developed.15 C. albicans is particularly susceptible to the presence of chitosan and the protecting effect of chitin and chitosan in experimentally induced murine candidiasis was reported. The aderence of C. albicans to human corneocytes was depressed in vitro.16-18 The antifungal properties of chitosan are of interest in the food industry especially because chitosan is a safe biopolymer suitable for oral administration.19 In apple juice, 15 yeasts and molds associated with food spoilage including Mucor racemosus and Byssoclamys spp. were inactivated by chitosan at various concentrations, pH values and temperatures.20 Similar results were obtained with Aspergillus niger and Aspergillus parasiticus in oriental food.21 Some inhibitory effect on the fish pathogenic Oomycete S. parasitica has been reported.22 The hyphae affected by chitosan at 500-600 mg/L were markedly shrunk and contracted. Chitosan derivatives were claimed as antimicrobials for fish and shellfish against infection from Vibrio anguillarum, Edwardsiella tarda, Pasteurella piscicida and several bacteria, in agreement with data obtained on brook trout.23,24 On the other hand chitin, chitosan, and cellulose are accepted diet supplements for cultured fish.25,26 Currently, sodium chloride, hydrogen peroxide, and Malachite green are common remedies when fungal infection affects rainbow trout eggs.27-29 The scope of this work was to compare the antifungal activities of chitosan and various modified chitosans in relation to S. parasitica growth in vitro. The study of a range of differently modified chitosans would permit to identify the modified chitosans endowed with maximum in vitro antifungal activity and to propose them for further in vivo trials. Methods Materials. Chitosan (degree of acetylation 0.18) was supplied by Primex, Bergen, Norway, and five derivatives,

10.1021/bm000091s CCC: $20.00 © 2001 American Chemical Society Published on Web 12/05/2000

166

Biomacromolecules, Vol. 2, No. 1, 2001

listed below, were prepared and characterized according to published methods.30-33 NPHM was prepared by adding phosphorous acid (4 g) to a chitosan (4 g) solution at 70 °C and then formaldehyde (37%, 10.8 g), and protracting heating for 6 h.34 The product was dialyzed and freeze-dried. Each modified chitosan was characterized by H NMR and FT-IR spectroscopy, to define the degree of substitution. The degrees of substitution of modified chitosans with the indicated functional groups were the following: MP, 5-methylpyrrolidinone chitosan,

0.27;30 NCM, N-carboxymethyl chitosan, -NH-CH2COONa, 0.27;31 DCM, N-dicarboxymethyl chitosan,-NHCH2-COONa, monosubstituted, 0.30; -N(CH2-COONa)2, disubstituted, 0.30;32 DMAP, N-dimethylaminopropyl chitosan, 0.40;19,33 NPHM, N-phosphonomethyl chitosan, -NHCH2-PO3Na2, monosubstituted, 0.24, disubstituted, -N(CH2PO3Na2)2, 0.14.34 S. parasitica was supplied by Carolina Biological Supply Co., Burlington, NC. Culture media were supplied by Difco; reagents were supplied by Aldrich, Milan, Italy. Chitosan-Bearing Broth. The Bacto YM broth (Difco) containing yeast, malt, peptone and dextrose was preferred (21 g/L), because the modified chitosans were fully soluble in it. Instead, the inclusion of the chitosans in the Sabouraud broth promoted turbidity or insolubilization that hindered the observation of the fungal growth. Modified chitosans in the freeze-dried form were directly dissolved at room temperature in the YM broth to obtain 0.4, 4, 6, and 8 mg/mL concentrations. Clear solutions with the same pH value were obtained in all cases, except for NCM chitosan-bearing solutions that showed a faint haze. In no case was broth viscosity altered, indicative of absence of interferences between chitosans and broth ingredients. A seeded Petri dish was washed with saline solution, and a suspension was obtained having optical density 1.3. From the latter, a dilute suspension with optical density 0.1 was obtained, and an aliquot (10 µL) was added to each broth (5 mL). Radial Growth Assays. Microbial assays were carried out on agar plates amended with modified chitosans dissolved in the YM broth (192 mg of freeze-dried polysaccharide in 12 mL); an aliquot (12 mL) of the YM broth containing 3% agar was added to obtain a modified chitosan concentration of 8 mg/mL. Five series of plates, one for each chitosan, with decreasing concentrations (8, 4, 2, 1 mg/mL) were prepared and inoculated in quadruplicate at the center with a 10 µL spore suspension. Fungal growth (colony diameter including the 8 mm inoculum diameter) was measured daily for 7 days at 20 °C and expressed as an average diameter (mm) with associated standard deviations. Morphological Analysis by Electron Microscopy. The ultrastructural morphological analysis was performed by transmission (TEM, Philips CM10) and scanning (SEM, Philips 505) electron microscopy. Samples were fixed in 2%

Muzzarelli et al. Table 1. Growth of Saprolegnia in Chitosan-Bearing Broth chitosan in physical form aspect of fungal growth, chitosan broth, g/L of chitosan broth, initial visual inspection MP NCM NPHM DCM DMAP control

0.4, 4, 6, 8 0.4, 4, 6, 8 0.4, 4, 6, 8 4, 6, 8 4, 6, 8 none

freeze-dried freeze-dried solution freeze-dried solution no

clear haze turbid clear clear clear

heavy precipitate precipitate precipitate regular growth partial growth regular growth

Figure 1. Scanning electron microscopy of Saprolegnia showing regular morphology (control).

glutaraldehyde in 0.1 M cacodylate buffer and postfixed in 1% osmium tetraoxide in the same buffer. They were then dehydrated in an ethanol gradient and embedded in Araldite for TEM observations, or subjected to critical point drying, mounted on aluminum stubs and gold-spattered for SEM analysis. Results Chitosan Bearing Broth. As for the chitosan-bearing broth assay, Saprolegnia did not grow normally; on the first day for MP and NPHM and on the second day for NCM, a tightly packed precipitate was present at the bottom of the test tubes, instead of the fluffy fungal material as in the control. On the contrary, DCMC seemed to favor fungal growth. The observations are summarized in Table 1. At the optical microscope, Saprolegnia grown in YM + DMAP and YM + MP exhibited mostly aggregated spores, accompanied by very few broken and empty hyphae. The S. parasitica control showed regular development of the hyphae in the broth (Figure 1), while NCM-chitosan, at 0.4 mg/mL broth, coated the fungal material and strongly depressed the development (Figure 2). With MP-chitosan at the same concentration in the broth, the hyphae were lesioned, flattened, distorted, and retracted (Figure 3). Remarkably, excess modified chitosans at 4 mg/ mL seemed to be precipitated together with the fungus. The wall was generally frayed, coated with chitosan, and expanded, while the internal organelles were morphologically altered. In fact the TEM provided evidence of various degrees of damage, ranging from frayed and expanded wall often coated with chitosan, to partial disappearance of the plasma

Fungistatic Activity of Modified Chitosans

Biomacromolecules, Vol. 2, No. 1, 2001 167

Figure 2. Scanning electron microscopy evidence of the inhibitory effect of NCM-chitosan (0.4 mg/mL) on Saprolegnia growth in Bacto YM broth. The fungal hyphae are heavily coated with NCM-chitosan. Figure 4. Transmission electron microscopy evidence of the damage produced by MP-chitosan on Saprolegnia. The upper part of the wall is frayed and heavily coated with MP-chitosan, and the plasma membrane is no longer identifiable; in the lower part, the wall is frayed and expanded, and the plasma membrane is only partly visible. Internal organelles are morphologically altered.

Figure 3. Scanning electron microscopy evidence of the inhibitory effect of MP-chitosan (0.4 mg/mL) on Saprolegnia growth in Bacto YM broth. The fungal structure appears to be damaged, and the morphology is deeply altered, leading to hyphal distortion and retraction.

membrane and damage to internal organelles, and to complete detachment of the wall and the membrane and severe alteration of the cytoplasmatic content (Figure 4). Similarly, NPHM-chitosan coated the fungus and locally damaged it (Figure 5). The damage was an alteration of the wall thickness and partial disappearance of the membrane and the alteration of the cytoplasm to the point that organelles were no longer identifiable (Figure 6). Certain specimens showed impressive cell distortion, plasma membrane disintegration, and organelle morphology destruction (Figure 7). Radial Growth Assays. The data relevant to fungal growth depression by modified chitosans, added to the agar at 2 mg/mL, are presented in Figure 8. MP, NCM, and NPHM were significantly effective in depressing the radial growth of Saprolegnia. Actually, no growth at all was observed in the case of NPHM in the initial 72 h period, while a real modest growth was noticeable after 50 h in the case of MP and NCM. Each colony was measured twice at 90°; based on the readings for the series of five experiments

Figure 5. Scanning electron microscopy evidence of the damage provided by NPHM-chitosan (0.4 mg/mL) to Saprolegnia growing in Bacto YM broth. Small amounts of NPHM-chitosan are deposited on the surface; flattened and damaged structures are clearly visible.

indicated above, standard deviations were calculated and represented as bars in Figure 8. Data for DMAP did not significantly differ from the control data, while DCMC was found to enhance the Saprolegnia growth since the beginning, and the diameter was the double of the control after 72 h. Discussion The data presented indicate that Saprolegnia growth can be depressed or inhibited by some modified chitosans even in the presence of a nutrient as the YM medium. The most effective modified chitosans are MP, NCM, and NPHM. While the latter is a new compound currently under study as a functional cosmetic ingredient,34 MP and NCM are the two most widely studied chitosan derivatives, proposed for

168

Biomacromolecules, Vol. 2, No. 1, 2001

Muzzarelli et al.

Figure 8. Radial growth of Saprolegnia on agar containing various modified chitosans, as a function of time at 20 °C. MP-chitosan, NPHM-chitosan, and NCM-chitosan exert fungistatic action against Saprolegnia. Bars indicate standard deviations accounting for reading errors and shape irregularities of the colonies.

Figure 6. Transmission electron microscopy evidence of the damage provided by NPHM-chitosan (0.4 mg/mL) to Saprolegnia growing in Bacto YM broth. The wall is either thinner or thicker than normal; the membrane is only partly visible and the cytoplasm is altered to the point that organelles are no longer identifiable.

Figure 7. Transmission electron microscopy evidence of the damage provided by NPHM-chitosan (0.4 mg/mL) to Saprolegnia growing in Bacto YM broth. The cell is distorted, disorganized and the internal organelles are hardly identifiable; the plasma membrane is disintegrating.

applications in wound healing and cosmetics, respectively. MP has also been qualified as the most biocompatible chitosan derivative.35 Therefore, these three chitosans are currently deemed to be nontoxic. On the other hand, under the present experimental conditions, DMAP chitosan did not provide clear evidence of efficacy against the S. parasitica growth. One of the five chitosans tested, DCMC, supported enhanced growth of S. parasitica.

It is not easy to provide an explanation for the above findings. DCMC carries EDTA-like functions, while NCM, monosubstituted to a much lower extent with the same -CH2COONa carboxymethyl group, has the opposite effect. The highly cationic DMAP is not particularly effective. Thus, the chelating ability and the cationicity of the biopolymers do not seem to be particularly important characteristics for Saprolegnia growth depression. Amphotericity might enable MP, NCM, and NPHM to interact effectively with some surface components of the S. parasitica cell wall, thus leading to increased permeability and consequent cellular leakage and ultimately to cell plasmolysis. More elaborated inhibitory mechanisms are also plausible: the chitin glucan β(1-4) transferase that links the reducing GlcNAc residue of chitin to the nonreducing glucose of glucan in fungi may be inhibited by certain N-acetyl hexosamine oligosaccharides.36 GlcNAc may prevent growth by inhibiting N-acetylglucosaminidase.37 While chitin in S. parasitica is low (0.7%), similar mechanisms involving glucan and cellulose formation might take place in the presence of certain modified chitosans.38 The structures of other enzymatic and nonenzymatic proteins might also be affected by the presence of a chitosan derivative. Immediate precipitation from amended broth and minimal if any radial growth in amended agar were easily perceivable aspects of a strong fungistatic action of some modified chitosans. From the present study it appears moreover that certain modified chitosans at 0.4 g/L broth can recognize the Saprolegnia tissues and react with them. Therefore, it might be possible that small nontoxic concentrations of MP, NCM, or NPHM prevent upsurge of infection simply because the chitosan would be collected by S. parasitica at the very early stage of infection, thus depressing development and spreading. Field trials will be carried out to verify this indication. Acknowledgment. This work was carried out in the frame of Progetto Cofinanziato 1997, MURST, Rome. Thanks are due to Mrs. Maria Weckx for handling the bibliography.

Fungistatic Activity of Modified Chitosans

References and Notes (1) Bruno, D. W.; Wood, B. P. In Fish Diseases and Disorders; Woo, P. T. K., Bruno, D. W., Eds.; CAB: New York; 1999, Vol. 3, pp 599-688. (2) Jolle`s, P.; Muzzarelli, R. A. A.; Eds. Chitin and Chitinases; Birkhauser: Basel, Switzerland, 1999. (3) Cuero, R.; Lillehoj, E. Biotechnol. Lett. 1990 4, 275-280. (4) Sosa, M. A.; Azley, F.; Koch, J. A.; Vercellotti, S. V.; Ruprecht, R. M. Biochem. Biophys. Res. Comm. 1991, 174, 489-491. (5) Muzzarelli, R. A. A.; Tarsi, R.; Filippini, O.; Giovanetti, E.; Biagini, G.; Varaldo, P. E. Antimicr. Agents Chemother. 1990, 34, 20192023. (6) Allan, C. R.; Hadwiger, L. A. Exp. Mycol. 1979, 3, 285-287. (7) Stossel, P.; Leuba, J. L. Physiopathol. Z. 1984, 111, 82-90. (8) Cuero, R. G.; Osuji, G.; Washington, A. N-Carboxymethyl chitosan inhibition of aflatoxin production: role of zinc. Biotechnol. Lett. 1991, 13, 441-444. (9) Cuero, R.; Duffus, E.; Osuji, G.; Pettit, R. J. Agric. Sci. 1991, 117, 165-169. (10) Laflamme, P.; Benhamou, N.; Bussieres, G.; Dessureault, M. Can. J. Bot. 1999, 77, 1460-1468. (11) El Ghaout, A. J.; Asselin, A. In AdVances in Chitin and Chitosan; Brine, C. J., Sandford, P. A., Zikakis, J. P., Eds.; Elsevier, Amsterdam, 1992; pp 440-452. (12) El Ghaout, A. J.; Wilson, C. L. Bioactive coating for harvested commodities. PCT, WO 96, 13985, 1996. (13) Freepons, D. In Applications of Chitin and Chitosan; Goosen, M. F. A., Ed.; Technomic: Basel, Switzerland, 1997; pp 129-140. (14) Hadwiger, L. A.; Fristensky, B.; Riggleman, R. In Chitin, Chitosan and Related Enzymes; Zikakis, J. P.; Ed.; Academic Press: New York, 1984; pp 292-301. (15) Nelson, J. D. U.S. Patent 4,957,908, 1990. (16) Suzuki, K.; Okawa, Y.; Hashimoto, K.; Suzuki, S.; Suzuki, M. Microbiol. Immunol. 1984, 28, 903-912. (17) Kahana, M.; Segal, E.; Millet, M. S.; Gov, Y. Acta Dermatol. Venereol. 1988, 68, 98-101. (18) Sandovsky-Losica, H.; Segal, E. Immunol. Infect. Diseases 1993, 3, 155-159. (19) Muzzarelli, R. A. A., Ed. Chitosan per os: from dietary supplement to drug carrier; Atec: Grottammare, Italy, 2000.

Biomacromolecules, Vol. 2, No. 1, 2001 169 (20) Roller, S.; Covill, N. Int. J. Food Microbiol. 1999, 47, 67-77. (21) Fang, S. W.; Li, C. F.; Shih, D. Y. C. J. Food Prot. 1994, 56, 136140. (22) Min, H. K.; Hatai, K.; Bai, S. Fish Pathol. 1994, 29, 73-77. (23) Kawai, T.; Naito, T.; Matadaira, Y.; Yura, H. Jpn. Kokai Tokkyo Koho JP 11 43,441, 1999. (24) Anderson, D. P.; Siwicki, A. K. Prog. Fish-Cult. 1994, 56, 258-261. (25) Kono, M.; Matsui, T.; Shimizu, C. Nippon Suisan Gakkaishi 1987, 53, 125-129. (26) Golovin, P. P.; Golovina, N. A.; Guseva, N. V.; Gamygin, E. A.; Romanova, N. N.; Suskov, I. D. In Chitin and Chitosan; Varlamov, P., Ed.; VINRO: Moscow, 1999. (27) Kitancharoen, N.; Ono, A.; Yamamoto, A.; Hatai, K. Fish Pathol. 1997, 32, 159-162. (28) Kitancharoen, N.; Yamamoto, A.; Hatai, K. Mycoscience 1997, 38, 375-378. (29) Kitancharoen, N.; Yamamoto, A.; Hatai, K. Biocontrol Sci. 1998, 3, 113-115. (30) Muzzarelli, R. A. A.; Ilari, P. L.; Tomasetti, M. Carbohydr. Polym. 1993, 20, 99-106. (31) Muzzarelli, R. A. A.; Tanfani, F.; Emanuelli, M.; Mariotti, S. Carbohydr. Res. 1982, 107, 199-219. (32) Muzzarelli, R. A. A.; Ilari, P. L.; Petrarulo, M. Int. J. Biol. Macromol. 1994, 16, 177-180. (33) Muzzarelli, R. A. A.; Frega, N.; Miliani. M.; Muzzarelli, C.; Cartolari, M. Carbohydr. Polym. 2000, 43, 263-268. (34) Baschong, W.; Huglin, D.; Maier, T.; Kulik, E. SO ¨ FW J. 1999, 125, 22-24. (35) Bersch, P. C.; Nies, B.; Liebendorfer, A.; Kreuter, J. J. Mater. Sci. Mater. Med. 1995, 6, 201-205. (36) Kollar, R.; Cabib, E.; Silverman, S.; Petrakova, E. PTC, WO 96/ 20717, 1996. (37) Giordani, R.; Cardenas, M. L.; Moulin-Traffori, J.; Regli, P. Mycosis 1996, 39, 103-110. (38) Gay, L.; Chanzy, H.; Bulone, V.; Girard, V.; Fevre, M. In Chitin Enzymology; Muzzarelli, R. A. A.; Ed.; Atec: Grottammare, Italy, 1993; Vol. 1, pp 57-66.

BM000091S