T. denticola) characterized bacteria strongly associated with periodontal disease, the orange complex (F. nucleatum, P. micra,and others) represent the core bacteria, certain oral streptococci were grouped in the yellow complex, V. parvula and A. odontolyticus formed the purple complex, and the green complex consisted of unrelated species [33]. The latest sequencing technologies allow deeper insights into the microbial composition of oral biofilms, revealing that a mature supragingival biofilm is dominated by Streptococcus sp. and Neisseria sp. Furthermore, Gracilibacteria, TM7, Bacteroides, Catonella, Porphyromonas, Tannerella, and others were identified, but not Archaea, yeasts, protozoa, or viruses [34].
The matrix of an oral biofilm contributes to the structural integrity and protects the biofilm against environmental insults [35]. Molecules penetrate through channels and pores, and microorganisms metabolize nutrients, resulting in very different conditions (e.g., pH, redox potential) in short distances, which allows the co-existence of different microorganisms [35]. However, the composition of the oral biofilm matrix has not been well studied. Glycoconjugates binding to 10 lectins were identified in pooled supragingival biofilm samples [36]. Recently, extracellular DNA was visualized in vivo in dental biofilms as an important component for biofilm stability [37]. Among the extracellular polysaccharides, synthesis and the role of glucans are best described, but how other bacterial-produced polysaccharides (polymers being rich in mannose and capsular polysaccharides) contribute to the extracellular matrix remains to be determined [38].
A synergistic interaction with other bacteria is also essential in the oral biofilms. Several periodontal pathogens can grow and multiply only in the presence of other bacteria [39]. Communication is of importance in biofilm formation and maintenance. Oral bacteria produce two major classes of signaling molecules, the competence-stimulating peptides (CSP) and autoinducer-2 [39]. CSP are synthesized by Gram-positive bacteria, they promote biofilm formation, DNA release, and may inhibit bacteriocins produced by S. mutans [39]. Autoinducer-2 production seems to be of importance in the communication of periodontopathogens [39].
Dental Biofilm and Diseases
Microbial homeostasis can break down and result in restructuring of the biofilm with a different composition [35]. The most prevalent oral diseases, dental caries, and periodontitis are multi-species biofilm-associated diseases.
For many years, dental caries has been strongly associated with S. mutans, which utilizes many carbohydrates to produce acids and to synthesize extracellular polysaccharides, an important constituent of the matrix of cariogenic biofilm [29]. Nowadays, caries is seen as a result of a dysbiosis induced by environmental factors [40]. Microorganisms metabolize sugar supplied by food and create an acidic environment where aciduric bacteria (including mutans streptococci) become dominant, which leads to the demineralization of enamel and dentine [41]. Then, the bacterial-induced acidification also activates host-derived proteases, which contribute to the degradation of the organic matrix of dentine and of the root surface [42]. Using the Shannon index as a measure of diversity and evenness in the microbiome, a higher diversity is found in healthy individuals than in those with caries disease in general, but streptococci were enriched in the cohort with caries [34]. In caries-positive adolescence, the presence of S. mutans is associated with a lower diversity, while if S. mutans is not present many other saccharolytic species occur [43]. Functional profiles detect more sugar uptake systems and more systems associated with antimicrobial resistance in the microbiomes of caries-positive than in healthy individuals [34]. When comparing samples with active dentine caries with enamel caries and healthy samples, diversity values were higher for dentine caries, although in a few samples only a very low diversity was found [44].
The pathogenesis of periodontitis is thought to be an inflammatory response to a dysbiotic microbiota in the subgingival biofilm [45]. P. gingivalis has been postulated as being a keystone pathogen, it drives the development of the microbial shifts, and causes dysbiosis and inflammation by modulating the host response [46]. A systematic review summarizing the published data of complex analyses of the periodontal microbiota found high evidence for P. gingivalis, T. forsythia, T. denticola, F. alocis, TM7 spp., and Desulfobulbus spp. being more abundant in periodontal disease than in periodontal health [47]. Shannon index results did not reveal clear differences between periodontal health and disease [48]. Functional analysis found a higher abundance of genes involved with bacterial motility, lipopolysaccharide biosynthesis, and peptidase in the subgingival metagenome of subjects with periodontitis [48].
In the oral cavity, biofilms are formed not only on natural teeth but also on restorative materials, prosthetic constructions, and dental implants. The biofilm formation is very similar, although in detail distinct differences occur. For example, a biofilm at an implant is less diverse than one at a tooth, and abundances of the species differ between both biofilms [49].
Candida Biofilms
Microorganisms in the biofilms mentioned above are mostly bacteria. However, Candida sp. may also be an important member of the oral biofilm. Candida biofilms both affect soft and hard tissue, they are complex biofilms, and Candida sp. interact with bacteria and host factors [50]. Candida might be an active member of a cariogenic biofilm and acts synergistically with S. mutans in biofilm matrix formation. The low pH generated by oral streptococci enables Candida albicans to grow in yeast form, and C. albicans lowers oxygen tension, which promotes streptococcal growth [50]. C. albicans is the most frequent yeast isolated in denture stomatitis, the hyphae form is more present and seems to be the more invasive form, while proteolytic and lipolytic enzymes induce inflammation at the palatum [50]. Also on dentures and at the palatum, the combination of C. albicans with oral streptococci strengthens the pathogenicity. The interaction of S. oralis with C. albicans increases the biomass of the biofilm and the inflammatory response [51]. Furthermore, it activates µ-calpain, an enzyme targeting E-cadherin that is an important epithelial cell adhesion molecule [51]. The biofilm formation on oral surfaces is summarized