USE OF PHOTO-ACTIVATED DISINFECTION (PAD) METHOD FOR THE MANAGEMENT OF DENTAL CARIES BIOFILMS

Abstract

Background: Oral microbial diversity presents significant challenges to caries model and disinfection protocol development. The production of bacterial and fungal mixed biofilms is a particular concern. Notably, changing local environmental conditions is one of the primary activities of a biofilm. Therefore, for a better understanding of the interaction between fungi and bacteria in dental biofilms, it is important to design physiologically relevant multikingdom biofilms that mimic cariogenic activity and evaluate the impact of antimicrobial system on these cariogenic biofilm models. Objectives: The objectives in this study are divided into sections. Development of a cariogenic multispecies biofilm model in the presence and absence of the oral fungi Candida albicans using plaque and cariogenic species, and development of a cariogenic multispecies biofilm model in the presence and absence of plaque and cariogenic species. The influence of Candida albicans and sucrose on the biofilm models will next be evaluated, and the cariogenic potential of polymicrobial plaque biofilm models will be tested. Finally, the antimicrobial and post antimicrobial effect of PAD on the developed multispecies cariogenic model will be investigated. Materials and methods: Biofilms were developed using a combination of “commensal” (Streptococcus oralis, Actinomyces oris, Actinomyces viscosus) and caries-associated (Streptococcus mutans, Lactobacillus acidophilus) oral bacteria as well as the fungus Candida albicans. Bacteria and C. albicans were grown to stationary phase, then washed and resuspended in either RPMI or artificial saliva containing mucin. Bacteria were resuspended at 1X106 CFU/mL and C. albicans 1X105 CFU/mL. Single- and polymicrobial biofilms were then prepared in 24 well plates (1 microbe, 4 bacteria, 5 bacteria, 5 bacteria with L. acidophilus added 3rd day) with and without C. albicans and left for 5 or 10 days, with media changed daily for the first 5days. Then biomass and metabolic activity were measured using Crystal violet and XTT assays respectively to investigate the biofilms developed. For evaluating the impact of sucrose and C. albicans on the developed multispecies biofilm a selection of biofilm communities was prepared (4 bacteria (4), 5 bacteria (5L), 5 bacteria with L. acidophilus added third day (5L3)) with/without C. albicans and grown on hydroxyapatite discs for 10 days, with artificial saliva (AS) ± 0.2 % sucrose. AS was changed daily days 1-5, then left day 5-10. Biomass, metabolic activity, pH level and microbial abundance were measured using Crystal violet, XTT assays, pH meter and qPCR respectively. To determine cariogenic activity, 5L3 biofilms ± C. albicans were grown on polished human enamel and characterised by non-contact laser profilometry to measure surface roughness (Sa), Raman spectroscopy and Knoop microhardness (KHN). Further, to evaluate the Photoactive disinfection (PAD) impact, the multispecies biofilms were grown on hydroxyapatite discs in 24 opaque well plates. The media on the biofilms was changed daily for five days and then was not changed for five days as previously mentioned, resulting in biofilms that were 10 days old. Each experiment for biomass and metabolic activity, was conducted in triplicate, while microbial composition utilising qPCR was conducted twice. On day 10, all analyses were conducted. Biofilm biomass, metabolic activity and the species composition of biofilm was analysed utilising Crystal violet, XTT assays and quantitative polymerase chain reaction qPCR respectively. Results: Significantly higher biomass was observed in multikingdom biofilms aged 10 days grown on polystyrene substrate in artificial saliva compared to RPMI-culture media. While no difference in metabolic activity was observed in the same group. When multispecies biofilms grown on polished enamel substrate in AS media with and without 0.2 % sucrose, biofilms formed successfully. Sucrose affected the species composition, with S. mutans and C. albicans being more abundant in biofilms with sucrose versus non-sucrose media. However, in the absence of sucrose, S. mutans was reduced in Candida-containing versus Candida-free biofilms. A. oris and A. viscosus were reduced in Candida-containing biofilms in sucrose versus non-sucrose media. S. oralis abundance was higher in Candida-containing versus Candida-free biofilms and further increased in sucrose-containing media. In all biofilms and media, there was an increase surface roughness (Sa) of enamel. However, the 5L3 community with sucrose but without Candida resulted in significantly higher surface roughness (Sa). Raman spectroscopy indicated a significant loss of mineral content. The surface Knoop microhardness (KHN) was decreased in sucrose-containing biofilms. Furthermore, Candida-free biofilms with no sucrose had lower Knoop microhardness level (KHN) than Candida-containing biofilms. All biofilm media examined had a lower pH on day 10 than on day 1, with the sucrose groups more acidic medium (low pH value) than non-sucrose and the Candida-containing biofilms less acidic than Candida-free biofilms. PAD affected the biomass of all multikingdom biofilms while in Candida-free biofilms only biofilm 4 was affected significantly. Viability of all biofilm models were reduced significantly after PAD treatment and PAD showed an effect after 5 days of treatment. PAD showed an effect on the composition of all groups treated, resulting in biofilms composed mainly of Actinomyces seen in most biofilms 4, 5, 5, 5C and 5L3C. Conclusions: Artificial saliva containing mucin can provide a favourable environment for the growth of multikingdom biofilms in vitro. The presence of C. albicans with sucrose induces changes in the composition of biofilm species, which affects biofilm cariogenicity. The polymicrobial biofilms developed in this study formed cariogenic communities on the enamel surfaces within 10 days. Multikingdom biofilms and sucrose influence changes in biofilm species composition, which has an effect on biofilm cariogenicity. C. albicans decreases demineralisation while sucrose increases it. PAD therapy showed an effect on multispecies biofilm viability reduction. Tolonium chloride was worth mentioning since it had an effect on some biofilms when applied alone.