Candida species in intact in vivo biofilm from carious lesions.

OBJECTIVES: The aim of this study was to investigate the occurrence and spatial localization of Candida species in intact in vivo biofilm from caries lesions in root and occlusal surfaces. MATERIALS AND METHODS: Biofilm from 7 teeth with root caries and 9 teeth with occlusal caries were analyzed. The teeth were fixed, embedded, sectioned and decalcified before fluorescence in situ hybridization using oligonucleotide probes against all bacteria (EUB338), all yeast (PF2), streptococci (STR405, MUT590) and C. albicans (Du.al 1249). Sections were analyzed using fluorescence microscopy. RESULTS: Yeasts, most of which were C. albicans or C. dubliniensis, colonized occlusal and root surface sites and exhibited both yeast and hyphal forms. Two characteristic colonization patterns were apparent: In one pattern, the biofilm presented defined areas comprising Candida hyphal networks mixed with filamentous, rod-like and coccoid bacteria, often extending through the entire biofilm thickness. In the other pattern, Candida formed corncob configurations with non-mutans streptococci in the biofilm surface layer. Mutans streptococci formed isolated colonies and did not typically appear in close proximity with Candida. CONCLUSION: For the first time, we showed that C. albicans and C. dubliniensis are integrated components of caries biofilm architecture. Co-localization of Candida and non-mutans streptococci indicates a beneficial interaction between these organisms. Diverse metabolic properties of Candida spp. suggest that these organisms may influence the ecology of cross-kingdom microbial communities in caries.

Effect of photodynamic therapy in combination with various irrigation protocols on an endodontic multispecies biofilm ex vivo.

AIM: To analyse the antibacterial effect of photodynamic therapy (PDT) in combination with various irrigation protocols on a multispecies biofilm in root canals ex vivo. METHODOLOGY: A total of 160 extracted human single-rooted teeth were divided into four groups (n = 40). In group G1, root canals were instrumented up to size 60 (control group), whereas in G2 to G4 canals were enlarged up to size 40. All root canals were inoculated with a multispecies biofilm (Enterococcus faecalis, Streptococcus oralis, Prevotella intermedia) for 5 days. In G2 to G4, instrumentation up to size 60 was performed using 0.9% sodium chloride (NaCl) (G2), 1% sodium hypochlorite (NaOCl) (G3), 1% NaOCl and a final irrigation with 2% chlorhexidine (CHX) (G4), respectively. In all groups half of the specimens received adjunctive PDT using phenothiazine chloride as photosensitizer and a diode laser (wavelength 660 nm). Counts of colony-forming units (CFUs) in each group were analysed separately for planktonic and dentine-adherent bacteria immediately after therapy (T1) and after 5 days of further incubation (T2). Descriptive statistics and two-way analysis of variance were carried out to analyse reduction of planktonic bacteria and nonparametric tests were used to analyse dentine-adherent bacteria. RESULTS: CFU reduction in planktonic bacteria was significantly affected by the irrigation protocol at T1 and T2 (P < 0.0001), but PDT significantly reduced CFUs only at T2 (P = 0.01; anova). Irrigation using NaOCl, CHX and adjunctive PDT significantly reduced CFUs at T2 (P < 0.0001; Tukey HSD) compared to the control group. In 85.6% of all samples the same categories of CFU counts in both planktonic and dentine-adherent bacteria were detected at T1 and T2. CONCLUSIONS: Adjunctive photodynamic therapy in combination with an irrigation protocol including NaOCl and CHX was an effective method for reduction of bacterial biofilm inside the root canals of extracted teeth.

Randomized Clinical Trials on Deep Carious Lesions: 5-Year Follow-up.

Deep caries presents a dilemma in terms of which treatment that will render an optimal prognosis by maintaining pulp vitality with absence of apical pathology. Previously, 2 randomized clinical trials were performed testing the short-term effects of stepwise carious tissue removal versus nonselective carious removal to hard dentin with or without pulp exposure. The aim of this article was to report the 5-y outcome on these previously treated patients having radiographically well-defined carious lesions extending into the pulpal quarter of the dentin but with a well-defined radiodense zone between the carious lesion and the pulp. In this long-term study, 239 of 314 (76.2%) patients were analyzed. The stepwise removal group had a significantly higher proportion of success (60.2%) at 5-y follow-up compared with the nonselective carious removal to hard dentin group (46.3%) ( P = 0.031) when pulp exposures per se were included as failures. Pulp exposure rate was significantly lower in the stepwise carious removal group (21.2% vs. 35.5%; P = 0.014). Irrespective of pulp exposure status, the difference (13.3%) was still significant when sustained pulp vitality without apical radiolucency and unbearable pain was considered (95% confidence interval, 3.1-26.3, P = 0.045). After pulp exposure, only 9% ( n = 4) of the analyzed patients were assessed as successful, indicating that the prognosis is highly dubious following conventional pulp-capping procedures (direct pulp capping or partial pulpotomy) in deep carious lesions in adults. In conclusion, the stepwise carious removal group had a significantly higher proportion of pulps with sustained vitality without apical radiolucency versus nonselective carious removal of deep carious lesions in adult teeth at 5-y follow-up ( ClinicalTrials.gov NCT00187837 and NCT00187850).

Colonization of the oral cavity by probiotic bacteria.

The aim of this study was to investigate if three probiotic bacteria present in the milk product Cultura Dofilus® naturell could be detected in saliva and on oral mucosal surfaces, and if they colonized dental surfaces in situ in 8 caries-inactive individuals after 8 daily exposures to the milk product for up to 3 days. Bacteria were identified by fluorescence in situ hybridization and confocal laser scanning microscopy. While probiotic bacteria were present sporadically in the oral cavity on mucosal surfaces and in saliva after 3 days of frequent use of the probiotic milk, they were not detected on dental surfaces. Probiotic bacteria may thus contribute to general oral health, but their potential role in biofilm-induced dental diseases remains unclear.

Actinomyces naeslundii in initial dental biofilm formation.

The combined use of confocal laser scanning microscopy (CLSM) and fluorescent in situ hybridization (FISH) offers new opportunities for analysis of the spatial relationships and temporal changes of specific members of the microbiota of intact dental biofilms. The purpose of this study was to analyse the patterns of colonization and population dynamics of Actinomyces naeslundii compared to streptococci and other bacteria during the initial 48 h of biofilm formation in the oral cavity. Biofilms developed on standardized glass slabs mounted in intra-oral appliances worn by ten individuals for 6, 12, 24 and 48 h. The biofilms were subsequently labelled with probes against A. naeslundii (ACT476), streptococci (STR405) or all bacteria (EUB338), and were analysed by CLSM. Labelled bacteria were quantified by stereological tools. The results showed a notable increase in the number of streptococci and A. naeslundii over time, with a tendency towards a slower growth rate for A. naeslundii compared with streptococci. A. naeslundii was located mainly in the inner part of the multilayered biofilm, indicating that it is one of the species that attaches directly to the acquired pellicle. The participation of A. naeslundii in the initial stages of dental biofilm formation may have important ecological consequences.

Application of stereological principles for quantification of bacteria in intact dental biofilms.

INTRODUCTION: Quantative confocal laser scanning microscopy (CLSM) in combination with fluorescent in situ hybridization (FISH) may help to increase our knowledge about biofilm formation. The purpose of this study was to develop and evaluate a stereological method for quantification of bacteria in intact biofilm. The method was applied in a quantitative study of the proportion of streptococci relative to other bacteria in initial in-situ-grown dental biofilms as a function of time. METHODS: Biofilms were collected on standardized glass slabs mounted in intra-oral appliances and worn by 10 individuals for 6, 12, 24, and 48 h. Biofilms were analysed using CLSM. Quantification of bacteria labelled with 16S ribosomal RNA oligonucleotide probes was performed with stereological tools: the unbiased counting frame and the two-dimensional fractionator. RESULTS: Results showed a notable increase in the total number of bacteria and streptococci over time, with a considerable inter-individual variation at each time-point. After 48 h there was a 12.5-fold difference between individuals in the total number of bacteria and a 12.6-fold difference in the number of streptococci. The number of streptococci exceeded that of other bacteria and over the examination period there was a relatively constant relationship between the number of streptococci and other bacteria (streptococci vs. non-streptococci: median 15.2; minimum 1.0; maximum 89.3). CONCLUSION: The study demonstrates that the combined use of FISH and stereology is a relevant and reliable tool for obtaining unbiased information about the numerical contributions of specific bacterial populations during early biofilm formation.