Effect of berberine on Staphylococcus epidermidis biofilm formation

https://doi.org/10.1016/j.ijantimicag.2008.10.033Get rights and content

Abstract

Staphylococcus epidermidis is one of the main causes of medical device-related infections owing to its adhesion and biofilm-forming abilities on biomaterial surfaces. Berberine is an isoquinoline-type alkaloid isolated from Coptidis rhizoma (huang lian in Chinese) and other herbs with many activities against various disorders. Although the inhibitory effects of berberine on planktonic bacteria have been investigated in a few studies, the capacity of berberine to inhibit biofilm formation has not been reported to date. In this study, we observed that berberine is bacteriostatic for S. epidermidis and that sub-minimal inhibitory concentrations of berberine blocked the formation of S. epidermidis biofilm. Using viability assays and berberine uptake testing, berberine at a concentration of 15–30 μg/mL was shown to inhibit bacterial metabolism. Data from this study also indicated that modest concentrations of berberine (30–45 μg/mL) were sufficient to exhibit an antibacterial effect and to inhibit biofilm formation significantly, as shown by the tissue culture plate (TCP) method, confocal laser scanning microscopy and scanning electron microscopy for both S. epidermidis ATCC 35984 and a clinical isolate strain SE243. Although the mechanisms of bacterial killing and inhibition of biofilm formation are not fully understood, data from this investigation indicated a potential application for berberine as an adjuvant therapeutic agent for the prevention of biofilm-related infections.

Introduction

In recent years, coagulase-negative Staphylococcus epidermidis has become the leading cause of infections related to indwelling medical devices such as vascular catheters, prosthetic joints and artificial heart valves [1], [2], [3]. The ability of these bacteria to produce biofilm on the surfaces of biomaterials used during surgery is one of the main causes of this difficult-to-cure infection. It has been estimated that ca. 65% of human bacterial infections involve biofilms [4].

Biofilms are communities of surface-associated microorganisms embedded in a self-produced extracellular polymeric matrix that are notoriously difficult to eradicate and are a source of many recalcitrant infections. Bacteria in biofilms can be up to 1000-fold more resistant to antibiotic treatment than the same organism growing planktonically [5]. Standard antibiotic therapy is only able to eliminate planktonic cells, leaving the sessile forms to propagate within the biofilm and to continue to disseminate when therapy is terminated. In biofilms, microbes are protected from antimicrobial agents and the host immune system. However, the mechanisms by which the biofilm-grown bacteria attain this resistance are still unknown. The ability of biofilm-embedded bacteria to resist clearance by antimicrobial agents points to the importance of a continuous search for novel agents that are effective against bacteria or that work in synergy with the currently available antimicrobial agents. Novel strategies are therefore required to deal with these biofilm-mediated infections.

Currently, a renewed interest in natural substances has focused attention on plants rich in bioactive compounds well known for their antimicrobial properties. Plants produce an enormous array of secondary metabolites and it is commonly accepted that a significant part of this chemical diversity serves to protect plants against microbial pathogens [6].

Berberine is an isoquinoline-type alkaloid isolated from Coptidis rhizoma (huang lian in Chinese) and other herbs [7]. Because berberine has antidiarrhoeal, antimicrobial and anti-inflammatory effects, it has been used for centuries in the treatment of liver disease, skin inflammation, diarrhoea and other disorders [8], [9]. Moreover, the toxicity and mutagenicity of berberine to human cells were relatively low in in vivo and in vitro experiments [8], [10]. Recently, berberine has also been used as a novel agent against a number of pathogenic microorganisms such as bacteria, fungi and viruses [11], [12], [13], [14].

In most studies of the anti-infective activities of berberine, the focus has been on its antimicrobial effects [11], [12], [13], [14], and little or no attention has been paid to the effects of berberine on the biofilm formation of S. epidermidis. Here we investigated the effect of berberine on S. epidermidis biofilm formation in order to ascertain its importance to biofilms.

Section snippets

Bacterial strains, media and reagents

Staphylococcus epidermidis 243 (SE243) used in this study was isolated from a patient with periprosthetic infection who was undergoing treatment at the Ninth People's Hospital, Shanghai Jiao Tong University (Shanghai, China). Two standard S. epidermidis strains, ATCC 35984 and ATCC 12228, were kindly provided by Prof. Di Qu (Laboratory of Medical Molecular Virology, Shanghai Medical College, Fudan University, Shanghai, China). The strains were stored at −80 °C in glycerol stocks and used as

MIC and MBC determination

The susceptibilities of S. epidermidis ATCC 12228, ATCC 35984 and clinical strain SE243 to berberine were determined in vitro by methods recommended by the CLSI [15]. The MICs and MBCs are given in Table 1. The MIC of ATCC 12228 was the lowest (64 μg/mL) among the three strains. The MICs of ATCC 35984 and SE243 were 128 μg/mL and 256 μg/mL, respectively. The MBC of clinical isolate SE243 (1024 μg/mL) was higher than those of the other two strains (256 μg/mL).

Effect of berberine on metabolic activity

AlamarBlue, an oxidation–reduction

Discussion

Owing to the increasing involvement of S. epidermidis in foreign-body-related infections, the rapid development and exhibition of multiple antibiotic resistances, as well as its great propensity to lead to persistent, chronic and recurrent infections, this pathogen has continued to receive significant attention [18], [19], [20], [21]. These types of infections are mediated by the ability of S. epidermidis to form biofilms. Bacteria that grow as a biofilm encased in a self-produced matrix are

Acknowledgment

The authors are grateful to Prof. Di Qu (Laboratory of Medical Molecular Virology, Shanghai Medical College, Fudan University, Shanghai, China) for providing S. epidermidis standard strains (ATCC 12228 and ATCC 35984) used in this study.

Funding: This work was supported by a grant from the National Natural Science Foundation of China (No. 30700852) and a grant from the National High Technology Research and Development Program of China (No. 2006AA02A137). The study sponsors were not involved in

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