Vol. 34, No. 10

Antimicrobial Properties of N-Carboxybutyl ChitosanRICCARDO MUZZARELLI,i* RENATO TARSI,2 OSCAR FILIPPINI,1 ELEONORA GIOVANETTI,3

GRAZIELLA BIAGINI,3 AND PIETRO E. VARALDO2

Institute of Biochemistry,1 Institute of Microbiology,2 and Institute ofAnatomy,Faculty of Medicine, University ofAncona, I-60100 Ancona, Italy

Received 26 March 1990/Accepted 16 July 1990

N-Carboxybutyl chitosan, a modified chitin of crustacean origin, displayed inhibitory, bactericidal, andcandidacidal activities when tested against 298 cultures of various pathogens. Examination by electronmicroscopy showed that microbial cells exposed to N-carboxybutyl chitosan underwent marked morphologicalalterations. The data are of importance in defining the suitability of N-carboxybutyl chitosan as a wounddressing.

A number of polysaccharides, mainly cross-linked dex-tran, calcium alginate, carboxymethyl starch, modified agar,and carboxymethyl cellulose, are used in wound treatment(13, 17). These polysaccharides, however, do not seem tocombine favorable physical forms with functional proper-ties; rather, besides the hemostatic action of oxycellulose,no functional property has been reported (17). The same istrue for gelatin and collagen (15). On the other hand, chitinderivatives are not in widespread use as wound dressings,and relevant studies on wound healing have been confined toa few recent articles (1, 11). Interestingly, some antibacterialand antifungal activities have been described with chitosanand modified chitosans (5, 8, 9, 18).N-Carboxybutyl chitosan is more versatile than any other

polysaccharide currently used in wound management andlends itself to the manufacture of wound dressings withpeculiar characteristics. Such dressings, in particular, canstimulate ordered regeneration (3) and vascularization (10) oftissue and allow gaseous exchange and turn into a gel formwhen they are in contact with body fluids (14), thus permit-ting dressing removal without disturbing the newly formedtissues. We undertook the present study to define theantimicrobial properties of N-carboxybutyl chitosan and totest it in different physical forms in view of its use in woundmanagement.N-Carboxybutyl chitosan, which was prepared from crus-

tacean chitosan (degree of deacetylation, 0.73) accordingto a proprietary procedure (R. Muzzarelli, U.S. patent4,835,265, June 1986) and as described previously (12, 14),was tested against a variety of gram-positive and gram-

negative pathogens and Candida spp. A total of 298 strainswere used, all of which were freshly isolated from clinicalmaterial and identified according to conventional laboratorycriteria.Two different methods were used to assess the antimicro-

bial activity of N-carboxybutyl chitosan. The first methodwas a quantitative assay based on conventional agar dilutiontests (20), with final concentrations being 2, 4, 6, 8, and 9mg/ml (concentrations above 9 mg/ml did not permit fullsolubilization of N-carboxybutyl chitosan into the test me-

dium). The inoculum suspensions were prepared from freshbroth cultures and adjusted to obtain a concentration ofapproximately 107 CFU/ml. Test plates were inoculated byusing an automatic replicating device (Titertek; Flow Labo-

* Corresponding author.

ratories, Inc., McLean, Va.) that delivered 1 1.l of bacterialsuspension per spot. A control plate with no N-carboxybutylchitosan was inoculated first, and a second control plate wasseeded last to ensure that viable organisms were presentthroughout the procedure and there was no antimicrobialcarry-over. The inoculated plates were allowed to stand untilthe inoculum spots were completely absorbed and were thenincubated at 37°C for 20 h. Complete growth inhibition was

considered as the cutoff point.The second method was a qualitative and empiric assay

based on the use of soft and flexible thin pads (thickness, 1

mm; ca. 3.3 mg/cm2) obtained by pressing freeze-driedN-carboxybutyl chitosan between steel plates. A portion ofca. 2 cm2 was deposited in the central part of the surface ofa plate inoculated as in standard agar diffusion assays (2) andbecame transparent in a few minutes beCause of wateruptake. Under these conditions, N-carboxybutyl chitosanwas virtually incapable of diffusing through the adjacent agar

medium. Microbial growth under the thin pad of N-carboxy-butyl chitosan was assessed after 20 h of incubation at 37°C.The complete transparency of the N-carboxybutyl chitosan-coated part of the agar, which was associated with regulargrowth in the remainder of the plate surface, was taken as an

indication of inhibitory activity exerted by N-carboxybutylchitosan (Fig. 1). In this case, a portion of the pad andmedium underneath was transferred to a test tube with 5 mlof nutrient broth and incubated for 24 h at 37°C. Subculturesin chitosan-free broth and streaks on suitable agar plateswere then performed from clear tubes. After 48 h of incuba-tion at 37°C, microbial growth denoted by broth turbidity,growth on the solid medium, or both was indicative ofbacteriostatic activity; broth transparency, confirmed by theabsence of growth on the streaks on solid medium, was

considered to be indicative of bactericidal activity of N-car-boxybutyl chitosan.As shown in Table 1, N-carboxybutyl chitosan was par-

ticularly active against candidae and gram-positive bacteria.The majority of the strains tested (including all streptococci,enterococci, and coagulase-negative staphylococci) were

inhibited by 8 mg/ml. When a thin pad of N-carboxybutylchitosan was used, growth of all strains was inhibited. Allcandidae and most staphylococci were killed, while no

bactericidal activity was observed with streptococci andenterococci. In most gram-negative organisms, approxi-mately one-half to three-quarters of the strains were inhib-ited by 6 mg/ml and 90 to 100% were inhibited by 9 mg/ml

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FIG. 1. Empiric assay used to assess qualitatively the antimicro-bial activity of N-carboxybutyl chitosan used as a freeze-dried thinpad deposited on the central part of an inoculated plate. (A)Inhibitory effect is denoted by full transparency of the N-carboxy-butyl chitosan-coated part of the agar, where some air bubbles(entrapped in the freeze-dried material) remain visible; (B) lack of aninhibitory effect is denoted by the growth that occurred under theN-carboxybutyl chitosan pad. The freeze-dried material becametransparent by gel formation consequent to water uptake from theagar.

(Table 2). At a concentration of 9 mg/ml, lower percentagesof inhibition were observed only with Haemophilus influ-enzae (75%), Acinetobacter anitratus (60%), Serratia spp.(53%), and Salmonella spp. (none of the three strains test-

TABLE 1. Activity of N-carboxybutyl chitosan againstgram-positive bacteria and candidae as determined

by the agar dilution and thin pad techniques% Inhibition at the % Activityfollowing polymer under thin pad

Organism concn (mg/ml):(no. tested)

Inhibitory Bacte-2 4 6 8 9 activitY rricidalactivity

Staphylococcus aureus (64) 11 87 92 94 100 86Coagulase-negative staph- 71 92 100 100 92

ylococci (24)aStreptococcus spp. (10)b 20 100 100 0Enterococcus faecalis (11) 9 100 100 0Candida spp. (18)C 6 33 44 89 100 100

a Including 12 strains of S. epidermidis and 12 strains of S. haemolyticus.b Including two strains of S. mutans, two strains of S. mitis, and six strains

of S. pyogenes.c Including 15 strains of C. albicans, 2 strains of C. glabrata, and 1 strain of

C. tropicalis.

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FIG. 2. Ultrastructural appearance of S. haemolyticus (A) closeto the N-carboxybutyl chitosan thin pad. The outer part of the cellwall is irregularly structured and frayed (arrowheads). Magnifica-tion, x161,500. (B) Control taken from the peripheral area of thesame plate. Magnification, x 166,250.

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FIG. 3. Ultrastructural appearance of Escherichia ccli close to

N-carboxybutyl chitosan (A). The periplasmic space is abnormally

expanded (arrowhead). The endocellular structures are densely

packed. Magnification, x32,760. (B) Control taken from the periph-

eral area of the same plate shown in panel A. Magnification,

x34,580.

FIG. 4. Ultrastructural aspect of C. albicans (A) within theN-carboxybutyl chitosan. Cellular degradation is visible. Portions ofN-carboxybutyl chitosan adhering to the wall (arrow) are alsoobservable. Magnification, x45,000. (B) Control taken from theperipheral area of the same plate shown in panel A. Magnification,x45,000.

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TABLE 2. Activity of N-carboxybutyl chitosan againstgram-negative bacteria as determined by the agar

dilution and the thin pad techniques

% Inhibition atthe following % Activitypolymer concn under thin pad

Organism (mg/ml):(no. tested)

Inhibitory Bacte-4 6 8 9 ricidactivityalactivity

Escherichia coli (21) 19 52 81 90 95 65Klebsiella spp. (28)a 39 82 96 96 48Enterobacter spp. (5)b 40 60 80 100 100 60Serratia spp. (19)c 15 31 53 58 45Citrobacterfreundii (2) 50 100 100 100Proteus spp. (24)d 25 46 96 100 83 45Morganella morganii (3) 33 66 100 33Salmonella spp. (3)e 0 0Pseudomonas aeruginosa (25) 40 76 88 96 88 32Acinetobacter anitratus (5) 40 60 100 40Haemophilus influenzae (36) 6 47 72 75 100 78

a Including 19 strains of K. pneumoniae, 5 strains of K. oxytoca, and 4strains of K. ozoenae.

b Including three strains of E. cloacae and two strains of E. aerogenes.c Including 13 strains of S. marcescens and 6 strains of S. liquefaciens.d Including 22 strains of P. mirabilis and 2 strains of P. vulgaris.Including two strains of S. typhi and one strain of S. typhimurium.

ed). When N-carboxybutyl chitosan was applied as a thinpad, the inhibitory activity was considerable (80 to 100%o)against most species, whereas respective percentages of thebactericidal activities were lower; again, the three Salmo-nella strains did not respond at all to N-carboxybutyl chito-san.

Electron microscopy studies were performed on agarslices taken from the N-carboxybutyl chitosan pad and inperipheral areas of the plate. The specimens were fixed in2.5% glutaraldehyde and postfixed in OS04 in cacodylatebuffer, dehydrated with alcohol, and embedded in araldite.Ultrathin sections were observed with an electron micro-scope (CM10; Philips Industries, Eindhoven, The Nether-lands). In staphylococci, the presence of N-carboxybutylchitosan caused fraying and weakening of the outer part ofthe cell wall, which locally appeared thicker than in controls(Fig. 2); duplication was also depressed. In gram-negativeorganisms an abnor,mally expanded periplasmic space wasobserved in cells close to the N-carboxybutyl chitosan pad(Fig. 3). The intracellular material in gram-negative organ-isms appeared more tightly packed than it did in controls.Fragments of cell wall and bacterial "sltadows" lacking anyintracellular organization were also detected. Candida albi-cans strains close to N-carboxybutyl chitosan showed celldamage to various extents (Fig. 4). In general, their cell wallswere still identifiable, but intracellular structures had eitherdisappeared or changed their normal characteristics or dis-tributions.The mechanism of the antimicrobial aptivity of N-carboxy-

butyl chitosan is probably complex, and its elucidation waslargely beyond the scope of this study. However, given thepolycationic nature of N-carboxybutyl chitosan, it is likelythat it can interact and form polyelectrolyte complexes withacidic polymers produced at the bacterial cell surface (e.g.,lipopolysaccharides, teichoic and teichuronic acids, or cap-sular polysaccharides). In candidae, N-carboxybutyl chito-san might also produce disturbances of membrane functionssimilar to those caused by polycations in other fungi (9). It is

noteworthy that chitosan can agglutinate a number of bac-teria, as well as mammalian cells (10), and chitosan-goldcomplexes have been used as polycationic probes for thedetection of cell surface anionic sites (7). Moreover, poly-electrolyte and macromolecular complexes of chitosan withglycosaminoglycans have been reported (6, 11), and alginicacid, which forms complexes with glycol chitosan (16), hasbeen studied in conjunction with biotechnological applica-tions of chitosan-alginate capsules (4, 19).The antibacterial and anticandidal actions exerted by

N-carboxybutyl chitosan appears to be an important addi-tional feature of N-carboxybutyl chitosan-based wounddressings because they could contribute to the prevention ofsecondary infections, resulting in limited scar formation.

Part of this work was performed under the auspices of contract89.00072.79 from the Consiglio Nazionale Ricerche, Progetto Final-izzato Chimica Fine II, Rome, Italy.

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10. Muzzarelli, R. A. A. 1977. Chitin. Pergamon Press, Oxford.11. Muzzarelli, R. A. A. 1987. Chitin. In 0. Aspinall (ed.), The

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and their biological significance. In P. Sandford (ed.), Proceed-ings of the 4th International Conference on Chitin/Chitosan.Elsevier, Amsterdam.

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14. Muzzarelli, R. A. A., M. Weckx, 0. Filippini, and C. Lough.1989. Characteristic properties of N-carboxybutyl chitosan.Carbohydr. Polymers 11:307-320.

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15. Scher, K. S., and J. A. Coil. 1982. Effects of oxidised celluloseand microfibrillar collagen on infection. Surgery 91:301-304.

16. Srinivasan, R., and R. Kamalam. 1982. Polyelectrolyte com-plexes of glycol chitosan with some polysaccharides. Biopoly-mers 21:265-275.

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Gekkan Fudo Kemikaru 4:22-29.19. Vorlop, K. D., and J. Klein. 1981. Formation of spherical

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