Marleen M. Janus

On the ecosystemic network of saliva in healthy young adults

A dysbiotic state is believed to be a key factor in the onset of oral disease. Although oral diseases have been studied for decades, our understanding of oral health, the boundaries of a healthy oral ecosystem and ecological shift toward dysbiosis is still limited. Here, we present the ecobiological heterogeneity of the salivary ecosystem and relations between the salivary microbiome, salivary metabolome and host-related biochemical salivary parameters in 268 healthy adults after overnight fasting. Gender-specific differences in the microbiome and metabolome were observed and were associated with salivary pH and dietary protein intake. Our analysis grouped the individuals into five microbiome and four metabolome-based clusters that significantly related to biochemical parameters of saliva. Low salivary pH and high lysozyme activity were associated with high proportions of streptococcal phylotypes and increased membrane-lipid degradation products. Samples with high salivary pH displayed increased chitinase activity, higher abundance of Veillonella and Prevotella species and higher levels of amino acid fermentation products, suggesting proteolytic adaptation. An over-specialization toward either a proteolytic or a saccharolytic ecotype may indicate a shift toward a dysbiotic state. Their prognostic value and the degree to which these ecotypes are related to increased disease risk remains to be determined.

In vitro phenotypic differentiation towards commensal and pathogenic oral biofilms

Commensal oral biofilms, defined by the absence of pathology-related phenotypes, are ubiquitously present. In contrast to pathological biofilms commensal biofilms are rarely studied. Here, the effect of the initial inoculum and subsequent growth conditions on in vitro oral biofilms was studied. Biofilms were inoculated with saliva and grown anaerobically for up to 21 days in McBain medium with or without fetal calf serum (FCS) or sucrose. Pathology-related phenotypes were quantified and the community composition was determined. Biofilms inoculated with pooled saliva or individual inocula were similar. Denaturing gradient gel electrophoresis (DGGE) analysis allowed differentiation of biofilms grown with sucrose, but not with FCS. Lactate production by biofilms was significantly increased by sucrose and protease activity by FCS. McBain grown biofilms showed low activity for both phenotypes. Three clinically relevant in vitro biofilm models were developed and could be differentiated based on pathology-related phenotypes but not DGGE analysis. These models allow analysis of health-to-disease shifts and the effectiveness of prevention measures.

Candida albicans alters the bacterial microbiome of early in vitro oral biofilms

ABSTRACT The yeast Candida albicans is an oral commensal microorganism, occurring in the oral cavity of 50–70% of healthy individuals. Its effect on oral ecology has mostly been studied using dual-species models, which disregards the complex nature of oral biofilms. The aim of this study was to culture C. albicans in a complex model to study its effect on oral biofilms. Biofilms, inoculated using pooled stimulated saliva with or without addition of C. albicans, were grown under anaerobic, aerobic, or aerobic +5% CO2 conditions. Red autofluorescence was quantified using a spectrophotometer and visualized in fluorescence photographs. The microbiome of 5 h biofilms was determined using 16S rDNA sequencing. C. albicans was only able to proliferate in biofilms grown under aerobic conditions. After 48 h, C. albicans did not induce differences in total biofilm formation, lactic acid accumulation (cariogenic phenotype) or protease activity (periodontitis phenotype). In vitro, anaerobically grown biofilms developed red autofluorescence, irrespective of inoculum. However, under aerobic conditions, only C. albicans–containing biofilms showed red autofluorescence. Facultative or strict anaerobic Veillonella, Prevotella, Leptotrichia, and Fusobacterium genera were significantly more abundant in biofilms with C. albicans. Biofilms without C. albicans contained more of the aerobic and facultative anaerobic genera Neisseria, Rothia, and Streptococcus. The presence of C. albicans alters the bacterial microbiome in early in vitro oral biofilms, resulting in the presence of strictly anaerobic bacteria under oxygen-rich conditions. This in vitro study illustrates that C. albicans should not be disregarded in healthy oral ecosystems, as it has the potential to influence bacteria significantly.

Candida albicans in Multispecies Oral Communities; A Keystone Commensal?

The complexity of the oral cavity, in which many hundreds of microbial species interact represents a challenge for modern microbiologists. What are all these species doing there? And why do we accept so many opportunistic pathogens to be part of our health (commensal) microflora? While the role of bacteria are often being studied, the role of fungi in the interactions within the oral cavity are understudied. This is partly because fungi in the oral cavity are generally considered as pathogens and related to diseases. In this chapter we will explore mechanisms of interaction between bacteria and fungi in the oral cavity that are involved in maintenance of oral health. We will argue that fungi in general and C. albicans specifically, should be regarded a keystone commensal in the oral cavity.

Red and Green Fluorescence from Oral Biofilms

Red and green autofluorescence have been observed from dental plaque after excitation by blue light. It has been suggested that this red fluorescence is related to caries and the cariogenic potential of dental plaque. Recently, it was suggested that red fluorescence may be related to gingivitis. Little is known about green fluorescence from biofilms. Therefore, we assessed the dynamics of red and green fluorescence in real-time during biofilm formation. In addition, the fluorescence patterns of biofilm formed from saliva of eight different donors are described under simulated gingivitis and caries conditions. Biofilm formation was analysed for 12 hours under flow conditions in a microfluidic BioFlux flow system with high performance microscopy using a camera to allow live cell imaging. For fluorescence images dedicated excitation and emission filters were used. Both green and red fluorescence were linearly related with the total biomass of the biofilms. All biofilms displayed to some extent green and red fluorescence, with higher red and green fluorescence intensities from biofilms grown in the presence of serum (gingivitis simulation) as compared to the sucrose grown biofilms (cariogenic simulation). Remarkably, cocci with long chain lengths, presumably streptococci, were observed in the biofilms. Green and red fluorescence were not found homogeneously distributed within the biofilms: highly fluorescent spots (both green and red) were visible throughout the biomass. An increase in red fluorescence from the in vitro biofilms appeared to be related to the clinical inflammatory response of the respective saliva donors, which was previously assessed during an in vivo period of performing no-oral hygiene. The BioFlux model proved to be a reliable model to assess biofilm fluorescence. With this model, a prediction can be made whether a patient will be prone to the development of gingivitis or caries.

Fungal mitochondrial oxygen consumption induces the growth of strict anaerobic bacteria

Fungi are commonly encountered as part of a healthy oral ecosystem. Candida albicans is the most often observed and investigated fungal species in the oral cavity. The role of fungi in the oral ecosystem has remained enigmatic for decades. Recently, it was shown that C. albicans, in vitro, influences the bacterial composition of young oral biofilms, indicating it possibly plays a role in increasing diversity in the oral ecosystem. C. albicans favored growth of strictly anaerobic species under aerobic culture conditions. In the present study, the role of mitochondrial respiration, as mechanism by which C. albicans modifies its environment, was investigated. Using oxygen sensors, a rapid depletion of dissolved oxygen (dO2) was observed. This decrease was not C. albicans specific as several non-albicans Candida species showed similar oxygen consumption. Heat inactivation as well as addition of the specific mitochondrial respiration inhibitor Antimycin A inhibited depletion of dO2. Using 16S rDNA sequencing, it is shown that mitochondrial activity, more than physical presence of C. albicans is responsible for inducing growth of strictly anaerobic oral bacteria in aerobic growth conditions. The described mechanism of dO2 depletion may be a general mechanism by which fungi modulate their direct environment.

A novel compound to maintain a healthy oral plaque ecology in vitro

Objective Dental caries is caused by prolonged episodes of low pH due to acid production by oral biofilms. Bacteria within such biofilms communicate via quorum sensing (QS). QS regulates several phenotypic biofilm parameters, such as biofilm formation and the production of virulence factors. In this study, we evaluated the effect of several QS modifiers on growth and the cariogenic potential of microcosm oral biofilms. Methods Biofilms were inoculated with pooled saliva and cultured in the presence of sucrose for 48 and 96 h. QS modifiers (or carrier controls) were continuously present. Lactic acid accumulation capacities were compared to evaluate the cariogenic potential of the biofilms. Subsequently, biofilm growth was quantified by determining colony forming unit counts (CFUs) and their ecology by 16S rDNA-based microbiome analyses. The minimal inhibitory concentration (MIC) for several Streptococcus spp. was determined using microbroth dilution. Results Of the tested QS modifiers only 3-oxo-N-(2-oxocyclohexyl)dodecanamide (3-Oxo-N) completely abolished lactic acid accumulation by the biofilms without affecting biofilm growth. This compound was selected for further investigation. The active range of 3-Oxo-N was 10–100 µM. The homologous QS molecule, acyl homoserine lactone C12, did not counteract the reduction in lactic acid accumulation, suggesting a mechanism other than QS inhibition. Microbial ecology analyses showed a reduction in the relative abundance of Streptococcus spp. in favor of the relative abundance of Veillonella spp. in the 3-Oxo-N exposed biofilms. The MIC of 3-Oxo-N for several streptococcal species varied between 8 and 32 µM. Conclusion 3-Oxo-N changes the ecological homeostasis of in vitro dental plaque. It reduces its cariogenic potential by minimizing lactic acid accumulation. Based on our in vitro data, 3-Oxo-N represents a promising compound in maintaining a healthy, non-cariogenic, ecology in in vivo dental plaque.

Metabolic Interactions between Bacteria and Fungi in Commensal Oral Biofilms

Oral health is more than just the absence of disease. The key to oral health is a diverse microbiome in an ecological balance. The oral microbiota is one of the most complex and diverse microbial communities in the human body. To maintain oral health, balance between the human host and the intrinsic microorganisms is essential. The healthy oral cavity is represented by a great microbial diversity, including both bacteria and fungi. The bacterial microbiome is very well studied. In contrast, fungi inhabiting the oral cavity are often overlooked. All microbial species in the oral cavity form communities which establish a variety of micro-niches and inter- and intra-species interactions. These interactions can be classified into three main groups: physical, chemical and metabolic interactions. Different metabolic interactions are reviewed in this report, among which are the metabolism of sugars, carbon, lactate and oxygen. This review set out with the aim of assessing the importance of metabolic interactions between fungi and bacteria in the healthy oral cavity.

Multi-species oral biofilm promotes reconstructed human gingiva epithelial barrier function

Since the oral mucosa is continuously exposed to abundant microbes, one of its most important defense features is a highly proliferative, thick, stratified epithelium. The cellular mechanisms responsible for this are still unknown. The aim of this study was to determine whether multi-species oral biofilm contribute to the extensive stratification and primed antimicrobial defense in epithelium. Two in vitro models were used: 3D reconstructed human gingiva (RHG) and oral bacteria representative of multi-species commensal biofilm. The organotypic RHG consists of a reconstructed stratified gingiva epithelium on a gingiva fibroblast populated hydrogel (lamina propria). Biofilm was cultured from healthy human saliva, and consists of typical commensal genera Granulicatella and major oral microbiota genera Veillonella and Streptococcus. Biofilm was applied topically to RHG and host–microbiome interactions were studied over 7 days. Compared to unexposed RHG, biofilm exposed RHG showed increased epithelial thickness, more organized stratification and increased keratinocyte proliferation. Furthermore biofilm exposure increased production of RHG anti-microbial proteins Elafin, HBD2 and HBD3 but not HBD1, adrenomedullin or cathelicidin LL-37. Inflammatory and antimicrobial cytokine secretion (IL-6, CXCL8, CXCL1, CCL20) showed an immediate and sustained increase. In conclusion, exposure of RHG to commensal oral biofilm actively contributes to RHG epithelial barrier function.

Effect of erythritol on microbial ecology of in vitro gingivitis biofilms

ABSTRACT Gingivitis is one of the most common oral infections in humans. While sugar alcohols such as erythritol are suggested to have caries-preventive properties, it may also have beneficial effects in prevention of gingivitis by preventing maturation of oral biofilms. The aim of this study was to assess the effect of erythritol on the microbial ecology and the gingivitis phenotype of oral microcosms. Biofilms were inoculated with stimulated saliva from 20 healthy donors and grown in a gingivitis model in the continuous presence of 0 (control group), 5, and 10% erythritol. After 9 days of growth, biofilm formation, protease activity (gingivitis phenotype), and microbial profile analyses were performed. Biofilm growth was significantly reduced in the presence of erythritol, and this effect was dose dependent. Protease activity and the Shannon diversity index of the microbial profiles of the biofilms were significantly lower when erythritol was present. Microbial profile analysis revealed that presence of erythritol induced a compositional shift from periodontitis- and gingivitis-related taxa toward early colonizers. The results of this study suggest that erythritol suppresses maturation of the biofilms toward unhealthy composition. The gingivitis phenotype was suppressed and biofilm formation was reduced in the presence of erythritol. Therefore, it is concluded that erythritol may contribute to a healthy oral ecosystem in vitro.