Zinc enhances the phototoxic effect of blue light against malodour-producing bacteria in an experimental oral biofilm Nir Sterer, Uziel Jeffet, Aurel Dadoun, Ronit Bar-Ness Greenstein and David Kohavi Correspondence Nir Sterer drsterer@gmail.com Received 3 March 2014 Accepted 6 June 2014 Department of Prosthodontics, Goldschleger School of Dental Medicine, Tel Aviv University, PO Box 39796, Ramat Aviv, Tel Aviv 69978, Israel Oral malodour is thought to be caused mainly by the production of volatile sulfide compounds (VSCs) by anaerobic Gram-negative oral bacteria. Previous studies have shown that these bacteria are susceptible to blue light (400–500 nm wavelength). In the present study, we tested the effect of blue light in the presence of zinc, erythrosine B or both on malodour production in an experimental oral biofilm. Biofilms were exposed to a plasma-arc light source for 30, 60 and 120 s (equal to energy fluxes of 41, 82 and 164 J cm ”2 , respectively) with or without the addition of zinc acetate, erythrosine B or both. After the light exposure, biofilm samples were examined for malodour production (by an odour judge) and VSC production (with a Halimeter), and VSC- producing bacteria were quantified using a microscopy-based sulfide assay (MSA) and in situ confocal laser scanning microscopy (CLSM). Results showed that exposing experimental oral biofilm to both blue light and zinc reduced malodour production, which coincided with a reduction in VSC-producing bacteria in the biofilm. These results suggest that zinc enhances the phototoxicity of blue light against malodour-producing bacteria. INTRODUCTION The proteolytic activity of anaerobic Gram-negative oral bacteria, such as Porphyromonas, Fusobacterium and Prevo- tella species, has been associated with oral malodour and gum disease (Berg & Fosdick, 1946; McNamara et al., 1972; De Boever & Loesche, 1995). These bacteria break down oral proteins and glycoproteins into their constituent amino acids (Kleinberg & Codipilly, 1997). Some of these amino acids are further metabolized, yielding malodorous compounds such as volatile sulfide compounds (VSCs; e.g. H 2 S and methyl mercaptan) (Persson et al., 1989; Kleinberg & Codipilly, 1997; Tonzetich & Carpenter, 1971). Zinc salts have been reported to act as putative anti- malodour agents (Jonski et al., 2004). This trait was mainly attributed to the ability of zinc ions to bind sulfide ions and rendering them thus non-volatile by precipitation. However, some evidence also suggests that zinc has activity against anaerobic oral bacteria (Sheng et al., 2005) by enhancing susceptibility of the bacteria to peroxide. Various studies have demonstrated the antibacterial effect of visible light applied with or without exogenous photo- sensitizers (Wilson, 1994; Henry et al., 1995, 1996; Konig et al., 2000; Soukos et al., 2005; Sterer & Feuerstein, 2005). It was further suggested that light-induced production of reactive oxygen species (ROS) is the mechanism underlying this phototoxic effect (Gourmelon et al., 1994). The aim of the present study was to test the hypothesis that zinc may promote the phototoxic effect of high-intensity blue light against malodour-producing bacteria in an experimental model of oral biofilm. METHODS Light source. A high-intensity non-coherent visible light source, known in dentistry as the plasma-arc, i.e. a xenon light source supplemented with a filter (wavelengths, 400–500 nm) (Sapphire Supreme; DenMat) and fitted with a 9 mm diameter light-guiding tip was used. The average light power (1500 mW cm 22 , in ‘Standard Curing’ (SC) mode) was measured with the unit’s built-in power meter prior to each experiment. Biofilm formation. Samples (40 ml) of fresh whole saliva from a single donor positive for oral malodour were placed into wells of a 48-well microplate (Nunc) with one-well spaces between samples. After 3 min of adsorption of salivary proteins, 1.2 ml decarboxylase medium (0.5% Peptone, 0.3% yeast extract, 0.1% dextrose, 0.002% bromocresol purple) was gently added to each well. Samples were incubated at 37 uC for 96 h under anaerobic conditions to allow experimental biofilm formation (Sterer et al., 2009). Experimental protocol. After incubation, the supernatant was discarded and replaced with 200 ml PBS with or without zinc acetate Abbreviations: CLSM, confocal laser scanning microscopy; LED, light- emitting diode; MSA, microscopy-based sulfide assay; ROS, reactive oxygen species; VSC, volatile sulfide compounds. Journal of Medical Microbiology (2014), 63, 1071–1075 DOI 10.1099/jmm.0.075044-0 075044 G 2014 The Authors Printed in Great Britain 1071