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Streptococcus mutans colonizing on tooth surfaces is one of the major causative agents of human dental caries. Despite numerous studies conducted on lasers and oral tissue interactions, little is known about the effect of laser energy on S. mutans gene expression in a biofilm form. The aim of this study was to investigate the effect of sublethal energies of CO2 laser on biofilm and gene expression of the oral bacteria S. mutans immobilized in biofilm. S. mutans biofilm was irradiated with CO2 laser. Vitality and construction of the biofilm were observed by confocal laser scanning microscopy and scanning electron microscopy. The effect of laser irradiation on gene expression was evaluated by DNA microarray. CO2 laser irradiation had a dose effect on the viability of S. mutans immobilized in biofilm. A nonsignificant lethal effect was observed at 31 J/cm2 while at higher energy of 70 and 144 J/cm2 an antibacterial effect was recorded. The mode of antibacterial action seems to be from the inner layers toward the outer layer of the biofilm, indicating the influence of the surface on the killing effect. At 31 J/cm2, microarray analysis indicated a moderate effect on S. mutans gene expression due to CO2 laser irradiation, mainly down-regulating genes related to bacterial stress response. In conclusion, laser irradiation at sublethal energy had an effect on gene expression of S. mutans. Copyright © 2011 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]