Comparison of Different Assay Methods and Sample Preparation Procedures for the Evaluation of the Antibacterial Activity of Chitosan

Abstract

Chitosan is a natural biopolymer obtained by deacetylation of chitin, which is a major structural component in the exoskeleton of crustaceans. The antimicrobial activity of chitosan and chitosan derivatives has been widely researched and these polymers have been shown to be effective against many Gram-negative and Gram-positive bacteria as well as fungi. However, the reported activity, against a given microorganism, can vary significantly from one study to another. This is partially due to the lack of a standardized procedure for the testing and sample preparation. The aim of this study was to investigate and compare different microbial assay methods that have frequently been reported for chitosan as well as the sample preparation procedures. Factors that may influence activity such as pH, NaCl concentration and aggregation of the polymer were also investigated. The Gram-positive bacteria Staphylococcus aureus and the Gram-negative Escherichia coli were used as model organisms in the study.The study found that the commonly reported disk diffusion and well diffusion assays were not suitable for chitosan as the polymer cannot diffuse into the agar network. Broth microdilution assay was found to be suitable but neither acetic acid nor hydrochloric acid could be recommended as solvents because the pure solvents influenced the readout from the assay. Thus, chitosan samples were prepared by converting the poorly soluble chitosan polymer to water soluble chitosan-hydrochloride before performing the microdilution. When the assay was performed in this way it showed the best antibacterial activity at pH 5.5 – 6.5 for S. aureus and pH 4.5 - 6.5 for E. coli. NaCl concentration was tested against S. aureus and did not show much effect on the MIC of chitosan samples. Even though a solution with chitosan hydrochloride salts with various pH levels looked clear, there was an increase in aggregation of particles in sample with increasing pH level, above pH 6.5.These results provide an important foundation that a standardized procedure for antimicrobial testing of chitosan can be based upon. Such standardization gives rise to a reliable comparison between different laboratories, thus further advancing research of the antimicrobial activity of chitosan.