Insights into the mode of action of chitosan as an antibacterial compound

Abstract

Chitosan is a polysaccharide biopolymer that combines a unique set of versatile physicochemical and biological characteristics which allow for a wide range of applications. Although its antimicrobial activity is well documented, its mode of action has hitherto remained only vaguely defined. In this work we investigated the antimicrobial mode of action of chitosan using a combination of approaches, including in vitro assays, killing kinetics, cellular leakage measurements, membrane potential estimations, and electron microscopy, in addition to transcriptional response analysis. Chitosan, whose antimicrobial activity was influenced by several factors, exhibited a dose-dependent growth-inhibitory effect. A simultaneous permeabilization of the cell membrane to small cellular components, coupled to a significant membrane depolarization, was detected. A concomitant interference with cell wall biosynthesis was not observed. Chitosan treatment of Staphylococcus simulans 22 cells did not give rise to cell wall lysis; the cell membrane also remained intact. Analysis of transcriptional response data revealed that chitosan treatment leads to multiple changes in the expression profiles of Staphylococcus aureus SG511 genes involved in the regulation of stress and autolysis, as well as genes associated with energy metabolism. Finally, a possible mechanism for chitosan's activity is postulated. Although we contend that there might not be a single classical target that would explain chitosan's antimicrobial action, we speculate that binding of chitosan to teichoic acids, coupled with a potential extraction of membrane lipids (predominantly lipoteichoic acid) results in a sequence of events, ultimately leading to bacterial death.

Figures

FIG. 1.

Effect of chitosan on the growth kinetics of S. aureus SG511. Numbers of survivors (in log units) of S. aureus SG511 (starting inoculum of 1.15 × 107 CFU/ml) in CAMHB at 37°C in the presence of 0 (•), 0.5× (○), 1× (▴), 2× (—), 5× (▾), and 10× (▪) MIC of chitosan.

FIG. 2.

Cell leakage assays. (a) Potassium release from S. simulans 22 cells (—) increases with increasing amounts of chitosan: 5 μg/ml (⧫), 10 μg/ml (○), 20 μg/ml (+), 40 μg/ml (▴), and 60 μg/ml (▪). 100% potassium leakage was achieved by the addition of 1 μM of the pore-forming lantibiotic nisin (▾). (b) Leakage of UV-absorbing cellular components from S. simulans 22, upon treatment with chitosan (20 μg/ml) in CAMHB, measured at 260 nm (▴). Nisin (1 μM) was used to mark 100% leakage. Parallel optical density measurements were conducted and compared to the initial culture density (percent OD600, □).

FIG. 3.

Measurement of chitosan's ability to perturb the membrane potential (ΔΨ) using [3H]TPP+. Cells of S. simulans 22 in the late log phase were allowed to equilibrate with [3H]TPP+. Chitosan was then added (arrow) to a final concentration of 10 μg/ml.

FIG. 4.

Electron micrographs of S. simulans 22 cells (control) (a), treated with 10× MIC of chitosan for 5 min (b), 20 min (c), and 60 min (d). Insets show close-ups of single cells. Bars, 2 μm (panels a to d) and 200 nm (for insets of panels a to d).