Evaluation of diffusion and dilution methods to determine the antimicrobial activity of water-soluble chitosan derivatives

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Chitosan has gained wide applications in the food industry and biomedical field owing to its biodegradability, biocompatibility, nontoxicity and its antimicrobial activity against a wide spectrum of micro-organisms. However, the methods used to investigate antimicrobial effects of chitosan vary considerably among studies, making comparisons difficult.

Methods and Results

One diffusion (disc diffusion) and two dilution (agar dilution and broth microdilution) methods commonly used in clinical laboratories to assess microbial susceptibility/resistance to antimicrobial agents were comparatively used to determine the antimicrobial activity of two water-soluble chitosan derivatives (molecular weights of 43 and 67 kDa) against 31 representative foodborne pathogens. When tested at 1·6% for the 43-kDa chitosan and 3·2% for the 67-kDa chitosan, by disc diffusion, approximately 10- to 11-mm-diameter inhibition zones were observed for all of the bacterial groups, except for Salmonella tested for the 67-kDa chitosan where no inhibition zone was observed. By agar dilution and broth microdilution, the minimal inhibitory concentration (MIC) values varied largely dependent upon the molecular weight of chitosan, bacterial genus/species and the testing method. The agreement between MIC values obtained by the two methods was poor, with broth microdilution generally having lower MIC values than agar dilution. Regardless of the testing method, Salmonella strains were the least susceptible among Gram-negative strains for both chitosans, followed by Escherichia coli and Vibrio.


Besides chitosan's molecular weight and bacterial genus/species, the antimicrobial activity of chitosan was also influenced largely by the susceptibility testing method used.

Significance and Impact of the Study

This is the first study that comparatively evaluated these diffusion and dilution methods, particularly two quantitative methods (agar dilution and broth microdilution), to assess the antimicrobial activity of two water-soluble chitosans against a large number of foodborne pathogens. The study highlights the need for standardized methods to be used in evaluating chitosan's antimicrobial properties in future studies.

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