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SCIENTIFIC CORRESPONDENCE

Chitosan: A potential biomaterial effective against typhoid
Chitin, a polysaccharide of animal origin, is obtained from waste material of seafood industries. It occurs in the skeletal material of crustaceans such as crabs, lobsters, shrimps, prawns and crayfish. Chitosan is the deacetylated product formed by treatment of chitin with concentrated (50%) caustic alkali. The regulatory and toxicological status of Chitosan has already been established. The oral toxicity of Chitosan has been reported to be 16 g/kg body weight (LD50)1. Thus Chitosan is safe (nontoxic), biocompatible and biodegradable. Chitosan has been subjected to a series of pharmacological and clinical studies2–6. For the first time in 1978, Balassa and Prudden7 studied the use of chitin and its derivatives (including Chitosan) in woundhealing. These studies found that chitin and Chitosan are effective wound healing accelerators in both animal and human tests. Macroporous artificial skin containing antibiotics was prepared by lyophilization of Chitosan/PVA blendmer, which could protect the wound surfaces from bacterial invasions by suppressing bacterial proliferation effectively8. Recently, the antimicrobial activity of Chitosan has been studied extensively. It has been shown that Chitosan acts by disrupting the barrier properties of the outer membrane of Gram-negative bacteria9. Zivanovic et al.10, while studying antimicrobial efficiency of Chitosan, have shown that addition of 0.1% Chitosan polysaccharide would be sufficient to ensure the microbial safety of oil-in-water emulsions. It has also been found that the antimicrobial activity of Chitosan is influenced by its molecular weight. The water-soluble Chitosan hydrolysate, consisting mainly of low molecular weight Chitosan (LMWC), shows antimicrobial activity against Escherichia coli, while Chitooligosaccharides have much weaker antimicrobial activity11. Chitosan, obtained as a gift sample from India Seafoods, Cochin was used in this study. Its viscosity (1% solution in 2 M acetic acid) is 325 and the deacetylation degree is 80%. The bacterial strains were procured from Krishna Institute of Medical Sciences and Research Centre, Karad, India. The antibacterial activity was studied against different strains of bacteria, viz. Staphylococcus aureus, Bacillus subtilis (Gram-positive), Pseudomonas aeruginosa, E. coli, Salmonella enterica, S. enterica var. Paratyphi-A and S. enterica var. Paratyphi-B (Gram-negative) by agar diffusion method in particular, cup-plate method12,13. In this method cups of standard diameter are made into the nutrient agar medium containing standard bacterial inoculum. Next 0.1 ml of the test material (Chitosan solution in 2M citric acid) and reference standards (ciprofloxacin, sparfloxacin aqueous solution) were taken into the cups. After incubation of the plates at 37 ± 1°C for 24 h, the diameter of zone of inhibition in millimetres was measured. Minimum inhibitory concentration (MIC) is the highest dilution of an antimicrobial agent that inhibits the growth of a particular microorganism in a test period. The MIC of Chitosan against S. enterica, S. enterica var. Paratyphi-A and S. enterica var. Paratyphi-B was determined. The antimicrobial activity of Chitosan against typhoid organisms was then compared with the reference standard antibiotics. Ciprofloxacin and sparfloxacin were used as standard antibiotics. The zones of inhibition produced by MIC of Chitosan and standard antibiotics were measured. Antimicrobial susceptibility testing with discs for testing bacterial sensitivity to various antibiotics and Chitosan was selected as a method of study14,15. Span-combi discs from Span Diagnostics Ltd, Surat, India were used in this study. Different strains of S. enterica (4 strains), S. enterica var. Paratyphi-A (1 strain) and S. enterica var. Paratyphi-B (4 strains) resistant to certain antibiotics were procured from Krishna Institute of Medical Sciences and Research. Table 1 gives the list of such strains with their sensitivity to certain antibiotics. Using disc method the sensitivity of resistant strains (viz. S. enterica-2, S. enterica-4, S. enterica var. Paratyphi-B-3, S. enterica var. ParatyphiB-4, S. enterica var. Paratyphi-A-1) against standard antibiotics and Chitosan have been studied. The antibacterial study was carried out by agar diffusion technique, in particular,

Table 1. Resistant strains of typhoid and their sensitivity Diameter of zone of inhibition (mm) Ampicillin 30* 27 (R) R 25 (R) R 22 (R) 20 (R) R R 20 (R) Ceftazidime 18* 24 20 24 26 26 24 R R 22 Ceftriaxon 21* 24 24 30 24 24 26 12 (R) R 26 Chloramphenicol 18* 30 R R R 26 24 R 24 26 Cipro- Nalidixic floxacin acid 21* 19* 20 (R) 26 30 28 28 26 20 (R) R 18 (R) R 22 22 24 22 22 R R R Netylmycin 15* 24 22 20 22 20 20 12 (R) 10 (R) 20 Pefloxacin 16* 20 24 30 26 20 28 18 R 14(R) Piperacillin 18* 22 13 (R) 25 R 22 22 R R 22 Tetracyclin 19* 10 (R) R R R R 10 (R) R 10 (R) 15 (R)

Organism S. enterica

Resistant strain 1 2 3 4 1 2 3 4 1

Bactrim 16* 30 R 34 R 28 30 22 R 34

S. enterica var. Paratyphi-B

S. enterica var. Paratyphi-A-1

*Minimum zone of sensitivity (mm); R, Resistant.

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CURRENT SCIENCE, VOL. 87, NO. 9, 10 NOVEMBER 2004

S. enterica var. The MIC of Chitosan against typhoid producing organisms. Table 2. which is comparable to the standard antibiotics used in clinical practice today (Table 4). enterica var. Our results showed that with increase in dilution. Paratyphi-A S. enterica var. Comparison of antimicrobial activity of Chitosan and standard antibiotics against S. Paratyphi-A-1. making it leaky and thus confirmed the non-specific action of polymines on membrane integrity. in our study we have attempted to investigate whether the antimicrobial activity of Chitosan is due to its glucosamine units. enterica var. The MIC of Chitosan was found to be 50 µg. In our study we found that glucosamine does not show antimicrobial activity. Antibacterial activity of Chitosan Diameter of zone of inhibition (mm) at 100 µg concentration 40 20 18 16 32 32 25 Table 4. have concluded that polyamino acids interact with the electronegative bacterial cell surface resulting in displacement of Ca++ from anionic membrane sites. Antimicrobial susceptibility testing using discs was performed to assess the ability of Chitosan to act against resistant strains of typhoid-producing organisms and to compare it with the standard antibiotics (Table 5). enterica var. against selected Gram-positive and Gram-negative organisms. In our study we found that glucosamine does not show any antimicrobial activity. Paratyphi-B-4. and S. Paratyphi-B-3. Paratyphi-A and S. Our results reveal that Chitosan has good antibacterial activity against typhoid organisms. Antimicrobial activity of Chitosan against S. enterica var. the diameter of zone of inhibition decreased. 87. VOL.SCIENTIFIC CORRESPONDENCE Figure 1. S. 10 NOVEMBER 2004 . Paratyphi-A Salmonella enterica var. Paratyphi-B Table 3. while studying the antifungal activity and effect of Chitosan and other polyamines. Paratyphi-B Organism S. It has also been shown that Chitosan agglutinates a variety of bacteria and yeasts as well as cells of mammalian origin18.17 have shown that Chitosan affects the membrane permeability of plants and fungi. Chitosan had significant activity against these test organisms (Table 2). Further. enterica var. enterica has been determined (Table 3). Leuba and Stossel19. Young and co-workers16. S. Paratyphi-B Diameter of zone of inhibition (mm) Ciprofloxacin (25 µg) 43 43 45 Sparfloxacin (25 µg) 26 21 24 Chitosan (50 µg) 20 18 16 Chitosan (100 µg) 39 38 35 Organism Staphylococcus aureus Bacillus subtilis Escherichia coli Pseudomonas aeruginosa Salmonella enterica Salmonella enterica var. 9. enterica. enterica S. NO. For comparing the activity of Chitosan with standard antibiotics useful against typhoid organisms. the zones of inhibition produced by MIC of Chitosan and MIC of these antibiotics were measured. The polycationic nature of Chitosan might be responsible for interaction with the electronegative 1177 CURRENT SCIENCE. Antimicrobial activity of Chitosan (central disc) and other standard antibiotics against resistant strains of S. enterica Chitosan (µg) 1000 800 600 400 200 100 50 10 Diameter of zone of inhibition (mm) 40 40 38 35 30 29 15 Nil the cup plate method.

698. Cochin. R. Conf. G. glucosamine does not show such activity. Kim. as suggested by Helander et al.6 23 ± 1. the disruption of barrier properties of outer membrane of Gramnegative bacteria might be the possible mechanism of antimicrobial action of Chitosan. Jeuniaux and Gooday)..8 R R R Cefazolin (CF) 18* Ceftazi.Ceftriadime xone (CZ) (XO) 18* 21* R R R R R Ciprofloxacin (CI) 21* R R 24 ± 0. p. revised accepted 30 July 2004 A.. R. L. and Prudden. 11. India *For correspondence.. 1968. 24.81 R R Kanamycin (K) 18* 22 ± 0. of India. 90–94. M. enterica var. 1975. enterica-2 S. Pollimo. L. L. Resistant. Takai Reg. Y. D. and Kauss. IIA.949. E. Karad 415 124. p. Histochem. and Kauss. Leuba. However. 1. Susceptibility testing of resistant strains of typhoid organisms Diameter of zone of inhibition (mm) ± SD Amikacin (AK) 17* 17 ± 0. P. p. S. 1986. p.. 396–398. enterica var. 9. Jpn. p. First Int. Received 31 May 2004. Chitosan–glycerol–water gel as a drug carrier for wound care.. BHISE Government College of Pharmacy.. 1978. Int. British Pharmacopoeia. on chitin/Chitosan (ed. VOL. Ahvenainen. Department of Health. 6. Int edn. L. 13. Takagi. 15. 67. enterica2). 1962. Food Microbiol.. New York. 10. I. for supplying the gift sample of Glucosamine sulphate. 67.4 17 ± 1. Plant Physiol.. pp. and Stossel. H. 2004. 1983. C. and Weiss. 4. IIA–167. Cytochem. J. 14. enterica var.. We thank India Seafoods. however. Balassa. S. E. 1982. 1449. enterica-4). T.. 19. Our study confirms and supports the earlier findings regarding usefulness of Chitosan as a wound-healing accelerator. Zhang. Balassa. A. especially on the grounds of limitations such as development of resistance to side effects and toxicity of currently used antibiotics. it is quite effective against resistant strains of S. 952–959. Davidson. pp.. Muzzarelli..5 30 ± 1. 10 NOVEMBER 2004 . 3911116.. Scottish Home and Health Department Welsh. Chi. 12.SCIENTIFIC CORRESPONDENCE Table 5.. S. 17. Basurto. L. Food Prot. S. Y. 71.. 8. Paratyphi-A and S. enterica-4 S. H. 70. Ministry of Health and Family Welfare. P.804. NO. 2. and Krishna Institute of Medical Sciences and Research Centre. Rhoades. D. Numiahio-Lassila. L. Ericson. J. 527–533. enterica (S. 3.4 R R 20 ± 0. enterica var. YADAV* S.4 R R R 25 ± 1. D.. Sect. J. enterica (S. L. Plenum Press. Our study further reveals that Chitosan may act effectively against typhoid-producing microorganisms. J. Paratyphi-A-1 22 ± 1. 87. D. Scand. 9. J. 89–94. and Shieh. e-mail: avyadav03@rediffmail. 1985.. Panacea Biotec Ltd. J. Eur. Jackson. Young. Paratyphi-B-3 S. L. H. Tsai. Kinumati.. Balassa. Evans. Paratyphi-B (Figure 1). L. 1971. Punjab. M. Paratyphi-B-4 S..7 R R R R *Minimum zone of sensitivity (mm). ACKNOWLEDGEMENTS. 1993. US 4659700 A 21 April 1987. 14. 7. In Chitin in Nature and Technology (eds Muzzarelli. Lalru. for supplying the gift sample of Chitosan. 5. Chitosan shows great potential in this respect. 3. 13. J. R. S. 235– 244.81 R Ofloxacin (OF) 16* R R Chitosan (C) 15* 23 ± 0. H. Karad for supplying bacterial strains. P.. and Sherries. H.. and its effectiveness in protecting wound from bacterial invasion by suppressing bacterial proliferation. and Lee. 1974.7 R 16 ± 1. and Fujita. Zivanovic. F. 296–305. National Committee for Clinical Laboratory Standards.4 20 ± 0. US Patent No. Kokai Tokkyo koho JP 2268766 A2 Heisei. 3. Food Prot. J. E. I. Mosbey. vol.5 Resistant strain S.. vol.35 R R R R Cefaclor (CG) 18* 26 ± 0. K. US Patent No. we could establish that Chitosan shows antimicrobial activity.8 Cefadxine (CD) 18* R 18 ± 0. 1990. Michio. In Proc.. Moreover. H. 217.. B. 1985. Antimicrobial susceptibility testing using discs showed that Chitosan is not effective against one of the resistant strains of S.4 21 ± 0. 215.9.4 21 ± 0.). B.6 Netilmicin (NT) 15* 20 ± 1. and Roller. S. Patent Appl.. 1993. Pharmacopoeia of India..com 1178 CURRENT SCIENCE. Govt. Arai. C. C.. Thus. Microbiol. L. K. 73. 16..6 R R R 25 ± 0. Reference Standard for Antimicrobial Disc Susceptibility Test. T. Ep 356060 A2. V. S. enterica var.. Bull. M. bacterial cell surface. 2001. Kohle. 24. 4th edn. P. 11. Young. Further investigations need to be done to develop Chitosan into a useful therapeutic tool for treatment of typhoid. 10.7 Norfloxacin (NF) 17* 20 ± 0. 43. vol. 5.. 1990.47 R 20 ± 1. 2004. 3rd edn. T. A. S. 28 February 1990. Plant Physiol. Shigeharu and Kimita. 18. Helander.41 R R R R R 20 ± 0. and Kent. Fish Lab. J.. Acta Pathol.

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