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Previous studies have shown that Streptococcus oligofermentans inhibits the growth of cariogenic Streptococcus mutans in biofilms in vitro and is considered a probiotic candidate for caries prevention. This study aimed to examine the effects of various environmental factors on the competition between S. oligofermentans and S. mutans in a dual-species biofilm model. Single or dual S. oligofermentans and S. mutans biofilms were grown in a 96-well active attachment model for 48 h. Several growth conditions were examined in the model, namely: S. oligofermentans was inoculated 24 h before S. mutans or vice versa; the growth medium was supplemented with 0.2% sucrose or 0.4% glucose; biofilms were grown under a constantly neutral pH or pH-cycling condition, which included 8 h of neutral pH and 16 h of pH 5.5. The 48-h biofilms were examined for viable cell counts and lactic acid and hydrogen peroxide production ability. When S. oligofermentans was inoculated first, it clearly inhibited the growth of S. mutans and reduced the biofilm lactic acid production by up to 8-fold through hydrogen peroxide production, independently of sugar supply and pH conditions. When S. mutans was inoculated first, the level of inhibition by S. oligofermentans varied depending on the sugar supply and pH conditions. Thus, the inhibition efficacy of S. oligofermentans against S. mutans in dual-species biofilms is influenced by environmental factors. This study provides practical information on how to maximize the efficacy of S. oligofermentans.

Aim: To describe the application of a newly-developed in vitro model in which the diffusion of antimicrobials in oral biofilms can be studied. Methodology: In a flow chamber consisting of three parallel feeding channels connected with each other by eight perpendicular side channels, multispecies biofilms were grown from saliva of a single donor for 48 h. The dimensions of the side channels were 100 μm × 100 μm × 5130 μm (H × W × L). When one or more side channels were filled with biofilm, the biofilms were stained with fluorescent stains. Then, one side-channel biofilm was selected and treated with phosphate buffered saline, 2% sodium hypochlorite (NaOCl), 17% ethylenediaminetetra-acetic acid (EDTA) or modified salt solution (MSS). Diffusion of the irrigants was observed by acquiring fluorescence images at 10× objective every 15 s for 30 min. Results: It was possible to culture biofilms in the narrow (100 μm) channels. The biofilms varied in phenotype. In this model, no diffusion of NaOCl into the biofilms was seen after its application. Seventeen-percentage EDTA only diffused into the biofilm up to 200 μm in 30 min. MSS did diffuse in the biofilm over a distance of 450 μm within 2 min after a single application. Conclusions: This new model enables the investigation of the diffusion of antimicrobials in biofilms. Other applications to improve our understanding of the characteristics of biofilms are now possible.

AimTo investigate the cytotoxicity of a modified salt solution (MSS) and evaluate the antimicrobial properties of MSS on in vitro biofilm models.MethodologyIn a metabolic assay, fibroblasts derived from periodontal ligaments (PDL) of human extracted teeth were cultured and challenged with MSS or controls. Then, in active attachment biofilm models, the efficacy of MSS in the presence of dentine powder and in eliminating mature biofilms was investigated. In the dentine assay, a biofilm of Enterococcus faecalis was employed. For the final assay, microorganisms were retrieved from infected root canals and cultured to produce biofilms. After the treatments with MSS or the controls, the biofilms were collected, serially diluted and plated. The colony-forming units were counted. One-way anova was used to analyse the differences between the groups. A P < 0.05 was considered significant.ResultsThe PDL fibroblasts remained metabolically active after challenges with MSS. Dentine powder did not alter the efficacy of MSS (P > 0.05). In endodontic biofilms, the culturable bacteria were equally reduced by MSS, 2% chlorhexidine (CHX) or 2% sodium hypochlorite (NaOCl) (P > 0.05).ConclusionsModified salt solution is noncytotoxic in vitro and has good antimicrobial properties equal to CHX and NaOCl. Although the results are promising, ex vivo and in vivo studies are needed before its use as an interappointment root canal dressing can be considered.

The shift in microbial diversity from young to mature plaque, related to caries activity on sound and restored surfaces, was studied using denaturing gradient gel electrophoresis. During a 20-week in situ study on caries progression 8 subjects wearing restored and unrestored dentin and enamel sections, biofilm was sampled after 1 and 20 weeks (young or mature plaque). A higher microbial diversity (mature plaque) was seen in caries-active compared to caries-free subjects. Rothia dentocariosa and Scardovia inopinata were absent from all caries-free sites, but appeared in 50% of the caries-active sites.

During the past 25 years, oral microbiology has developed from researching a limited number of pathogenic microorganisms to a field in which the entire oral micro flora is studied. While 25 years ago, a limited number of species were studied, now the thousands of species that can be found in the mouth are the subject of research. We can now study the effects of bacteria and fungi on each other, but also make small steps in terms of features that microorganisms occupy in the network of the complex biofilm. The biofilm can be seen as a complex ecosystem, which may be unique to each individual and certainly should not always be treated antimicrobially. It is better to stimulate the healthy biofilm and to prevent transmission of pathogens by targeted infection prevention measures. These measures may only be applied if appropriate microbiological knowledge is present.De orale microbiologie heeft zich in 25 jaar ontwikkeld van het onderzoeken van een beperkt aantal pathogene micro-organismen tot een vakgebied waarin de hele orale microflora wordt bestudeerd. Toentertijd konden beperkte soorten worden bestudeerd, nu zijn de duizenden soorten die zich bevinden in de mond onderwerp van onderzoek. Inmiddels worden de effecten van bacteriën en schimmels op elkaar onderzocht, maar worden ook schoorvoetend stappen gezet op het gebied van functies die micro-organismen innemen in het netwerk van de complexe biofilm. De biofilm blijkt een complex ecosysteem, dat persoonsgebonden is en zeker niet altijd antimicrobieel moet worden behandeld. Het is beter de gezonde biofilm te stimuleren en overdracht van pathogenen te voorkomen door gerichte infectiepreventie maatregelen. Deze maatregelen kunnen alleen worden toegepast als adequate microbiologische kennis aanwezig is.

To investigate the effect of hyperosmotic hyperosmosis or alkaline stress on a dual-species biofilm of Enterococcus faecalis and Pseudomonas aeruginosa.Biofilms were grown on glass cover slips suspended in bacterial inoculate for 96 h, after which the cover slips with attached biofilms were immersed in brain heart infusion broth (BHI-broth) with 6 mol L(-1) sodium chloride (NaCl) representing the hyperosmotic group or Ca(OH)(2), pH 12.1, representing the alkaline group. Two per cent sodium hypochlorite and BHI- broth served as positive and negative controls, respectively. After treatment, the biofilms were washed, harvested and plated on blood-agar plates after serial dilution. The bactericidal effect was assessed by determining the colony-forming units (CFU). The effect on the biofilm mass was imaged with confocal laser scanning microscopy (CLSM).Hyperosmosis reduced the CFU of both species significantly after 72 h (P0.0001). After 168 h, P. aeruginosa was eradicated and the E. faecalis reduction was more than 99%. High pH could not induce a significant bacterial reduction. CLSM revealed dense flocculation of the biofilms incubated in alkaline broth.Hyperosmosis effectively reduced a dual-species biofilm of E. faecalis and P. aeruginosa, whilst high pH had limited bactericidal effect in this model.

An important initial step in biofilm development and subsequent establishment of fungal infections by the human pathogen Candida glabrata is adherence to a surface. Adherence is mediated through a large number of differentially regulated cell wall-bound adhesins. The fungus can modify the incorporation of adhesins in the cell wall allowing crucial adaptations to new environments. In this study, expression and cell wall incorporation of C. glabrata adhesins were evaluated in biofilms cultured in two different media: YPD and a semi-defined medium SdmYg. Tandem mass spectrometry of isolated C. glabrata cell walls identified 22 proteins including six adhesins: the novel adhesins Awp5 and Awp6, Epa3 and the previously identified adhesins Epa6, Awp2 and Awp4. Regulation of expression of these and other relevant adhesin genes was investigated using real-time qPCR analysis. For most adhesin genes, significant up-regulation was observed in biofilms in at least one of the culturing media. However, this was not the case for EPA6 and AWP2, which is consistent with their gene products already being abundantly present in planktonic cultures grown in YPD medium. Furthermore, most of the adhesin genes tested also show medium-dependent differential regulation. These results underline the idea that many adhesins in C. glabrata are involved in biofilm formation and that their expression is tightly regulated and dependent on environmental conditions and growth phase. This may contribute to its potential to form resilient biofilms and cause infection in various host tissues.