Antibacterial activity of chemically defined chitosans: influence of molecular weight, degree of acetylation and test organism

Chitosans, polysaccharides obtained from the exoskeleton of crustaceans, have been shown to exert antibacterial activity in vitro and their use as a food preservative is of growing interest. However, beyond a consensus that chitosan appears to disrupt the bacterial cell membrane, published data are inconsistent on the chemical characteristics that confer the antibacterial activity of chitosan. While most authors agree that the net charge density of the polymer (reflected in the fraction of positively charged amino groups at the C-2 position of the glucosamine unit) is an important factor in antibacterial activity, conflicting data have been reported on the effect of molecular weight and on the susceptibility among different bacterial species to chitosan. Therefore, we prepared batches of water-soluble hydrochloride salts of chitosans with weight average molecular weights (M_w) of 2-224 kDa and degree of acetylation of 0.16 and 0.48. Their antibacterial activity was evaluated using tube inhibition assays and membrane integrity assays (N-Phenyl-1-naphthylamine fluorescence and potassium release) against Bacillus cereus, Escherichia coli, Salmonella Typhimurium and three lipopolysaccharide mutants of E. coli and S. Typhimurium. Chitosans with lower degree of acetylation (F_A = 0.16) were more active than the more acetylated chitosans (F_A = 0.48). No trends in antibacterial action related to increasing or decreasing M_w were observed although one of the chitosans (M_w 28.4 kDa, F_A = 0.16) was more active than the other chitosans, inhibiting growth and permeabilizing the membrane of all the test strains included. The test strains varied in their susceptibility to the different chitosans with wild type S. Typhimurium more resistant than the wild type E. coli. Salmonellae lipopolysaccharide mutants were more susceptible than the matched wild type strain. Our results show that the chitosan preparation details are critically important in identifying the antibacterial features that target different test organisms.