Antimicrobial susceptibility testing of Citrox^® mouthwashes containing citrus bioflavonoids

Introduction: Citrox^® is a formulation of soluble bioflavonoids obtained from citrus fruits. The non-toxic and antimicrobial properties of natural bioflavonoids are well documented, and so such preparations have many potential applications.

Objectives: To determine the antimicrobial activity of a range of Citrox^®-formulations against a variety of pathogenic microorganisms.

Methods: Fourteen species of microorganism were selected, including bacteria associated with periodontal disease, Candida species, and the bacterium Clostridium difficile, associated with diarrhoeal infection in hospitalised patients. Initial evaluation of two Citrox^® formulations (BC30 and MDC30; dilution range 0.007 – 8% v/v) was based on in vitro Minimal Inhibitory Concentration (MIC) testing against an inoculum of ~10³ microbial cells. Comparative growth was assessed by measuring optical density after 24 h incubation. Serial dilutions of Citrox^® were also tested for their ability to inhibit microbial growth in a microtiter biofilm assay: suspensions of each microorganism (containing ~10⁴ cfu) were incubated for 24 h to allow biofilms to form. Media containing Citrox^® was then added and, following incubation, growth was estimated by measuring changes in absorbance. The biofilm assay was also used to test antimicrobial activity of 6 sample Citrox^® mouthwashes.

Results: Both Citrox^® formulations exhibited antimicrobial activity, although BC30 was found to be the most efficient, and significantly inhibited growth of all bacteria and yeasts at a working concentration of 1% (v/v) in both MIC and biofilm assays. Mouthwashes containing 1% (v/v) BC30 were also effective at inhibiting microbial growth, regardless of additional ingredients.

Conclusions: Citrox^® is an efficient broad-spectrum antimicrobial agent with great potential for use in mouthwashes, playing a role in the maintenance of good oral hygiene.