Combinatorial effects of trans-cinnamaldehyde with fluoride and chlorhexidine on Streptococcus mutans.

AIMS: The aim of this study was to investigate the effects of trans-cinnamaldehyde (TC) and its synergistic activity with chlorhexidine (CHX) and fluoride against Streptococcus mutans. METHODS AND RESULTS: Streptococcus mutans UA159 was treated with TC alone and in combination with CHX or sodium fluoride. The synergy profile was analysed using the Zero Interaction Potency model. TC showed strong synergism (synergy score of 21·697) with CHX, but additive effect (synergy score of 5·298) with fluoride. TC and the combinations were tested for acid production (glycolytic pH drop) and biofilm formation by S. mutans, and nitric oxide production in macrophages. TC significantly inhibited sucrose-dependent biofilm formation and acid production by S. mutans. Mechanistic studies were carried out by qRT-PCR-based transcriptomic studies which showed that TC acts by impairing genes related to metabolism, quorum sensing, bacteriocin expression, stress tolerance and biofilm formation. CONCLUSIONS: trans-Cinnamaldehyde potentiates CHX and sodium fluoride in inhibiting S. mutans biofilms and virulence through multiple mechanisms. This study sheds significant new light on the potential to develop TC as an anti-caries treatment. SIGNIFICANCE AND IMPACT OF THE STUDY: Oral diseases were classified as a 'silent epidemic' in the US Surgeon General's Report on Oral Health. Two decades later, >4 billion people are still affected worldwide by caries, having significant effects on the quality of life. There is an urgent need to develop novel compounds and strategies to combat dental caries. Here, we prove that TC downregulates multiple pathways and potentiates the CHX and fluoride to prevent S. mutans biofilms and virulence. This study sheds significant new light on the potential to develop TC in combination with CHX or fluoride as novel treatments to arrest dental caries.

The influence of substrate surface conditioning and biofilm age on the composition of Enterococcus faecalis biofilms.

AIM: To investigate the null hypothesis that neither the surface conditioning (collagen, serum, saliva) of hydroxyapatite (HA) discs, nor the biofilm age (3 days vs. 21 days) has a significant effect on the cellular and matrix composition of biofilms, using Enterococcus faecalis as the model organism. METHODOLOGY: Sterile HA discs were conditioned with collagen, saliva or serum, and inoculated with E. faecalis to form 3-day and 21-day-old biofilms. Unconditioned discs served as controls. The biofilms were analysed using culture-dependent and independent (confocal microscopy and biochemical analysis) methods, to determine the colony-forming units and the biofilm matrix composition (polysaccharides and proteins), respectively. Statistical analyses were performed using appropriate parametric and nonparametric tests (P = 0.05). RESULTS: Collagen conditioning significantly increased the number of CFUs in the 21-day biofilms, compared to the 3-day biofilms (P < 0.05). Although the biochemical analysis revealed that surface conditioning had no significant effect on the total carbohydrate content in the 21-day biofilms, confocal microscopic analysis revealed that collagen and saliva conditioning selectively increased the polysaccharide content of 21-day biofilms, compared to the 3-day biofilms (P < 0.05). CONCLUSIONS: The results of this study raise an important methodological concern that the substrate conditioning substances and biofilm age differentially influence the cellular and extracellular matrix components of E. faecalis biofilms.

Escape from the competence state in Streptococcus mutans is governed by the bacterial population density.

Horizontal gene transfer through natural DNA transformation is an important evolutionary mechanism among bacteria. Transformation requires that the bacteria are physiologically competent to take and incorporate free DNA directly from the environment. Although natural genetic transformation is a remarkable feature of many naturally competent bacteria, the process is energetically expensive for the cells. Consequently, a tight control of the competence state is necessary. The objective of the present work was to help decipher the molecular mechanisms regulating the escape from the competence state in Streptococcus mutans, the principal etiological agent responsible for tooth decay in humans. Our results showed that the cessation of competence in S. mutans was abrupt, and did not involve the accumulation of a competence inhibitor nor the depletion of a competence activator in the extracellular environment. The competence state was repressed at high cell population density via concomitant repression of sigX gene encoding the master regulator of the competence regulon. Co-culture experiments performed with oral and non-oral bacteria showed that S. mutans assesses its own population density and also the microbial density of its surroundings to regulate its competence escape. Interestingly, neither the intra-species and extra-species quorum-sensing systems nor the other 13 two-component regulatory systems identified in S. mutans were involved in the cell-density-dependent escape of the competence state. Altogether, our results suggest a complex mechanism regulating the competence shut-off involving cell-density-dependent repression of sigX through an as yet undefined system, and possibly SigX protein stability.

Involvement of Streptococcus mutans regulator RR11 in oxidative stress response during biofilm growth and in the development of genetic competence.

AIMS: To identify the genes regulated by RR11, the regulator of the Streptococcus mutans HK/RR11 two-component system. METHODS AND RESULTS: The S. mutans RR11-encoding gene was inactivated, and the effects of gene disruption on the cell's ability to form biofilms under stresses and acquire extracellular DNA were tested. Biofilm was reduced in cells lacking RR11 following exposure to oxidative stress. RR11-defective cells showed approx. 20-fold reduction in transformation efficiency. Microarray used to decipher the RR11-regulated genes in biofilm showed that approx. 5% of the UA159 genome underwent a significant change in expression. RR11 was found to regulate 174 genes, including genes involved in competence, stress-response and cell division. CONCLUSIONS: Target genes controlled by RR11during biofilm growth have been identified by a comparison of transcriptional profiles between an RR11 defective mutant and the parental strain. The results demonstrated that RR11 is involved in the control of diverse cellular processes, including the formation of biofilm under oxidative stress and development of genetic competence. SIGNIFICANCE AND IMPACT OF THE STUDY: The regulator of HK/RR11 system controls a large regulon and is an important regulator involved in stress response during S. mutans biofilm growth enabling the survival and persistence of its progeny in the microbial community.

Systemic inactivation and phenotypic characterization of two-component systems in expression of Streptococcus mutans virulence properties.

AIM: To assess potential function of each two-component signal transduction system in the expression of Streptococcus mutans virulence properties. METHODS AND RESULTS: For each two-component system (TCS), the histidine kinase-encoding gene was inactivated by a polymerase chain reaction (PCR)-based deletion strategy and the effects of gene disruption on the cell's ability to form biofilms, become competent, and tolerate acid, osmotic, and oxidative stress conditions were tested. Our results demonstrated that none of the mutations were lethal for S. mutans. The TCS-2 (CiaRH) is involved in biofilm formation and tolerance to environmental stresses, the TCS-3 (ScnRK-like) participates in the survival of cells at acidic pH, and the TCS-9 affects the acid tolerance response and the process of streptococcal competence development. CONCLUSIONS: Our results confirmed the physiological role of the TCS in S. mutans cellular function, in particular the SncRK-like TCS and TCS-9 as they may represent new regulatory systems than can be involved in S. mutans pathogenesis. SIGNIFICANCE AND IMPACT OF THE STUDY: Multiple TCS govern important biological parameters of S. mutans enabling its survival and persistence in the biofilm community.