Differential Actions of Chlorhexidine on the Cell Wall of Bacillus subtilis and Escherichia coli

Hon-Yeung Cheung,Yun-Wah Lam,Longman Yimin Liang,Matthew Man-Kin Wong,Sau-Ha Cheung,Sung-Kay Chiu,Tarek Msadek

doi:10.1371/journal.pone.0036659

Abstract

Chlorhexidine is a chlorinated phenolic disinfectant used commonly in mouthwash for its action against bacteria. However, a comparative study of the action of chlorhexidine on the cell morphology of Gram-positive and Gram-negative bacteria is lacking. In this study, the actions of chlorhexidine on the cell morphology were identified with the aids of electron microscopy. After exposure to chlorhexidine, numerous spots of indentation on the cell wall were found in both Bacillus subtilis and Escherichia coli. The number of indentation spots increased with time of incubation and increasing chlorhexidine concentration. Interestingly, the dented spots found in B. subtilis appeared mainly at the hemispherical caps of the cells, while in E. coli the dented spots were found all over the cells. After being exposed to chlorhexidine for a prolonged period, leakage of cellular contents and subsequent ghost cells were observed, especially from B subtilis. By using 2-D gel/MS-MS analysis, five proteins related to purine nucleoside interconversion and metabolism were preferentially induced in the cell wall of E. coli, while three proteins related to stress response and four others in amino acid biosynthesis were up-regulated in the cell wall materials of B. subtilis. The localized morphological damages together with the biochemical and protein analysis of the chlorhexidine-treated cells suggest that chlorhexidine may act on the differentially distributed lipids in the cell membranes/wall of B. subtilis and E. coli.

Highlights

Chlorhexidine (CHX) is one of the most favorable choices amongst all the disinfectants currently in use
The inhibitory action of CHX lasted for 6 h before the cells regained their ability to grow. Both B. subtilis and E. coli grew at a similar rate for another 4 h, but B. subtilis entered the stationary phase at a lower cell density than E. coli (Figure 1b and c, closed triangles)
The scanning electron microscopy on the CHXtreated cells showed that it caused morphological changes on the cell surfaces of the bacteria, we investigated whether the same concentration of CHX might affect the internal structure of these cells

Summary

Introduction

Chlorhexidine (CHX) is one of the most favorable choices amongst all the disinfectants currently in use. It has been reported that CHX is effective to prevent and control infectious diseases of the mouth by killing bacteria in saliva and tongue [1]; yet it has little undesirable effects [2]. It is a broad-spectrum bactericidal agent against both Gram (+) and (2) bacteria and fungi [3,4]. Its propensity to bind to the surface of tissues offers a long-lasting antimicrobial effect [7] This property of CHX makes it suitable for use as a preservative in some pharmaceutical or medical products, such as ophthalmic solutions, and as a disinfectant of medical instruments and hard surfaces

Methods

Results

Conclusion