Get 20M+ Full-Text Papers For Less Than $1.50/day. Start a 14-Day Trial for You or Your Team.

Learn More →

Oral fungal-bacterial biofilm models in vitro: a review

Oral fungal-bacterial biofilm models in vitro: a review Abstract Inclusion of fungi as commensals in oral biofilm is an important innovation in oral biology, and this work aimed to review the literature on the available biofilm and related disease in vitro models. Actually, thousands of bacterial and around one hundred of fungal phylotypes can colonize the oral cavity. Taxonomic profiling combined with functional expression analysis has revealed that Candida albicans, Streptococcus mutans and prominent periodontopathogens are not always present or numerically important in candidiasis, caries, or periodontitis lesions. However, C. albicans combined with Streptococcus spp. co-increase their virulence in invasive candidiasis, early childhood caries or peri-implantitis. As Candida species and many other fungi are also members of oral microcosms in healthy individuals, mixed fungal-bacterial biofilm models are increasingly valuable investigative tools, and new fungal-bacterial species combinations need to be investigated. Here we review the key points and current methods for culturing in vitro mixed fungal-bacterial models of oral biofilms. According to ecosystem under study (health, candidiasis, caries, periodontitis), protocol design will select microbial strains, biofilm support (polystyrene plate, cell culture, denture, tooth, implant), pre-treatment support (human or artificial saliva) and culture conditions. Growing mixed fungal-bacterial biofilm models in vitro is a difficult challenge. But reproducible models are needed, because oral hygiene products, food and beverage, medication, licit and illicit drugs can influence oral ecosystems. So, even though most oral fungi and bacteria are not cultivable, in vitro microbiological models should still be instrumental in adapting oral care products, dietary products and care protocols to patients at higher risk of oral diseases. Microbial biofilm models combined with oral epithelial cell cultures could also aid in understanding the inflammatory reaction. biofilms, Candida, co-culture, oral ecosystem, Streptococcus Introduction Inclusion of fungi as commensals in oral biofilm is an important innovation in oral biology, and this work aimed to review the literature on the available biofilm and related disease in vitro models. Actually, metagenomics and proteomics screening have revealed that thousands of bacterial and around one hundred of fungal phylotypes can colonise the oral cavity.1 Microbial communities colonising the mouth grow in a biofilm with a protective extracellular matrix. Specific biofilms colonise soft and hard oral surfaces, but all microbiome members are not biofilm formers. In addition to bacteria and fungi, they also contain archaea, parasites, and viruses infecting oral epithelia or vectored via saliva and respiratory secretions.2–7 In oral health, biofilms modulate the host immune system, which in turn tolerates them.8 Commensal bacteria and fungi and their polymeric and hydrated matrix constitute a first-line defense against pathogenic microorganisms. Any breakdown favors local infections (gingivitis and periodontitis, dental caries and endodontic infections, oral candidiasis, mucositis, peri-implantitis) as well as aspiration pneumonia and blood-borne infections (infectious endocarditis, deep abscesses).9–12 Oral infections are often complicated by oral pain and tooth loss, increasing the risk of anorexia and malnutrition.13 Evidence points to an association between oral infections, the resulting inflammation, and systemic diseases such as diabetes mellitus, rheumatoid arthritis, neurodegenerative diseases (Alzheimer's disease), atherosclerosis, cardiovascular disease, and stroke.12,14,15 Recent research has underlined the role of inter-kingdom microbial synergies or antagonisms in biofilms in health and oral diseases.16 For instance, oral Actinomyces and Lactobacillus spp. can inhibit Candida albicans biofilm formation.16,17 Conversely, C. albicans combined with Streptococcus spp. can co-increase their virulence in invasive candidiasis, early childhood caries or peri-implantitis.18–22 In vivo, the dental plaque typically presents a high microbial density, with approximately 1011 cells/g (wet weight). Approximately 700 oral bacterial species could be isolated and grown in vitro, and any given individual generally harbors 100 or more cultivable bacterial strains in its mouth.23 However, screening of various oral ecosystems has revealed that more than 19,000 noncultivable bacterial phylotypes could also colonise the human oral cavity.24–27 Similarly, more than 100 fungal species have been identified in the oral microbiome, most of them noncultivable.1,3,28 Animal models have been developed using mice, rats, and worms to mimic the human oral microcosm, but human oral ecosystems are different from animal ecosystems.17,19,29–31 In order to get closer to in vivo conditions, some biofilm models are grown with human epithelial cell cultures.18,32 In the model described by Ramage et al., the biofilm actually grows at a 0.5 mm distance from an epithelial monolayer.33 It is very likely that many noncultivable species play a role in health and disease.1,10,23,34 Among cultivable species, taxonomic profiling combined with functional expression analysis has recently revealed that C. albicans, Streptococcus mutans, and prominent periodontopathogens were not always present or numerically important in candidiasis, caries or periodontitis lesions.3,5,10,19,28 Thus, metagenomics and proteomics screening are driving new trends in oral disease prevention, diagnosis and treatment efficacy control based on individual follow-up.8,26,35–37 Future therapies will aim to replace cariogenic and periodontopathogenic microbiomes with the initial healthy microbiome of each subject.6,8,26,35 However, there is a need for reproducible in vitro models of mixed fungal and bacterial biofilms whenever it is necessary to compare different growth conditions or inhibitory substances at different concentrations.38–44 Multispecies culture with both fungal and bacterial strains is difficult because, in contrast to oral fungus Candida, most oral bacteria are slow-growing, nutritionally fastidious, and oxygen-sensitive. Oxygen is very important for Candida growth as well. Anaerobically, it grows yet very slowly, and it is out-competed by many bacterial species.45–47 In mixed biofilm models, strains can have two origins: some biofilms are grown with a defined consortium of bacteria and fungi, while others, called microcosms, are grown from saliva samples from donors. Co-cultures in defined consortiums are reproducible and easier to study because all the strains are known.48,49 In contrast, saliva sampling is closer to real-world conditions in terms of number and respective proportions of microbial species. One important advantage of using saliva is that the species and strains in the inoculum are better adapted to each other. Strain differences are probably important in dictating compatibility between species and kingdoms. However, saliva sampling suffers a lack of reproducibility due to the different saliva donors used.30,50–52 Moreover, only a limited number of bacterial and fungal species can be grown in vitro, and biofilms composition always differs from the initial inoculum.53,54 Choice of microbial strains or inoculum is pivotal. Co-culture conditions are critical too, as aerobic fungi are grown with strict anaerobic bacteria, hyphal formation is often required, and overgrowth of any one species is to be avoided. Here we review the key points and current methods for designing and culturing oral multispecies biofilms in vitro. Search criteria References in English were identified through PubMed and Science Direct searches for articles published since Jan 1, 2005. At first, the search terms used were “bacteria OR bacterial OR Streptococcus OR anaerobic bacteria” AND “fungi OR Candida” AND “biofilms OR models OR in vitro techniques.” Then second, search terms used were “dental caries OR periodontitis OR gingivitis OR abscess OR peri-implantitis OR denture stomatitis OR candidiasis OR mouth diseases OR oral health” AND “biofilms OR models OR in vitro techniques.” Finally, we retained only the studies with an in vitro model of bacterial and fungal cultures, in connection with the oral health or the diseases of the mouth. Stages of oral biofilm development In vivo and in vitro, biofilm development follows five stages: (1) adhesion to hard or soft tissues (adhesins and extracellular polysaccharides); (2) growth (microbial co-adhesion and co-aggregation, matrix formation); (3) maturation characterised by metabolic and genetic microbial exchanges, growth control by quorum-sensing molecules (auto-inducers) and antimicrobial peptides (bacteriocins); (4) tissue invasion/destruction (toxic metabolites and enzymes); and 5) surface detachment (enzymes). 21,35,55–57 Early biofilms Early microbial colonisers specifically adhere to cellular and to salivary receptors, such as mucins, proline-rich protein, statherin, salivary agglutinin (gp-340) and α-amylase.58,59 Organic salivary compounds are adsorbed to epithelial surfaces or hard surfaces (enamel, dentin, calculus, and restorative or prosthetic materials), and then provide static receptors for early colonizers.60–62 The salivary film coating dental enamel is called acquired salivary pellicle.62 The initial adhesion stage of oral bacteria lasts only a few seconds and is reversible. There are both nonspecific surface forces and recognition between microbial adhesins and their receptors. Hydrodynamic forces create either repulsive or attractive nonspecific connections via low-energy interactions (electrostatic, steric, hydrophobic, Van de Waals). Next step, the irreversible adhesion process is slower. Its duration depends on the microbial strains, its population density and the duration of its exponential growth phase.63,64 The taxonomic bacterial profile of early dental plaque, based on genomic data, has been recently detailed.23 A critical step is the choice of biofilm support (polystyrene plate, cell culture, acrylic resin for denture, tooth hydroxyapatite crystals, complete bovine or human teeth, titanium implant…) and pretreatment support (human or artificial saliva), adapted to early colonizers. Biofilm growth and maturation can take hours or days depending on the microbial species and environmental conditions involved, and, importantly, the frequency of nutrient replenishment. Bacteria and fungi multiply, colonise the support, and form aggregates (or microcolonies) that become confluent. The production of extracellular polymers varies according to microbial communities, local environment, and the biofilm maturity.65,66 Mature biofilms Mature biofilms are aggregates of microorganisms growing within an extracellular matrix. In vivo, Candida and streptococci form corn-cob-like structures.24 The matrix contains microbial metabolites, dead microbial and host cells (desquamated epithelial cells), other host components (mainly fibronectin, laminin, collagen and salivary constituents), food nutrients (sugars), and possibly also drugs. The matrix is well hydrated and crossed by channels conveying oxygen, nutrients and metabolites.31,67 Maturation of mixed biofilms depends on oxygen availability and metabolic interactions. End-chain products may be nutrients of different fungal and bacterial species, which may be either partners or competitors.68Streptococcus oralis combined with C. albicans synergistically increases both biofilm formation and virulence factor expression.18 In early-childhood caries, the presence of C. albicans and sucrose (but not glucose) synergistically increases S. mutans virulence, resulting in rapid onset of extensive caries lesions. C. albicans can tolerate acids in dental caries lesions.19,20,69 However, in a recent report, Willems and coworkers showed that while growth of S. muttons is increased by presence of Candida, and lactate production is also increased, the environmental pH increases to above the critical pH and Ca2+ release from hydroxyapatite disks is inhibited by the presence of Candida.70 Microorganisms are able to perceive various environmental parameters, either abiotic, such as physical-chemical signals (pH, osmolality, temperature), or biotic, such as signaling proteins.19,26,35,71–73 When signalling molecules reach a sufficient concentration, they can communicate and coordinate the formation of biofilm or the synthesis of virulence factors.71–73In vivo, quorum sensing coordinates the evolution from colonization stage to acute infection stage. In vitro, farnesol promotes the formation of Staphylococcus aureus biofilm at low levels (0.5–5 nM) and inhibits S. aureus growth at higher concentration (180 μM).57 In addition, some microbial species can produce antimicrobial peptides, although their direct microbe-killing effect is prevented in physiological conditions where they probably act as immunomodulators.73 In vitro, some studies aim to inhibit biofilm formation or to assay pre-formed biofilms, leading to models of early21,31,64 and mature biofilms,74 with corresponding culture duration, sugar pulses in caries models,75 sequential addition of strains, and progressive anaerobic conditions in peri-implantitis models.49 Late stages The invasion and destruction of soft tissues is mediated by microbial diffusible enzymes, such as lipases, proteases, nucleases and ureases. Enzymatic dissolution of mucosal barriers can also take place by activation of host enzymes triggered by C. albicans, for example, calpain.76 The destruction and invasion of hard dental tissues that results in tooth cavities starts with localised acid decalcification of the hydroxyapatite crystals constituting enamel and dentin, followed by an enzymatic lysis of organic structures.20 In vivo, the healthy adult's biofilm is bathed by a saliva flow of approximately 0.35 ml/min, which provides nutrients, moistens the mucus membranes, eliminates part of the bacteria and buffers the pH resulting in remineralization of the tooth surface.32,50 The biofilm's thickness is mechanically controlled by salivary flow, tongue and jaw movements, and by chewing solid food. The turnover of epithelial cells and the immune system (in saliva and epithelia) protect soft mucosal surfaces. In contrast, there is no cell turnover on the hard surfaces of teeth, calculus, dentures, and dental biomaterials.18,25 The late stage of biofilm development is the detachment of microbial cells or aggregates, which then colonise saliva and other supports. Detachment depends on support, microbial community, nutrient availability, hydrodynamics and physical-chemical conditions of the environment. Microbial aggregate detachment is facilitated by the lysis of extracellular polymers, such as by the production of dextranases by S. mutans and glucanases by C. albicans.65,77In vitro, sophisticated flux systems enable continuous renewal of the culture medium.18,30,51,78 Choice of microbial strains Consortium models Current consortium models contain cultivable species representative of ecosystems colonising oral mucosa, teeth, dentures, and peri-implantitis pockets. They can combine fungal and bacterial reference strains, wild-type strains or mutant strains. The number of species ranges from two31,69,79,80 to 11 strains.74 To our knowledge, neither archaea, viruses nor parasites have been used in multispecies bacterial biofilm models. Examples of consortium models combining fungal and bacterial strains are listed in Tables 1 and 2. Table 1. Oral candidiasis models. References Authors Model Microbial strains Supports Culture apparatus Main objective Assessment methods 18 Diaz 2012 Oral candidiasis Candida albicans Streptococcus gordonii Streptococcus oralis Streptococcus sanguinis Reconstructed oral and pharyngeal epithelium Flux system To demonstrate a synergistic interaction between commensal oral streptococci and Candida albicans Histology (FISH) CLSM RT-qPCR 21 Park 2014 Oral candidiasis Candida albicans Escherichia coli Proteus vulgaris Pseudomonas aeruginosa Staphylococcus aureus Streptococcus pyogenes Streptococcus salivarius Polystyrene plate 96-well plate To examine the influence of bacterial presence on biofilm formation of Candida albicans XTT cell viability assay SEM RT-qPCR 79 Schlecht 2015 Oral candidiasis Candida albicans Staphylococcus aureus Plastic Permanox slides 6-well plate To analyse how Staphylococcus aureus infection is mediated by Candida albicans hyphal invasion of mucosal tissue Phase-contrast microscopy Fluorescence microscopy CLSM 64 Matsubara 2016 Oral candidiasis Candida albicans Lactobacillus acidophilus Lactobacillus casei Lactobacillus rhamnosus Polystyrene plate 96-well plate To evaluate the inhibitory effects of the probiotic Lactobacillus species on different phases of Candida albicans biofilm development cfu count XTT cell viability assay SEM CLSM 32 Bertolini 2015 Xerostomia Candida albicans (and mutant strains) Streptococcus oralis Reconstructed oral epithelium Tissue-culture plate (not detailed) To analyse the structural and virulence characteristics of Candida-streptococcal mixed-biofilm models on reconstructed oral epithelium, in different conditions of moisture and nutrient availability CLSM Histology (FISH) 50 de Moraes 2012 Denture stomatitis Saliva microcosm Acrylic resin, soft denture liner 24-well plate To assess the antimicrobial activity of a triazine derivative added into denture liners cfu count 31 Barbosa 2016 Denture stomatitis Candida albicans Streptococcus mutans Polystyrene plate Acrylic resin 24-well plate To study the effects of Streptococcus mutans on biofilm formation and morphology of Candida albicans pH measure cfu count Bright-field microscopy: crystal violet and hyphae quantification SEM 74 Sherry 2016 Denture stomatitis Candida albicans Aggregatibacter actinomycetemcomitans Actinomyces naeslundii Fusobacterium nucleatum Fusobacterium nucleatum spp. vincentii Porphyromonas gingivalis Prevotella intermedia Streptococcus intermedius Streptococcus oralis Streptococcus mitis Veillonella dispar Acrylic resin 24-well plate To assess the activity of three protocols for cleaning removable dentures cfu count SEM CLSM Live-Dead PCR RT-qPCR 103 Cavalcanti 2015 Denture stomatitis Candida albicans Streptococcus mutans Streptococcus sanguinis Actinomyces viscosus Actinomyces odontolyticus Acrylic coupons Reconstructed human oral epithelium 24-well plate To examine the effect of a bacterial component on the virulence and pathogenicity of Candida biofilms and to assess epithelial cell responses to the different biofilm compositions Histology (FISH) CLSM RT-qPCR 78 Yassin 2016 Denture stomatitis Candida albicans Lactobacillus casei Streptococcus mutans Acrylic resin Flux system To assess the antimicrobial properties of a fluoride-releasing denture resin pH measure SEM CLSM RT-qPCR References Authors Model Microbial strains Supports Culture apparatus Main objective Assessment methods 18 Diaz 2012 Oral candidiasis Candida albicans Streptococcus gordonii Streptococcus oralis Streptococcus sanguinis Reconstructed oral and pharyngeal epithelium Flux system To demonstrate a synergistic interaction between commensal oral streptococci and Candida albicans Histology (FISH) CLSM RT-qPCR 21 Park 2014 Oral candidiasis Candida albicans Escherichia coli Proteus vulgaris Pseudomonas aeruginosa Staphylococcus aureus Streptococcus pyogenes Streptococcus salivarius Polystyrene plate 96-well plate To examine the influence of bacterial presence on biofilm formation of Candida albicans XTT cell viability assay SEM RT-qPCR 79 Schlecht 2015 Oral candidiasis Candida albicans Staphylococcus aureus Plastic Permanox slides 6-well plate To analyse how Staphylococcus aureus infection is mediated by Candida albicans hyphal invasion of mucosal tissue Phase-contrast microscopy Fluorescence microscopy CLSM 64 Matsubara 2016 Oral candidiasis Candida albicans Lactobacillus acidophilus Lactobacillus casei Lactobacillus rhamnosus Polystyrene plate 96-well plate To evaluate the inhibitory effects of the probiotic Lactobacillus species on different phases of Candida albicans biofilm development cfu count XTT cell viability assay SEM CLSM 32 Bertolini 2015 Xerostomia Candida albicans (and mutant strains) Streptococcus oralis Reconstructed oral epithelium Tissue-culture plate (not detailed) To analyse the structural and virulence characteristics of Candida-streptococcal mixed-biofilm models on reconstructed oral epithelium, in different conditions of moisture and nutrient availability CLSM Histology (FISH) 50 de Moraes 2012 Denture stomatitis Saliva microcosm Acrylic resin, soft denture liner 24-well plate To assess the antimicrobial activity of a triazine derivative added into denture liners cfu count 31 Barbosa 2016 Denture stomatitis Candida albicans Streptococcus mutans Polystyrene plate Acrylic resin 24-well plate To study the effects of Streptococcus mutans on biofilm formation and morphology of Candida albicans pH measure cfu count Bright-field microscopy: crystal violet and hyphae quantification SEM 74 Sherry 2016 Denture stomatitis Candida albicans Aggregatibacter actinomycetemcomitans Actinomyces naeslundii Fusobacterium nucleatum Fusobacterium nucleatum spp. vincentii Porphyromonas gingivalis Prevotella intermedia Streptococcus intermedius Streptococcus oralis Streptococcus mitis Veillonella dispar Acrylic resin 24-well plate To assess the activity of three protocols for cleaning removable dentures cfu count SEM CLSM Live-Dead PCR RT-qPCR 103 Cavalcanti 2015 Denture stomatitis Candida albicans Streptococcus mutans Streptococcus sanguinis Actinomyces viscosus Actinomyces odontolyticus Acrylic coupons Reconstructed human oral epithelium 24-well plate To examine the effect of a bacterial component on the virulence and pathogenicity of Candida biofilms and to assess epithelial cell responses to the different biofilm compositions Histology (FISH) CLSM RT-qPCR 78 Yassin 2016 Denture stomatitis Candida albicans Lactobacillus casei Streptococcus mutans Acrylic resin Flux system To assess the antimicrobial properties of a fluoride-releasing denture resin pH measure SEM CLSM RT-qPCR cfu, colony-forming unit; CLSM, confocal laser scanning electron microscopy; FISH, fluorescence in situ hybridisation; (RT)-Qpcr, quantitative (reverse transcription) polymerase chain reaction; SEM, scanning electron microscopy; XTT, (2,3-Bis-(2-Methoxy-4-Nitro-5-Sulfophenyl)-2H-Tetrazolium-5-Carboxanilide). View Large Table 1. Oral candidiasis models. References Authors Model Microbial strains Supports Culture apparatus Main objective Assessment methods 18 Diaz 2012 Oral candidiasis Candida albicans Streptococcus gordonii Streptococcus oralis Streptococcus sanguinis Reconstructed oral and pharyngeal epithelium Flux system To demonstrate a synergistic interaction between commensal oral streptococci and Candida albicans Histology (FISH) CLSM RT-qPCR 21 Park 2014 Oral candidiasis Candida albicans Escherichia coli Proteus vulgaris Pseudomonas aeruginosa Staphylococcus aureus Streptococcus pyogenes Streptococcus salivarius Polystyrene plate 96-well plate To examine the influence of bacterial presence on biofilm formation of Candida albicans XTT cell viability assay SEM RT-qPCR 79 Schlecht 2015 Oral candidiasis Candida albicans Staphylococcus aureus Plastic Permanox slides 6-well plate To analyse how Staphylococcus aureus infection is mediated by Candida albicans hyphal invasion of mucosal tissue Phase-contrast microscopy Fluorescence microscopy CLSM 64 Matsubara 2016 Oral candidiasis Candida albicans Lactobacillus acidophilus Lactobacillus casei Lactobacillus rhamnosus Polystyrene plate 96-well plate To evaluate the inhibitory effects of the probiotic Lactobacillus species on different phases of Candida albicans biofilm development cfu count XTT cell viability assay SEM CLSM 32 Bertolini 2015 Xerostomia Candida albicans (and mutant strains) Streptococcus oralis Reconstructed oral epithelium Tissue-culture plate (not detailed) To analyse the structural and virulence characteristics of Candida-streptococcal mixed-biofilm models on reconstructed oral epithelium, in different conditions of moisture and nutrient availability CLSM Histology (FISH) 50 de Moraes 2012 Denture stomatitis Saliva microcosm Acrylic resin, soft denture liner 24-well plate To assess the antimicrobial activity of a triazine derivative added into denture liners cfu count 31 Barbosa 2016 Denture stomatitis Candida albicans Streptococcus mutans Polystyrene plate Acrylic resin 24-well plate To study the effects of Streptococcus mutans on biofilm formation and morphology of Candida albicans pH measure cfu count Bright-field microscopy: crystal violet and hyphae quantification SEM 74 Sherry 2016 Denture stomatitis Candida albicans Aggregatibacter actinomycetemcomitans Actinomyces naeslundii Fusobacterium nucleatum Fusobacterium nucleatum spp. vincentii Porphyromonas gingivalis Prevotella intermedia Streptococcus intermedius Streptococcus oralis Streptococcus mitis Veillonella dispar Acrylic resin 24-well plate To assess the activity of three protocols for cleaning removable dentures cfu count SEM CLSM Live-Dead PCR RT-qPCR 103 Cavalcanti 2015 Denture stomatitis Candida albicans Streptococcus mutans Streptococcus sanguinis Actinomyces viscosus Actinomyces odontolyticus Acrylic coupons Reconstructed human oral epithelium 24-well plate To examine the effect of a bacterial component on the virulence and pathogenicity of Candida biofilms and to assess epithelial cell responses to the different biofilm compositions Histology (FISH) CLSM RT-qPCR 78 Yassin 2016 Denture stomatitis Candida albicans Lactobacillus casei Streptococcus mutans Acrylic resin Flux system To assess the antimicrobial properties of a fluoride-releasing denture resin pH measure SEM CLSM RT-qPCR References Authors Model Microbial strains Supports Culture apparatus Main objective Assessment methods 18 Diaz 2012 Oral candidiasis Candida albicans Streptococcus gordonii Streptococcus oralis Streptococcus sanguinis Reconstructed oral and pharyngeal epithelium Flux system To demonstrate a synergistic interaction between commensal oral streptococci and Candida albicans Histology (FISH) CLSM RT-qPCR 21 Park 2014 Oral candidiasis Candida albicans Escherichia coli Proteus vulgaris Pseudomonas aeruginosa Staphylococcus aureus Streptococcus pyogenes Streptococcus salivarius Polystyrene plate 96-well plate To examine the influence of bacterial presence on biofilm formation of Candida albicans XTT cell viability assay SEM RT-qPCR 79 Schlecht 2015 Oral candidiasis Candida albicans Staphylococcus aureus Plastic Permanox slides 6-well plate To analyse how Staphylococcus aureus infection is mediated by Candida albicans hyphal invasion of mucosal tissue Phase-contrast microscopy Fluorescence microscopy CLSM 64 Matsubara 2016 Oral candidiasis Candida albicans Lactobacillus acidophilus Lactobacillus casei Lactobacillus rhamnosus Polystyrene plate 96-well plate To evaluate the inhibitory effects of the probiotic Lactobacillus species on different phases of Candida albicans biofilm development cfu count XTT cell viability assay SEM CLSM 32 Bertolini 2015 Xerostomia Candida albicans (and mutant strains) Streptococcus oralis Reconstructed oral epithelium Tissue-culture plate (not detailed) To analyse the structural and virulence characteristics of Candida-streptococcal mixed-biofilm models on reconstructed oral epithelium, in different conditions of moisture and nutrient availability CLSM Histology (FISH) 50 de Moraes 2012 Denture stomatitis Saliva microcosm Acrylic resin, soft denture liner 24-well plate To assess the antimicrobial activity of a triazine derivative added into denture liners cfu count 31 Barbosa 2016 Denture stomatitis Candida albicans Streptococcus mutans Polystyrene plate Acrylic resin 24-well plate To study the effects of Streptococcus mutans on biofilm formation and morphology of Candida albicans pH measure cfu count Bright-field microscopy: crystal violet and hyphae quantification SEM 74 Sherry 2016 Denture stomatitis Candida albicans Aggregatibacter actinomycetemcomitans Actinomyces naeslundii Fusobacterium nucleatum Fusobacterium nucleatum spp. vincentii Porphyromonas gingivalis Prevotella intermedia Streptococcus intermedius Streptococcus oralis Streptococcus mitis Veillonella dispar Acrylic resin 24-well plate To assess the activity of three protocols for cleaning removable dentures cfu count SEM CLSM Live-Dead PCR RT-qPCR 103 Cavalcanti 2015 Denture stomatitis Candida albicans Streptococcus mutans Streptococcus sanguinis Actinomyces viscosus Actinomyces odontolyticus Acrylic coupons Reconstructed human oral epithelium 24-well plate To examine the effect of a bacterial component on the virulence and pathogenicity of Candida biofilms and to assess epithelial cell responses to the different biofilm compositions Histology (FISH) CLSM RT-qPCR 78 Yassin 2016 Denture stomatitis Candida albicans Lactobacillus casei Streptococcus mutans Acrylic resin Flux system To assess the antimicrobial properties of a fluoride-releasing denture resin pH measure SEM CLSM RT-qPCR cfu, colony-forming unit; CLSM, confocal laser scanning electron microscopy; FISH, fluorescence in situ hybridisation; (RT)-Qpcr, quantitative (reverse transcription) polymerase chain reaction; SEM, scanning electron microscopy; XTT, (2,3-Bis-(2-Methoxy-4-Nitro-5-Sulfophenyl)-2H-Tetrazolium-5-Carboxanilide). View Large Table 2. Caries and periodontitis models. Referen-ces Authors Model Microbial strains Supports Culture apparatus Main objective Assessment methods 75 Reese 2007 Dental plaque Candida albicans Actinomyces naeslundii Fusobacterium nucleatum Streptococcus oralis Streptococcus sobrinus Veillonella dispar Bovine enamel 24-well plate To observe the biofilm matrix in the dental plaque, before and after glucose-sucrose feeding TEM 86 Pereira-Cenci 2008 Dental plaque Candida albicans Candida glabrata Streptococcus mutans Hydroxyapatite Acrylic resin Soft denture liner 24-well plate To investigate how oral bacteria modulate the development and characteristics of Candida biofilms cfu count SEM CLSM 94 Sampaio 2009 Dental plaque Candida albicans Lactobacillus casei Streptococcus mutans Streptococcus oralis Streptococcus salivarius Bovine enamel Propylene tubes To assess the antimicrobial activity of a plant extract cfu count 80 Montelongo-Jauregui 2016 Dental plaque Candida albicans Streptococcus gordonii Polystyrene plate 96-well plate To assess the activity of antifungal and antibacterial drugs Bright-field microscopy: crystal violet Fluorescent plate reader: Presto Blue™ SEM CLSM 20 Falsetta 2014 Dental caries Candida albicans Streptococcus mutans (and mutant strains) Hydroxyapatite 24-well plate To study synergy (virulence) of Streptococcus mutans and Candida albicans in dental caries biofilm pH measure cfu count CLSM RT-qPCR 51 Koopman 2015 Dental caries Saliva microcosm Glass coverslips Multistation artificial mouth: drip system To assay the pH-raising effect of arginine pH measure RT-qPCR 101 Mistry 2015 Endodontic infection Candida albicans Enterococcus faecalis Staphylococcus aureus Streptococcus mutans Human dentin Eppendorf vials To assess the antibacterial activity of two plant extracts Cfu SEM 49 Thurnheer 2016 Periodontitis Candida albicans Actinomyces oris Campylobacter rectus Fusobacterium nucleatum Porphyromonas gingivalis Prevotella intermedia Streptococcus anginosus Streptococcus oralis Streptococcus mutans Tannerella forsythia Treponema denticola Veillonella dispar Hydroxyapatite 24-well plate To investigate changes in biofilm composition and structure during the shift from a ‘supragingival’ aerobic profile to a ‘subgingival’ anaerobic profile CLSM RT-qPCR 62 Cavalcanti 2014 Peri-implantitis Candida albicans Actinomyces naeslundii Fusobacterium nucleatum Streptococcus mutans Streptococcus oralis Veillonella dispar Titanium 24-well plate To assess surface properties of titanium disks nitrided by cold plasma Cfu SEM CLSM RT-qPCR 30 Sousa 2016 Peri-implantitis Saliva microcosm Titanium Flux system in vitro and secondly rabbit in vivo To assess three disinfection protocols with an experimental model for contamination of titanium surfaces cfu count Combined in vitro/in vivo animal model 69 Junka 2015 Bone destruction Candida albicans Streptococcus mutans Hydroxyapatite 24-well plate To assess bone hydroxyapatite destruction pH measure cfu count SEM CLSM Referen-ces Authors Model Microbial strains Supports Culture apparatus Main objective Assessment methods 75 Reese 2007 Dental plaque Candida albicans Actinomyces naeslundii Fusobacterium nucleatum Streptococcus oralis Streptococcus sobrinus Veillonella dispar Bovine enamel 24-well plate To observe the biofilm matrix in the dental plaque, before and after glucose-sucrose feeding TEM 86 Pereira-Cenci 2008 Dental plaque Candida albicans Candida glabrata Streptococcus mutans Hydroxyapatite Acrylic resin Soft denture liner 24-well plate To investigate how oral bacteria modulate the development and characteristics of Candida biofilms cfu count SEM CLSM 94 Sampaio 2009 Dental plaque Candida albicans Lactobacillus casei Streptococcus mutans Streptococcus oralis Streptococcus salivarius Bovine enamel Propylene tubes To assess the antimicrobial activity of a plant extract cfu count 80 Montelongo-Jauregui 2016 Dental plaque Candida albicans Streptococcus gordonii Polystyrene plate 96-well plate To assess the activity of antifungal and antibacterial drugs Bright-field microscopy: crystal violet Fluorescent plate reader: Presto Blue™ SEM CLSM 20 Falsetta 2014 Dental caries Candida albicans Streptococcus mutans (and mutant strains) Hydroxyapatite 24-well plate To study synergy (virulence) of Streptococcus mutans and Candida albicans in dental caries biofilm pH measure cfu count CLSM RT-qPCR 51 Koopman 2015 Dental caries Saliva microcosm Glass coverslips Multistation artificial mouth: drip system To assay the pH-raising effect of arginine pH measure RT-qPCR 101 Mistry 2015 Endodontic infection Candida albicans Enterococcus faecalis Staphylococcus aureus Streptococcus mutans Human dentin Eppendorf vials To assess the antibacterial activity of two plant extracts Cfu SEM 49 Thurnheer 2016 Periodontitis Candida albicans Actinomyces oris Campylobacter rectus Fusobacterium nucleatum Porphyromonas gingivalis Prevotella intermedia Streptococcus anginosus Streptococcus oralis Streptococcus mutans Tannerella forsythia Treponema denticola Veillonella dispar Hydroxyapatite 24-well plate To investigate changes in biofilm composition and structure during the shift from a ‘supragingival’ aerobic profile to a ‘subgingival’ anaerobic profile CLSM RT-qPCR 62 Cavalcanti 2014 Peri-implantitis Candida albicans Actinomyces naeslundii Fusobacterium nucleatum Streptococcus mutans Streptococcus oralis Veillonella dispar Titanium 24-well plate To assess surface properties of titanium disks nitrided by cold plasma Cfu SEM CLSM RT-qPCR 30 Sousa 2016 Peri-implantitis Saliva microcosm Titanium Flux system in vitro and secondly rabbit in vivo To assess three disinfection protocols with an experimental model for contamination of titanium surfaces cfu count Combined in vitro/in vivo animal model 69 Junka 2015 Bone destruction Candida albicans Streptococcus mutans Hydroxyapatite 24-well plate To assess bone hydroxyapatite destruction pH measure cfu count SEM CLSM cfu, colony forming unit; CLSM, confocal laser scanning electron microscopy; HIV, human immunodeficiency virus; RT-qPCR, quantitative reverse transcription polymerase chain reaction; SEM: scanning electron microscopy; TEM: transmission electron microscopy. View Large Table 2. Caries and periodontitis models. Referen-ces Authors Model Microbial strains Supports Culture apparatus Main objective Assessment methods 75 Reese 2007 Dental plaque Candida albicans Actinomyces naeslundii Fusobacterium nucleatum Streptococcus oralis Streptococcus sobrinus Veillonella dispar Bovine enamel 24-well plate To observe the biofilm matrix in the dental plaque, before and after glucose-sucrose feeding TEM 86 Pereira-Cenci 2008 Dental plaque Candida albicans Candida glabrata Streptococcus mutans Hydroxyapatite Acrylic resin Soft denture liner 24-well plate To investigate how oral bacteria modulate the development and characteristics of Candida biofilms cfu count SEM CLSM 94 Sampaio 2009 Dental plaque Candida albicans Lactobacillus casei Streptococcus mutans Streptococcus oralis Streptococcus salivarius Bovine enamel Propylene tubes To assess the antimicrobial activity of a plant extract cfu count 80 Montelongo-Jauregui 2016 Dental plaque Candida albicans Streptococcus gordonii Polystyrene plate 96-well plate To assess the activity of antifungal and antibacterial drugs Bright-field microscopy: crystal violet Fluorescent plate reader: Presto Blue™ SEM CLSM 20 Falsetta 2014 Dental caries Candida albicans Streptococcus mutans (and mutant strains) Hydroxyapatite 24-well plate To study synergy (virulence) of Streptococcus mutans and Candida albicans in dental caries biofilm pH measure cfu count CLSM RT-qPCR 51 Koopman 2015 Dental caries Saliva microcosm Glass coverslips Multistation artificial mouth: drip system To assay the pH-raising effect of arginine pH measure RT-qPCR 101 Mistry 2015 Endodontic infection Candida albicans Enterococcus faecalis Staphylococcus aureus Streptococcus mutans Human dentin Eppendorf vials To assess the antibacterial activity of two plant extracts Cfu SEM 49 Thurnheer 2016 Periodontitis Candida albicans Actinomyces oris Campylobacter rectus Fusobacterium nucleatum Porphyromonas gingivalis Prevotella intermedia Streptococcus anginosus Streptococcus oralis Streptococcus mutans Tannerella forsythia Treponema denticola Veillonella dispar Hydroxyapatite 24-well plate To investigate changes in biofilm composition and structure during the shift from a ‘supragingival’ aerobic profile to a ‘subgingival’ anaerobic profile CLSM RT-qPCR 62 Cavalcanti 2014 Peri-implantitis Candida albicans Actinomyces naeslundii Fusobacterium nucleatum Streptococcus mutans Streptococcus oralis Veillonella dispar Titanium 24-well plate To assess surface properties of titanium disks nitrided by cold plasma Cfu SEM CLSM RT-qPCR 30 Sousa 2016 Peri-implantitis Saliva microcosm Titanium Flux system in vitro and secondly rabbit in vivo To assess three disinfection protocols with an experimental model for contamination of titanium surfaces cfu count Combined in vitro/in vivo animal model 69 Junka 2015 Bone destruction Candida albicans Streptococcus mutans Hydroxyapatite 24-well plate To assess bone hydroxyapatite destruction pH measure cfu count SEM CLSM Referen-ces Authors Model Microbial strains Supports Culture apparatus Main objective Assessment methods 75 Reese 2007 Dental plaque Candida albicans Actinomyces naeslundii Fusobacterium nucleatum Streptococcus oralis Streptococcus sobrinus Veillonella dispar Bovine enamel 24-well plate To observe the biofilm matrix in the dental plaque, before and after glucose-sucrose feeding TEM 86 Pereira-Cenci 2008 Dental plaque Candida albicans Candida glabrata Streptococcus mutans Hydroxyapatite Acrylic resin Soft denture liner 24-well plate To investigate how oral bacteria modulate the development and characteristics of Candida biofilms cfu count SEM CLSM 94 Sampaio 2009 Dental plaque Candida albicans Lactobacillus casei Streptococcus mutans Streptococcus oralis Streptococcus salivarius Bovine enamel Propylene tubes To assess the antimicrobial activity of a plant extract cfu count 80 Montelongo-Jauregui 2016 Dental plaque Candida albicans Streptococcus gordonii Polystyrene plate 96-well plate To assess the activity of antifungal and antibacterial drugs Bright-field microscopy: crystal violet Fluorescent plate reader: Presto Blue™ SEM CLSM 20 Falsetta 2014 Dental caries Candida albicans Streptococcus mutans (and mutant strains) Hydroxyapatite 24-well plate To study synergy (virulence) of Streptococcus mutans and Candida albicans in dental caries biofilm pH measure cfu count CLSM RT-qPCR 51 Koopman 2015 Dental caries Saliva microcosm Glass coverslips Multistation artificial mouth: drip system To assay the pH-raising effect of arginine pH measure RT-qPCR 101 Mistry 2015 Endodontic infection Candida albicans Enterococcus faecalis Staphylococcus aureus Streptococcus mutans Human dentin Eppendorf vials To assess the antibacterial activity of two plant extracts Cfu SEM 49 Thurnheer 2016 Periodontitis Candida albicans Actinomyces oris Campylobacter rectus Fusobacterium nucleatum Porphyromonas gingivalis Prevotella intermedia Streptococcus anginosus Streptococcus oralis Streptococcus mutans Tannerella forsythia Treponema denticola Veillonella dispar Hydroxyapatite 24-well plate To investigate changes in biofilm composition and structure during the shift from a ‘supragingival’ aerobic profile to a ‘subgingival’ anaerobic profile CLSM RT-qPCR 62 Cavalcanti 2014 Peri-implantitis Candida albicans Actinomyces naeslundii Fusobacterium nucleatum Streptococcus mutans Streptococcus oralis Veillonella dispar Titanium 24-well plate To assess surface properties of titanium disks nitrided by cold plasma Cfu SEM CLSM RT-qPCR 30 Sousa 2016 Peri-implantitis Saliva microcosm Titanium Flux system in vitro and secondly rabbit in vivo To assess three disinfection protocols with an experimental model for contamination of titanium surfaces cfu count Combined in vitro/in vivo animal model 69 Junka 2015 Bone destruction Candida albicans Streptococcus mutans Hydroxyapatite 24-well plate To assess bone hydroxyapatite destruction pH measure cfu count SEM CLSM cfu, colony forming unit; CLSM, confocal laser scanning electron microscopy; HIV, human immunodeficiency virus; RT-qPCR, quantitative reverse transcription polymerase chain reaction; SEM: scanning electron microscopy; TEM: transmission electron microscopy. View Large Choice of bacterial strains The choice of bacterial strains commonly selected for caries and periodontitis models warrants update. Peterson et al. (2014) used microarrays and high-throughput sequencing to investigate biofilm physiology and microbial interactions in dental caries27 and demonstrated that taxonomic profile was not predictive of dental caries. In particular, S. mutans was not always prominent or present in caries ecosystems. Conversely, functional analysis based on RNA expression was more informative. Different bacterial species were shown to display similarities in gene expression patterns, and functional redundancy was common. Extensive listings of cultivable and noncultivable oral bacteria have recently been published. New prominent species have been identified in samples of healthy saliva,51,81,82 dental plaque,23,27,81,82 healthy gingiva,27 and periodontitis.27,81,83 The salivary flora is more similar to the tongue flora than to dental plaque.27 It is impossible to achieve the dynamics of the oral cavity using predefined combinations of laboratory or wild-type strains, but the choice of strains must also take into account co-occurrence and co-exclusion patterns in oral communities.10 For a single species, biofilm formation can be also strain-dependant.84 Even in simplified in vitro models, the source of inoculum may influence the model used. For instance, different results are anticipated when wild-type strains obtained from healthy young adults, healthy elderly people or specifically diseased individuals will be used instead of laboratory strains. Choice of fungal strains Knowledge on oral fungi is more limited, but several prominent genera have been identified in oral saliva (Table 3). To date, C. albicans is the most common species used in oral consortium studies as it is the most amenable to isolation, identification, and culture.6 Bertolini et al. (2015) also used three strains of mutant C. albicans.32,85 Pereira-Cenci et al. (2008) described a co-culture model with C. albicans and Candida glabrate,86 while Chew et al. (2015) recently developed a model of vulvovaginal candidiasis containing C. glabrata combined with two probiotic lactobacilli, Lactobacillus router and Lactobacillus rhamnosus.22 Furthermore, Pichia species have been identified in oral rinse samples of patients infected with human immunodeficiency virus (HIV), and this species could be antagonist to Candida, Aspergillus and Fusarium species.3 Table 3. Consensus members in the oral mycobiome. References Authors Oral ecosystem Main genera Prominent Candida species 1 Ghannoum 2010 20 oral rinse samples Frequency in the 20 subjects: Candida (75%) Cladosporium (65%) Aureobasidium (50%) Saccharomycetales (50%) Non-cultivable categories (36%) Aspergilllus (35%) Fusarium (30%) Cryptococcus (20%). Frequency of Candida species in the 20 subjects: Candida albicans (40%) Candida paraphilics (15%) Candida tropicalis (15%) Candida khmerensis (5%) Candida metapsilosis (5%) 28 Dupuy 2014 6 saliva samples Frequency of genera occurring in at least 50% of the six subjects: Malassezia (38%) Epicoccum (33%) Candida/Pichia (10%) Fusarium/Gibberella (4%) Cladosporium/Davidiella (3%) Alternaria/Lewia (2%) Aspergilllus/Emericella/Eurotium (2%) Cryptococcus/Filobasidiella (1%) Candida albicans Candida utilis/Pichia jadinii 3 Mukherjee 2014 24 oral rinse samples Frequency in the 12 subjects of each group: 12 HIV-noninfected patients: Candida (58%) Pichia (33%) Fusarium (33%) Cladosporium Penicillium 12 HIV-infected patients: Candida (92%) Epicoccum (33%) Alternaria (25%) Penicillium Trichosporon Main species identified in the Candida genus: Candida albicans (58%) Candida dublinensis (17%) Candida intermedia Candida albicans (83%) Candida dublinensis (17%) Candida sake References Authors Oral ecosystem Main genera Prominent Candida species 1 Ghannoum 2010 20 oral rinse samples Frequency in the 20 subjects: Candida (75%) Cladosporium (65%) Aureobasidium (50%) Saccharomycetales (50%) Non-cultivable categories (36%) Aspergilllus (35%) Fusarium (30%) Cryptococcus (20%). Frequency of Candida species in the 20 subjects: Candida albicans (40%) Candida paraphilics (15%) Candida tropicalis (15%) Candida khmerensis (5%) Candida metapsilosis (5%) 28 Dupuy 2014 6 saliva samples Frequency of genera occurring in at least 50% of the six subjects: Malassezia (38%) Epicoccum (33%) Candida/Pichia (10%) Fusarium/Gibberella (4%) Cladosporium/Davidiella (3%) Alternaria/Lewia (2%) Aspergilllus/Emericella/Eurotium (2%) Cryptococcus/Filobasidiella (1%) Candida albicans Candida utilis/Pichia jadinii 3 Mukherjee 2014 24 oral rinse samples Frequency in the 12 subjects of each group: 12 HIV-noninfected patients: Candida (58%) Pichia (33%) Fusarium (33%) Cladosporium Penicillium 12 HIV-infected patients: Candida (92%) Epicoccum (33%) Alternaria (25%) Penicillium Trichosporon Main species identified in the Candida genus: Candida albicans (58%) Candida dublinensis (17%) Candida intermedia Candida albicans (83%) Candida dublinensis (17%) Candida sake View Large Table 3. Consensus members in the oral mycobiome. References Authors Oral ecosystem Main genera Prominent Candida species 1 Ghannoum 2010 20 oral rinse samples Frequency in the 20 subjects: Candida (75%) Cladosporium (65%) Aureobasidium (50%) Saccharomycetales (50%) Non-cultivable categories (36%) Aspergilllus (35%) Fusarium (30%) Cryptococcus (20%). Frequency of Candida species in the 20 subjects: Candida albicans (40%) Candida paraphilics (15%) Candida tropicalis (15%) Candida khmerensis (5%) Candida metapsilosis (5%) 28 Dupuy 2014 6 saliva samples Frequency of genera occurring in at least 50% of the six subjects: Malassezia (38%) Epicoccum (33%) Candida/Pichia (10%) Fusarium/Gibberella (4%) Cladosporium/Davidiella (3%) Alternaria/Lewia (2%) Aspergilllus/Emericella/Eurotium (2%) Cryptococcus/Filobasidiella (1%) Candida albicans Candida utilis/Pichia jadinii 3 Mukherjee 2014 24 oral rinse samples Frequency in the 12 subjects of each group: 12 HIV-noninfected patients: Candida (58%) Pichia (33%) Fusarium (33%) Cladosporium Penicillium 12 HIV-infected patients: Candida (92%) Epicoccum (33%) Alternaria (25%) Penicillium Trichosporon Main species identified in the Candida genus: Candida albicans (58%) Candida dublinensis (17%) Candida intermedia Candida albicans (83%) Candida dublinensis (17%) Candida sake References Authors Oral ecosystem Main genera Prominent Candida species 1 Ghannoum 2010 20 oral rinse samples Frequency in the 20 subjects: Candida (75%) Cladosporium (65%) Aureobasidium (50%) Saccharomycetales (50%) Non-cultivable categories (36%) Aspergilllus (35%) Fusarium (30%) Cryptococcus (20%). Frequency of Candida species in the 20 subjects: Candida albicans (40%) Candida paraphilics (15%) Candida tropicalis (15%) Candida khmerensis (5%) Candida metapsilosis (5%) 28 Dupuy 2014 6 saliva samples Frequency of genera occurring in at least 50% of the six subjects: Malassezia (38%) Epicoccum (33%) Candida/Pichia (10%) Fusarium/Gibberella (4%) Cladosporium/Davidiella (3%) Alternaria/Lewia (2%) Aspergilllus/Emericella/Eurotium (2%) Cryptococcus/Filobasidiella (1%) Candida albicans Candida utilis/Pichia jadinii 3 Mukherjee 2014 24 oral rinse samples Frequency in the 12 subjects of each group: 12 HIV-noninfected patients: Candida (58%) Pichia (33%) Fusarium (33%) Cladosporium Penicillium 12 HIV-infected patients: Candida (92%) Epicoccum (33%) Alternaria (25%) Penicillium Trichosporon Main species identified in the Candida genus: Candida albicans (58%) Candida dublinensis (17%) Candida intermedia Candida albicans (83%) Candida dublinensis (17%) Candida sake View Large In vitro antagonisms between fungal and bacterial strains According to the data currently available, in vitro, some bacterial species decrease C. albicans biofilm formation and viability within biofilms, whereas C. albicans increases both bacterial growth and biofilm formation. For instance, C. albicans growth is inhibited by Escherichia coli, Pseudomonas aeruginosa, Proteus vulgaris, Staphylococcus aureus, Streptococcus pyogenes, Prevotella nigrescens, Porphyromonas gingivalis, and Streptococcus salivarius. Motile Gram-negative species display a greater inhibitory effect than Gram-positive species.21 The presence of Streptococcus intermedius seemed to have no effect.87 In contrast, both S. mutans20,86 and S. gorodki88 enhanced C. albicans hyphal development and biofilm formation, partially via the modulation of its signalling pathways. Conversely, C. albicans promoted the growth of S. aureus57 and the growth of Clostridium perfringens and Bacteroides fragilis, which are two strict anaerobes.2 This article published by Fox et al. (2014) showed that preformed Candida biofilms allowed cultivation of strict anaerobes. A more recent article published by van Leeuwen et al. (2016) showed that co-culturing does a similar thing.89 Furthermore, C. albicans synergistically promoted S. gordonii, S. mutans, S. oralis, S. sanguinis, and biofilm formation.18,76 The presence of sucrose greatly increased this synergistic effect of C. albicans and oral streptococci on biofilm formation.69 Similarly, Ramírez Granillo et al. (2015) developed a mixed Aspergillus fumigatus and S. aureus biofilm. Independently of bacterial concentration, they observed a low abundance of A. fumigatus biofilm production and abnormal fungal structures (hyphae and conidia).90 Microcosm models In microcosm models, the inoculum is constituted of saliva or dental plaque collected from donors.30,50,51 Rudney et al. (2012) recommended pooling saliva or plaque samples from multiple donors and then freezing aliquots in order to reproduce experiments.54 The resulting in vitro microcosms are much more diverse than consortia, but microcosms are difficult to characterise. Ex vivo and in vitro, culture-independent methodologies are expensive as they involve metagenomics data combined with sophisticated software analysis. Note too that many bacterial species, mostly fastidious anaerobic bacteria, are lost when the taxonomic profile of the microcosm is compared to the initial sample collected in vivo.54 There are few examples of oral microcosm models (Table 1 and 2). De Moraes et al. (2012) validated a model in 24-well tissue cultures plates.50 A commonly used model is the AAA-model first described by Exterkate et al. (2010).91 More recently, Koopman et al. (2015) and Sousa et al. (2016) developed microcosm models in a sophisticated flux system apparatus.30,51 Zijnge et al. (2010) showed that F. nucleateum and yeasts form aggregates in vivo.24 Krom et al. (2014) hypothesized that fungi could act as bridging organisms in analogy to Fusobacterium.10 Rudney et al. (2015) validated a reproducible oral microcosm model for experiments using plaque with sucrose pulsing, but taxonomic profiles were limited to bacteria and did not include fungal investigation.23 Culture conditions Biofilm support Solid supports and cell culture supports Biofilms need to grow onto a support, which can either be a solid material or an air-liquid surface (abiotic) or a cell culture (biotic). Oral biofilms are usually developed onto solid supports such as bovine enamel blocks, human enamel disks, human dentine, hydroxyapatite disks, polystyrene microtiter plates, methacrylate resin disks, soft denture liners, glass coverslips, and titanium disks (Table 1 and 2). Diaz et al. (2012) successfully developed fungal-bacterial biofilms onto reconstructed non-keratinised epithelium, using oral cells (OKF6/Tert-2) and oesophageal cells (EPC2) seeded on collagen type I-embedded fibroblasts (3T3 cells) (Table 2).18 Non-oral cell cultures have also been used, with either bacterial or fungal inoculum. For instance, Krishnamurthy & Kyd (2014) demonstrated that contact with lung epithelial cells (A549/CCL-185) increased the formation of a two-bacterial species biofilm compared to a no-contact model.92 Kavanaugh et al. (2014) worked with mucus-secreting cells derived from a human colorectal adenocarcinoma (HT29-MTX) and showed that mucins downregulated fungal genes involved in surface attachment (Als1, Als3) and suppressed surface adhesion of C. albicans.8 Recently, Townsend et al. (2016) developed a polymicrobial inter-kingdom in vitro wound biofilm model on complex hydrogel-based cellulose substrata to test commonly used topical treatments.93 Pre-treatment of the support Solid supports and epithelial cell cultures are frequently pre-incubated with human saliva to reproduce the salivary pellicle and provide receptor binding sites for bacteria.78,86,94 Diaz et al. (2012) used the culture medium of the biofilm (defined mucin medium) supplemented with saliva.18 The main limitation of using saliva is that its composition varies both intra- and inter-individually. To achieve an acceptably reproducible biofilm model, the saliva collection conditions must be standardised (no antibiotics during the previous weeks, hour of sampling, stimulated- or non-stimulated saliva, pooling various donors’ samples). The saliva is then filtered, aliquoted, and frozen, ready to perform all the experiments with the same saliva pool.62 The use of artificial saliva allows other authors to reproduce the model. Culture medium The culture medium used to grow the final multispecies mix is a key factor that influences the growth, stability, thickness, and composition of the biofilm.95 Consortium models: pre-culture of separate fungal and bacterial strains C. albicans can be pre-grown aerobically at 25°C, 35°C, or 37°C, with or without shaking. In different models, the starter culture broth is either a semi-defined medium with 18 mM glucose,86 Sabouraud Dextrose broth,94 yeast nitrogen base broth (YND) with 100 mM glucose,31 or yeast peptone dextrose medium (YPD).80 The final culture medium can contain the mix of every bacterial and fungal pre-culture broth, saliva, or fluid universal medium.96 In parallel, single bacterial species are grown separately in the most appropriate culture broth. There is no consensual medium. Pre-culture broth can be brain-heart infusion (BHI),18,69,94 BHI broth supplemented with 5% sucrose,31 ultrafiltered tryptone-yeast extract broth (UFTYE; 2.5% tryptone and 1.5% yeast extract, pH 7.0) with 1% glucose,20 Todd-Hewitt broth with 0.02% yeast extract media (THB + 0.02% YE),80 universal medium broth (thioglycolate medium supplemented with 67 mMl/l of Sorensen's solution (UM),94 or modified fluid universal medium (mFUM)97 with 0.3% glucose.75 Bacterial strains are pre-grown for 4 h to 18 h at 37°C, in aerobic, microaerophilic (5% CO2) or anaerobic (5% CO2, 95% N at 200 bar) conditions. Pre-cultures are grown to the exponential growth phase, and an aliquot is diluted in the final culture mix. The optimal inoculum concentration for bacteria and Candida strains generally ranges from 106 to 107 colony-forming units (cfu)/ml. The proportion of each species culture in the final mix must be carefully determined according to strains, support and culture conditions, to prevent any one species from overgrowing.35 Microcosm models In microcosm models, the culture medium must be directly adapted to fungal and bacterial strains. In a model of peri-implantitis, Sousa et al. (2016) developed a medium adapted to epithelial cell, fungal and bacterial growth. This unique medium mimicking the peri-implant sulcular fluid contained a tissue culture medium supplemented with horse serum, menadione, and haemin.30 Culture duration Protocols of current models are summarised in Table 1 and 2. Every protocol has its own unique design, with rinses and steps to promote Candida adhesion to the support, Candida filamentation, or biofilm formation, for instance.79 Culture duration ranges from 4 hours18 to 16 weeks.98 In studies designed to evaluate the efficacy of methods or compounds to eliminate bacteria in biofilm, the incubation time generally ranges from 2 to 4 days.94,99 Culture apparatus Batch cultures Batch culture models are grown in polystyrene plates, such as 96-well microtiter plates and 24-well tissue culture plates100 or more rarely in bottles (Fig. 1A). They are inexpensive and allow to test in parallel a large number of conditions.21,50 The biofilm is grown in the bottom of wells or onto solid samples placed into the wells (hydroxyapatite, resin or titanium disks, for instance). Extracted teeth specimens can be processed in Eppendorf tubes.101 In batch cultures, the culture is stopped at given times and submitted to rinses, new medium and/or sugar pulses or addition of antiseptics. Plates are well suited to the screening of several molecules at various concentrations or contact times. Mixed models contain facultative and/or obligate anaerobic bacteria as well as Candida sp. Some are grown in a microaerophilic atmosphere20,31,32,80 or an anaerobic atmosphere.62,74In vitro, agitation is important.102 Shaker speed usually ranges from 75 to 180 rpm.21,31,57 Figure 1. View largeDownload slide (A) Schematic representation of batch culture system: microtiter plate or bottle. (B) Schematic representation of flow cell system. This Figure is reproduced in color in the online version of Medical Mycology. Figure 1. View largeDownload slide (A) Schematic representation of batch culture system: microtiter plate or bottle. (B) Schematic representation of flow cell system. This Figure is reproduced in color in the online version of Medical Mycology. Flux systems Flux systems have been designed to continuously renew the culture medium, such as in a flow cell18,78 (Fig. 1B), an artificial mouth system,51 and a constant depth film fermentor.30 The model described by Diaz et al. was grown under aerobic conditions.18 Sousa et al. (2016) managed to create a peri-implantitis model in rabbits.30 The inoculum was a saliva microcosm, grown onto titanium discs. It was pumped by a peristaltic pump at the rate of 1 ml/min for 8 h. To simulate a subgingival environment, an anaerobic environment was progressively created as follows: day 0, aerobic atmosphere; days 1 to 9, microaerophilic environment (2% O2, 3% CO2, 95% N); days 10 to 30, anaerobic environment (5% CO2, 95% N at 200 bar). After a 30-day incubation period, the discs were surgically positioned onto rabbit tibial bone. Qualitative and quantitative methods to assess in vitro biofilm models The benefits and limitations of each method are presented in Table 4. A flow chart is also available (Fig. 2) to choose the suitable method. Figure 2. View largeDownload slide Flow chart to choose the suitable method. Figure 2. View largeDownload slide Flow chart to choose the suitable method. Table 4. Benefits and limitations of each method presented. Benefits Limitations Solid supports • Simplicity • Less close to the reality • Reproducibility • Moderate cost • No specific equipment required Biofilm support • Large number of samples at the same time Cell culture supports • Allow to take into account partially the immune response of the host • Difficulty of development • Not adapted for a large number of samples • Moderate cost Human saliva • To reproduce the salivary pellicle and provide receptor binding sites for bacteria • Composition varies both intra- and inter-individually Pre-treatment of the support • Closer to reality • To achieve an acceptable reproducibility of a biofilm model, the saliva collection conditions must be standardized (no antibiotics during the previous weeks, hour of sampling, pooling various donors’ samples) Artificial saliva • To reproduce the salivary pellicle and provide receptor binding sites for bacteria • Lack of potentially relevant molecules such as enzymes Batch cultures • Simplicity • Reproducibility • No renewal or renewal rough of the nutriments and oxygen • Moderate cost • No specific equipment required Culture apparatus • Large number of samples at the same time Flux systems • Reproducibility • Require scientific equipment • Moderate cost • Control of parameters (pH, flux, nutriments…) Numeration of viable cells (Colony Forming Units) • Simplicity • No consideration of viable but noncultivable bacteria • Reproducibility • Moderate cost • No specific equipment required Crystal violet staining • Simplicity • Moderate cost • Poor reproducibility • Inability to differentiate live or dead bacteria Qualitative and quantitative methods to assess in vitro biofilm models Routine laboratory methods Colorimetric XTT assay • Simplicity • Metabolic activity can be altered by a restricted access to nutrients and oxygen • Reproducibility • Limited to fungal cells • Moderate cost Fluorescence microscopy (i.e. LIVE/DEAD Baclight® method) • Simplicity • Limited applications and essentially for bacteria: (i.e: Gram stain kit, LIVE/DEAD viability kit…) • Multi-purpose method: a quantitative method for counting CFU/mL with a fluorescence microscope or a qualitative method to visualize biofilm colonization with a CSLM • Few fluorescent stains can be employed simultaneously • Less toxic than conventional assays Imaging Confocal scanning laser microscopy (CSLM) • Multi-purpose method: to estimate the biomass, to quantify extracellular polysaccharides and microbial cells • Only a semi-quantitative investigation • Few fluorescent stains can be employed simultaneously allowing the visualization of just a couple of components in the same image • Do not disrupt the biofilm Scanning electron microscopy (SEM) • High-resolution, • Samples preparation does not preserve the vitality of the biofilm • Three-dimensional images provide topographical, morphological and compositional information • Requires expensive scientific equipment RT-qPCR • Powerful and sensitive gene analysis techniques • Strict rules of sample preparation (No contaminants or PCR inhibitors; choice of the primers sequence …) • High costs • Difficulty of execution requiring expensive scientific equipment and skilled technical staff Genetic assays Fluorescence in situ hybridization (FISH) • Capacity to quantify nonculturable organisms • Cost • Inability to differentiate viable and nonviable cells • Impossibility to work on live material • Only qualitative method Microarrays • Allow the investigation of the biofilm physiology and microbial interactions • Powerful and sensitive gene analysis techniques • Strict rules of sample preparation • High costs • Difficulty of execution and expensive scientific equipment often lead to the use of platform of sequencing Benefits Limitations Solid supports • Simplicity • Less close to the reality • Reproducibility • Moderate cost • No specific equipment required Biofilm support • Large number of samples at the same time Cell culture supports • Allow to take into account partially the immune response of the host • Difficulty of development • Not adapted for a large number of samples • Moderate cost Human saliva • To reproduce the salivary pellicle and provide receptor binding sites for bacteria • Composition varies both intra- and inter-individually Pre-treatment of the support • Closer to reality • To achieve an acceptable reproducibility of a biofilm model, the saliva collection conditions must be standardized (no antibiotics during the previous weeks, hour of sampling, pooling various donors’ samples) Artificial saliva • To reproduce the salivary pellicle and provide receptor binding sites for bacteria • Lack of potentially relevant molecules such as enzymes Batch cultures • Simplicity • Reproducibility • No renewal or renewal rough of the nutriments and oxygen • Moderate cost • No specific equipment required Culture apparatus • Large number of samples at the same time Flux systems • Reproducibility • Require scientific equipment • Moderate cost • Control of parameters (pH, flux, nutriments…) Numeration of viable cells (Colony Forming Units) • Simplicity • No consideration of viable but noncultivable bacteria • Reproducibility • Moderate cost • No specific equipment required Crystal violet staining • Simplicity • Moderate cost • Poor reproducibility • Inability to differentiate live or dead bacteria Qualitative and quantitative methods to assess in vitro biofilm models Routine laboratory methods Colorimetric XTT assay • Simplicity • Metabolic activity can be altered by a restricted access to nutrients and oxygen • Reproducibility • Limited to fungal cells • Moderate cost Fluorescence microscopy (i.e. LIVE/DEAD Baclight® method) • Simplicity • Limited applications and essentially for bacteria: (i.e: Gram stain kit, LIVE/DEAD viability kit…) • Multi-purpose method: a quantitative method for counting CFU/mL with a fluorescence microscope or a qualitative method to visualize biofilm colonization with a CSLM • Few fluorescent stains can be employed simultaneously • Less toxic than conventional assays Imaging Confocal scanning laser microscopy (CSLM) • Multi-purpose method: to estimate the biomass, to quantify extracellular polysaccharides and microbial cells • Only a semi-quantitative investigation • Few fluorescent stains can be employed simultaneously allowing the visualization of just a couple of components in the same image • Do not disrupt the biofilm Scanning electron microscopy (SEM) • High-resolution, • Samples preparation does not preserve the vitality of the biofilm • Three-dimensional images provide topographical, morphological and compositional information • Requires expensive scientific equipment RT-qPCR • Powerful and sensitive gene analysis techniques • Strict rules of sample preparation (No contaminants or PCR inhibitors; choice of the primers sequence …) • High costs • Difficulty of execution requiring expensive scientific equipment and skilled technical staff Genetic assays Fluorescence in situ hybridization (FISH) • Capacity to quantify nonculturable organisms • Cost • Inability to differentiate viable and nonviable cells • Impossibility to work on live material • Only qualitative method Microarrays • Allow the investigation of the biofilm physiology and microbial interactions • Powerful and sensitive gene analysis techniques • Strict rules of sample preparation • High costs • Difficulty of execution and expensive scientific equipment often lead to the use of platform of sequencing View Large Table 4. Benefits and limitations of each method presented. Benefits Limitations Solid supports • Simplicity • Less close to the reality • Reproducibility • Moderate cost • No specific equipment required Biofilm support • Large number of samples at the same time Cell culture supports • Allow to take into account partially the immune response of the host • Difficulty of development • Not adapted for a large number of samples • Moderate cost Human saliva • To reproduce the salivary pellicle and provide receptor binding sites for bacteria • Composition varies both intra- and inter-individually Pre-treatment of the support • Closer to reality • To achieve an acceptable reproducibility of a biofilm model, the saliva collection conditions must be standardized (no antibiotics during the previous weeks, hour of sampling, pooling various donors’ samples) Artificial saliva • To reproduce the salivary pellicle and provide receptor binding sites for bacteria • Lack of potentially relevant molecules such as enzymes Batch cultures • Simplicity • Reproducibility • No renewal or renewal rough of the nutriments and oxygen • Moderate cost • No specific equipment required Culture apparatus • Large number of samples at the same time Flux systems • Reproducibility • Require scientific equipment • Moderate cost • Control of parameters (pH, flux, nutriments…) Numeration of viable cells (Colony Forming Units) • Simplicity • No consideration of viable but noncultivable bacteria • Reproducibility • Moderate cost • No specific equipment required Crystal violet staining • Simplicity • Moderate cost • Poor reproducibility • Inability to differentiate live or dead bacteria Qualitative and quantitative methods to assess in vitro biofilm models Routine laboratory methods Colorimetric XTT assay • Simplicity • Metabolic activity can be altered by a restricted access to nutrients and oxygen • Reproducibility • Limited to fungal cells • Moderate cost Fluorescence microscopy (i.e. LIVE/DEAD Baclight® method) • Simplicity • Limited applications and essentially for bacteria: (i.e: Gram stain kit, LIVE/DEAD viability kit…) • Multi-purpose method: a quantitative method for counting CFU/mL with a fluorescence microscope or a qualitative method to visualize biofilm colonization with a CSLM • Few fluorescent stains can be employed simultaneously • Less toxic than conventional assays Imaging Confocal scanning laser microscopy (CSLM) • Multi-purpose method: to estimate the biomass, to quantify extracellular polysaccharides and microbial cells • Only a semi-quantitative investigation • Few fluorescent stains can be employed simultaneously allowing the visualization of just a couple of components in the same image • Do not disrupt the biofilm Scanning electron microscopy (SEM) • High-resolution, • Samples preparation does not preserve the vitality of the biofilm • Three-dimensional images provide topographical, morphological and compositional information • Requires expensive scientific equipment RT-qPCR • Powerful and sensitive gene analysis techniques • Strict rules of sample preparation (No contaminants or PCR inhibitors; choice of the primers sequence …) • High costs • Difficulty of execution requiring expensive scientific equipment and skilled technical staff Genetic assays Fluorescence in situ hybridization (FISH) • Capacity to quantify nonculturable organisms • Cost • Inability to differentiate viable and nonviable cells • Impossibility to work on live material • Only qualitative method Microarrays • Allow the investigation of the biofilm physiology and microbial interactions • Powerful and sensitive gene analysis techniques • Strict rules of sample preparation • High costs • Difficulty of execution and expensive scientific equipment often lead to the use of platform of sequencing Benefits Limitations Solid supports • Simplicity • Less close to the reality • Reproducibility • Moderate cost • No specific equipment required Biofilm support • Large number of samples at the same time Cell culture supports • Allow to take into account partially the immune response of the host • Difficulty of development • Not adapted for a large number of samples • Moderate cost Human saliva • To reproduce the salivary pellicle and provide receptor binding sites for bacteria • Composition varies both intra- and inter-individually Pre-treatment of the support • Closer to reality • To achieve an acceptable reproducibility of a biofilm model, the saliva collection conditions must be standardized (no antibiotics during the previous weeks, hour of sampling, pooling various donors’ samples) Artificial saliva • To reproduce the salivary pellicle and provide receptor binding sites for bacteria • Lack of potentially relevant molecules such as enzymes Batch cultures • Simplicity • Reproducibility • No renewal or renewal rough of the nutriments and oxygen • Moderate cost • No specific equipment required Culture apparatus • Large number of samples at the same time Flux systems • Reproducibility • Require scientific equipment • Moderate cost • Control of parameters (pH, flux, nutriments…) Numeration of viable cells (Colony Forming Units) • Simplicity • No consideration of viable but noncultivable bacteria • Reproducibility • Moderate cost • No specific equipment required Crystal violet staining • Simplicity • Moderate cost • Poor reproducibility • Inability to differentiate live or dead bacteria Qualitative and quantitative methods to assess in vitro biofilm models Routine laboratory methods Colorimetric XTT assay • Simplicity • Metabolic activity can be altered by a restricted access to nutrients and oxygen • Reproducibility • Limited to fungal cells • Moderate cost Fluorescence microscopy (i.e. LIVE/DEAD Baclight® method) • Simplicity • Limited applications and essentially for bacteria: (i.e: Gram stain kit, LIVE/DEAD viability kit…) • Multi-purpose method: a quantitative method for counting CFU/mL with a fluorescence microscope or a qualitative method to visualize biofilm colonization with a CSLM • Few fluorescent stains can be employed simultaneously • Less toxic than conventional assays Imaging Confocal scanning laser microscopy (CSLM) • Multi-purpose method: to estimate the biomass, to quantify extracellular polysaccharides and microbial cells • Only a semi-quantitative investigation • Few fluorescent stains can be employed simultaneously allowing the visualization of just a couple of components in the same image • Do not disrupt the biofilm Scanning electron microscopy (SEM) • High-resolution, • Samples preparation does not preserve the vitality of the biofilm • Three-dimensional images provide topographical, morphological and compositional information • Requires expensive scientific equipment RT-qPCR • Powerful and sensitive gene analysis techniques • Strict rules of sample preparation (No contaminants or PCR inhibitors; choice of the primers sequence …) • High costs • Difficulty of execution requiring expensive scientific equipment and skilled technical staff Genetic assays Fluorescence in situ hybridization (FISH) • Capacity to quantify nonculturable organisms • Cost • Inability to differentiate viable and nonviable cells • Impossibility to work on live material • Only qualitative method Microarrays • Allow the investigation of the biofilm physiology and microbial interactions • Powerful and sensitive gene analysis techniques • Strict rules of sample preparation • High costs • Difficulty of execution and expensive scientific equipment often lead to the use of platform of sequencing View Large Routine laboratory methods Routine laboratory methods are a first step in observing consortium and microcosm models. C. albicans yeast-hyphal transition and fungal-bacterial interactions are observable directly in biofilms formed on glass coverslips and in the wells of polystyrene plates, with bright-field and phase-contrast microscopes.79 Light microscopy with x400 magnification is adapted to hyphae/yeast count.31 In dental plaque and caries models, pH evolution20,31,51,69,78 or metabolite releases51 are monitored in the culture medium bathing the biofilms. In-biofilm cell viability is commonly determined by scraping the microbial deposits formed onto solid supports followed by serial dilutions on appropriate agar plates for cfu count,30,31,50,62,64,69,74,86,94,101 but Falsetta et al. (2014) highlighted the limitations of cfu count data for C. albicans, as most hyphae are multicellular with a large biomass compared to yeasts yet like yeasts they form a single cfu.20 Alternatively, in-well biomass formation can be quantified with colorimetric methods needing a plate spectrophotometer.80 The XTT assay is based on the reduction of the tetrazolium salt of XTT in formazan by the succinate dehydrogenase system of the mitochondrial respiratory chain in fungal cells but not bacterial cells.21,64 The crystal violet assay dies in violet the total biomass, including fungal cells, bacterial cells and exopolysaccharides,31,80 but it quantifies both live and dead cells in the biofilm.74 Finally, routine histology are suitably adapted to analysing biofilms grown onto epithelial cell cultures.18,32,103 Imaging The LIVE/DEAD® Biofilm viability kit (BacLight, Invitrogen, Paisley, UK) method utilizes mixtures of green-fluorescent (SYTO9) and red-fluorescent (propidium iodide) nucleic acid stains for bacteria. The difference in stain penetration of bacterial and fungal cells allows making difference between healthy cells (green) and bacteria with damaged membranes (red). BacLight® can be used as a quantitative method or as a qualitative method.98,104 Figure 3A is an example of LIVE/DEAD® method, applied to a single species C. albicans biofilm. Figure 3. View largeDownload slide Comparison of different methods of imaging applied to a single species C. albicans biofilm. (A) Biofilm was stained using SYTO-9 (BacLight®) to stain live biofilm cells green and examined by fluorescence microscopy (x63); (B) Biofilm observed with SEM (Bar, 10 μm). (C) Biofilm 3D visualization after z-stack acquisition with CSLM (Bar, 50 μm) (Courtesy Pr. G. Ramage). This Figure is reproduced in color in the online version of Medical Mycology. Figure 3. View largeDownload slide Comparison of different methods of imaging applied to a single species C. albicans biofilm. (A) Biofilm was stained using SYTO-9 (BacLight®) to stain live biofilm cells green and examined by fluorescence microscopy (x63); (B) Biofilm observed with SEM (Bar, 10 μm). (C) Biofilm 3D visualization after z-stack acquisition with CSLM (Bar, 50 μm) (Courtesy Pr. G. Ramage). This Figure is reproduced in color in the online version of Medical Mycology. Scanning electron microscopy (SEM) is suitably adapted to observing fungal-bacterial organisation in a biofilm. It is also useful to observe the surface of the biofilm support before and after microbial colonisation, such as enamel acid attack in caries models.21,31,62,64,69,74,78,80,86,101 Figure 3B is an example of a C. albicans biofilm observed with SEM. Confocal laser scanning microscopy (CLSM) is suitably adapted to 2D and 3D analysis of the biofilm, combined with fluorescent staining of specific microbial cells and matrix components.18,20,32,64,69,74,78–80,103 Figure 3C is an example of CSLM method, applied to a single species C. albicans biofilm. After image reconstruction on appropriate software, it is possible to measure average thickness of the biofilm and to characterise its microbial species and their respective percentages in the biomass.49 Some fluorophore combinations are also adapted to identify and quantify live and dead cells in the biofilm.74 Genetic assays Quantitative reverse transcription polymerase chain reaction (RT-qPCR) is suitably adapted to characterising the taxonomic and functional profile of microcosm models based on selected genes,20,21,49,51,62,74,78 including cell viability74 and hyphal morphology18; however, RT-qPCR requires expensive equipment.54 Fluorescence in situ hybridization (FISH) is based on oligonucleotide probes labeled with fluorescent dyes. FISH can be used to determine the bacterial and fungal composition of a biofilm,105 to visualize spatial distribution in combination with confocal laser scanning microscopy (CLSM),106 or the colonization of gingival epithelia by subgingival biofilm.97 A microarray is a miniaturized solid support displaying a very large set of oligonucleotide probes, allowing the screening of >30.000 genes during a two-day protocol. For instance, microarrays allow the detection of variations in a gene sequence expression, the comparison of a bacterial genome expression at different times of growth or different culture conditions, or the comparison of two bacterial consortia grown in similar culture conditions.107 In a fungal biofilm model, Cao et al.108 used microarrays to show the influence of farnesol on C. albicans biofilm: some hyphal-formation-associated genes (including TUP1) were differentially expressed in farnesol-treated biofilms. Conclusion There is increasing interest in in vitro models of mixed fungal-bacterial biofilms designed to mimic various oral ecosystems. Protocol designs, culture broths and culture conditions are highly diverse, but the supports used to develop biofilms are relatively consensual, as is the choice of C. albicans and bacterial species in consortium models. S. mutans and oral streptococci are near-standard bacterial species in caries, periodontitis, and candidiasis models, but new bacterial and fungal combinations could be explored. Species selection could take into account genomic and proteomic results obtained in vivo, as recent studies have revealed new taxonomy profiles, unexpected quantitative compositions, and functional expressions in oral microcosms. This review also revealed a broad difference between culture apparatuses and assessment methods, ranging from microtiter plates to custom-made flux systems, and from cfu counts to CLSM and RT-qPCR. These sophisticated and expensive technologies may ultimately lead to therapies designed to clean up oral microcosms or improve oral health at molecular level. However, microtiter plate models still warrant attention, as they are well adapted to the development of new therapeutic agents. Many populations of patients still need basic oral hygiene education, first-line oral care, healthy diet, and medical help to reduce polymedication and combat addictions to alcohol, tobacco, and illicit drugs. In vivo, these conditions influence oral ecosystems, particularly in children and teenagers, in chronically ill, poly-medicated or malnourished patients, and in frail elderly people. In vitro assays with appropriate models could help improve oral care products, drug formulations, or the composition of foods, beverages, and oral nutritional supplements. Acknowledgements This work was supported by grants from the Association Française du Gougerot-Sjögren et des syndromes secs and the Fondation de l’Avenir (gAFGS-2013 and AP-RMA-2015-025). For manuscript revision, we thank Chetan S. Patil DDS, PhD, Periodontal Associate (LLC, Englewood, NJ, USA) and Clinical instructor (Columbia University College of Dental Medicine, NY, USA). The authors greatly acknowledge the CCMA (Centre Commun de Microscopie Appliquée, Université Côte d’Azur, Microscopy and Imaging platform Côte d’Azur, MICA) and its personnel. We also thank Pr. Gordon Ramage (Infection and Immunity Research Group, University of Glasgow, UK) for CLS micrograph. Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and the writing of the paper. References 1. Ghannoum MA , Jurevic RJ , Mukherjee PK et al. Characterization of the oral fungal microbiome (mycobiome) in healthy individuals . PLoS Pathog . 2010 ; 6 : e1000713 . Google Scholar CrossRef Search ADS PubMed 2. Fox EP , Cowley ES , Nobile CJ , Hartooni N , Newman DK , Johnson AD . Anaerobic bacteria grow within Candida albicans biofilms and induce biofilm formation in suspension cultures . Curr Biol . 2014 ; 24 : 2411 – 2416 . Google Scholar CrossRef Search ADS PubMed 3. Mukherjee PK , Chandra J , Retuerto M et al. Oral mycobiome analysis of HIV-infected patients: identification of Pichia as an antagonist of opportunistic fungi . PLoS Pathog . 2014 ; 10 : e1003996 . Google Scholar CrossRef Search ADS PubMed 4. Huynh HTT , Pignoly M , Nkamga VD , Drancourt M , Aboudharam G . The repertoire of archaea cultivated from severe periodontitis . PLoS ONE . 2015 ; 10 : e0121565 . Google Scholar CrossRef Search ADS PubMed 5. Diaz PI , Strausbaugh LD , Dongari-Bagtzoglou A . Fungal-bacterial interactions and their relevance to oral health: linking the clinic and the bench . Front Cell Infect Microbiol . 2014 ; 4 : 101 . Google Scholar CrossRef Search ADS PubMed 6. Xu H , Dongari-Bagtzoglou A . Shaping the oral mycobiota: interactions of opportunistic fungi with oral bacteria and the host . Curr Opin Microbiol . 2015 ; 26 : 65 – 70 . Google Scholar CrossRef Search ADS PubMed 7. Vincent-Bugnas S , Vitale S , Mouline CC et al. EBV infection is common in gingival epithelial cells of the periodontium and worsens during chronic periodontitis . PloS One . 2013 ; 8 : e80336 . Google Scholar CrossRef Search ADS PubMed 8. Kavanaugh NL , Zhang AQ , Nobile CJ , Johnson AD , Ribbeck K . Mucins suppress virulence traits of Candida albicans . mBio . 2014 ; 5 : e0 1911. Google Scholar CrossRef Search ADS 9. Ewan V , Perry JD , Mawson T et al. Detecting potential respiratory pathogens in the mouths of older people in hospital . Age Ageing . 2010 ; 39 : 122 – 125 . Google Scholar CrossRef Search ADS PubMed 10. Krom BP , Kidwai S , ten Cate JM . Candida and other fungal species: forgotten players of healthy oral microbiota . J Dent Res . 2014 ; 93 : 445 – 451 . Google Scholar CrossRef Search ADS PubMed 11. Ortega O , Sakwinska O , Combremont S et al. High prevalence of colonization of oral cavity by respiratory pathogens in frail older patients with oropharyngeal dysphagia . Neurogastroenterol Motil Off J Eur Gastrointest Motil Soc . 2015 ; 12 : 1804 – 1816 . Google Scholar CrossRef Search ADS 12. Scannapieco FA , Cantos A . Oral inflammation and infection, and chronic medical diseases: implications for the elderly . Periodontol 2000 . 2016 ; 72 : 153 – 175 . Google Scholar CrossRef Search ADS PubMed 13. Saarela RKT , Lindroos E , Soini H et al. Dentition, nutritional status and adequacy of dietary intake among older residents in assisted living facilities . Gerodontology . 2016 ; 33 : 225 – 232 . Google Scholar CrossRef Search ADS PubMed 14. Otomo-Corgel J , Pucher JJ , Rethman MP , Reynolds MA . State of the science: chronic periodontitis and systemic health . J Evid Based Dent Pract . 2012 ; 12 : 20 – 28 . Google Scholar CrossRef Search ADS PubMed 15. Gil-Montoya JA , Ferreira de Mello AL , Barrios R , Gonzalez-Moles MA , Bravo M . Oral health in the elderly patient and its impact on general well-being: a nonsystematic review . Clin Interv Aging . 2015 ; 10 : 461 – 467 . Google Scholar CrossRef Search ADS PubMed 16. Guo Y , Wei C , Liu C et al. Inhibitory effects of oral Actinomyces on the proliferation, virulence and biofilm formation of Candida albicans . Arch Oral Biol . 2015 ; 60 : 1368 – 1374 . Google Scholar CrossRef Search ADS PubMed 17. Vilela SFG , Barbosa JO , Rossoni RD et al. Lactobacillus acidophilus ATCC 4356 inhibits biofilm formation by C. albicans and attenuates the experimental candidiasis in Galleria mellonella. Virulence . 2015 ; 6 : 29 – 39 . Google Scholar CrossRef Search ADS PubMed 18. Diaz PI , Xie Z , Sobue T et al. Synergistic Interaction between Candida albicans and commensal oral streptococci in a novel in vitro mucosal model . Infect Immun . 2012 ; 80 : 620 – 632 . Google Scholar CrossRef Search ADS PubMed 19. Xu H , Sobue T , Thompson A et al. Streptococcal co-infection augments Candida pathogenicity by amplifying the mucosal inflammatory response . Cell Microbiol . 2014 ; 16 : 214 – 231 . Google Scholar CrossRef Search ADS PubMed 20. Falsetta ML , Klein MI , Colonne PM et al. Symbiotic relationship between Streptococcus mutans and Candida albicans synergizes virulence of plaque biofilms in vivo . Infect Immun . 2014 ; 82 : 1968 – 1981 . Google Scholar CrossRef Search ADS PubMed 21. Park SJ , Han K-H , Park JY , Choi SJ , Lee K-H . Influence of bacterial presence on biofilm formation of Candida albicans . Yonsei Med J . 2014 ; 55 : 449 – 458 . Google Scholar CrossRef Search ADS PubMed 22. Chew SY , Cheah YK , Seow HF , Sandai D , Than LTL . In vitro modulation of probiotic bacteria on the biofilm of Candida glabrata . Anaerobe . 2015 ; 34 : 132 – 138 . Google Scholar CrossRef Search ADS PubMed 23. Rudney JD , Jagtap PD , Reilly CS et al. Protein relative abundance patterns associated with sucrose-induced dysbiosis are conserved across taxonomically diverse oral microcosm biofilm models of dental caries . Microbiome . 2015 ; 3 : 69 . Google Scholar CrossRef Search ADS PubMed 24. Zijnge V , van Leeuwen MBM , Degener JE et al. Oral biofilm architecture on natural teeth . PloS One . 2010 ; 5 : e9321 . Google Scholar CrossRef Search ADS PubMed 25. Frias-Lopez J , Duran-Pinedo A . Effect of periodontal pathogens on the metatranscriptome of a healthy multispecies biofilm model . J Bacteriol . 2012 ; 194 : 2082 – 2095 . Google Scholar CrossRef Search ADS PubMed 26. Palmer RJ. Composition and development of oral bacterial communities . Periodontol 2000 . 2014 ; 64 : 20 – 39 . Google Scholar CrossRef Search ADS PubMed 27. Peterson SN , Meissner T , Su AI et al. Functional expression of dental plaque microbiota . Front Cell Infect Microbiol . 2014 ; 4 : 108 . Google Scholar CrossRef Search ADS PubMed 28. Dupuy AK , David MS , Li L et al. Redefining the human oral mycobiome with improved practices in amplicon-based taxonomy: discovery of Malassezia as a prominent commensal . PloS One . 2014 ; 9 : e90899 . Google Scholar CrossRef Search ADS PubMed 29. Vanhoecke BWA , De Ryck TRG , De boel K et al. Low-dose irradiation affects the functional behavior of oral microbiota in the context of mucositis . Exp Biol Med Maywood NJ . 2016 ; 241 : 60 – 70 . Google Scholar CrossRef Search ADS 30. Sousa V , Mardas N , Spratt D , Boniface D , Dard M , Donos N . Experimental models for contamination of titanium surfaces and disinfection protocols . Clin Oral Implants Res . 2016 ; 10 : 1233 – 1242 . Google Scholar CrossRef Search ADS 31. Barbosa JO , Rossoni RD , Vilela SFG et al. Streptococcus mutans can modulate biofilm formation and attenuate the virulence of Candida albicans . PloS One . 2016 ; 11 : e0150457 . Google Scholar CrossRef Search ADS PubMed 32. Bertolini MM , Xu H , Sobue T , Nobile CJ , Del Bel Cury AA , Dongari-Bagtzoglou A . Candida-streptococcal mucosal biofilms display distinct structural and virulence characteristics depending on growth conditions and hyphal morphotypes . Mol Oral Microbiol . 2015 ; 30 : 307 – 322 . Google Scholar CrossRef Search ADS PubMed 33. Ramage G , Lappin DF , Millhouse E et al. The epithelial cell response to health and disease associated oral biofilm models . J Periodontal Res . 2016 ; 52 : 325 – 333 . Google Scholar CrossRef Search ADS PubMed 34. Wade WG. Characterisation of the human oral microbiome . J Oral Biosci . 2013 ; 55 : 143 – 148 . Google Scholar CrossRef Search ADS 35. Kreth J , Merritt J , Qi F . Bacterial and host interactions of oral streptococci . DNA Cell Biol . 2009 ; 28 : 397 – 403 . Google Scholar CrossRef Search ADS PubMed 36. Jorth P , Turner KH , Gumus P , Nizam N , Buduneli N , Whiteley M . Metatranscriptomics of the human oral microbiome during health and disease . mBio . 2014 ; 5 : e01012 – 01014 . Google Scholar CrossRef Search ADS PubMed 37. Rams TE , Degener JE , van Winkelhoff AJ . Antibiotic resistance in human chronic periodontitis microbiota . J Periodontol. 2014 ; 85 : 160 – 169 . Google Scholar CrossRef Search ADS PubMed 38. Brändle N , Zehnder M , Weiger R , Waltimo T . Impact of growth conditions on susceptibility of five microbial species to alkaline stress . J Endod . 2008 ; 34 : 579 – 582 . Google Scholar CrossRef Search ADS PubMed 39. Yamazaki H , Ohshima T , Tsubota Y , Yamaguchi H , Jayawardena JA , Nishimura Y . Microbicidal activities of low frequency atmospheric pressure plasma jets on oral pathogens . Dent Mater J . 2011 ; 30 : 384 – 391 . Google Scholar CrossRef Search ADS PubMed 40. Ramalingam K , Amaechi BT , Ralph RH , Lee VA . Antimicrobial activity of nanoemulsion on cariogenic planktonic and biofilm organisms . Arch Oral Biol . 2012 ; 57 : 15 – 22 . Google Scholar CrossRef Search ADS PubMed 41. Muhammad OH , Chevalier M , Rocca J-P , Brulat-Bouchard N , Medioni E . Photodynamic therapy versus ultrasonic irrigation: Interaction with endodontic microbial biofilm, an ex vivo study . Photodiagnosis Photodyn Ther . 2014 ; 11 : 171 – 181 . Google Scholar CrossRef Search ADS PubMed 42. Salli KM , Ouwehand AC . The use of in vitro model systems to study dental biofilms associated with caries: a short review . J Oral Microbiol . 2015 ; 7 : 26149 . Google Scholar CrossRef Search ADS PubMed 43. Rahmani-Badi A , Sepehr S , Babaie-Naiej H . A combination of cis-2-decenoic acid and chlorhexidine removes dental plaque . Arch Oral Biol . 2015 ; 60 : 1655 – 1661 . Google Scholar CrossRef Search ADS PubMed 44. Soares GMS , Teles F , Starr JR et al. Effects of azithromycin, metronidazole, amoxicillin, andmetronidazole plus amoxicillin on an in vitro polymicrobial subgingival biofilm model . Antimicrob Agents Chemother . 2015 ; 59 : 2791 – 2798 . Google Scholar CrossRef Search ADS PubMed 45. Periasamy S , Kolenbrander PE . Aggregatibacter actinomycetemcomitans builds mutualistic biofilm communities with Fusobacterium nucleatum and Veillonella species in saliva . Infect Immun . 2009 ; 77 : 3542 – 3551 . Google Scholar CrossRef Search ADS PubMed 46. Li H , Zhang C , Liu P , Liu W , Gao Y , Sun S . In vitro interactions between fluconazole and minocycline against mixed cultures of Candida albicans and Staphylococcus aureus . J Microbiol Immunol Infect . 2015 ; 48 : 655 – 661 . Google Scholar CrossRef Search ADS PubMed 47. Shen Y , Stojicic S , Qian W , Olsen I , Haapasalo M . The synergistic antimicrobial effect by mechanical agitation and two chlorhexidine preparations on biofilm bacteria . J Endod . 2010 ; 36 : 100 – 104 . Google Scholar CrossRef Search ADS PubMed 48. Fröjd V , Chávez de Paz L , Andersson M , Wennerberg A , Davies JR , Svensäter G . In situ analysis of multispecies biofilm formation on customized titanium surfaces . Mol Oral Microbiol . 2011 ; 26 : 241 – 252 . Google Scholar CrossRef Search ADS PubMed 49. Thurnheer T , Bostanci N , Belibasakis GN . Microbial dynamics during conversion from supragingival to subgingival biofilms in an in vitro model . Mol Oral Microbiol . 2016 ; 31 : 125 – 135 . Google Scholar CrossRef Search ADS PubMed 50. de Moraes AP Barwaldt CK , Nunes TZ et al. Effect of triazine derivative added to denture materials on a microcosm biofilm model . J Biomed Mater Res B Appl Biomater . 2012 ; 100 : 1328 – 1333 . Google Scholar CrossRef Search ADS PubMed 51. Koopman JE , Röling WFM , Buijs MJ et al. Stability and resilience of oral microcosms toward acidification and Candida outgrowth by arginine supplementation . Microb Ecol . 2015 ; 69 : 422 – 433 . Google Scholar CrossRef Search ADS PubMed 52. Verardi G , Cenci MS , Maske TT , Webber B , Santos LR dos . Antiseptics and microcosm biofilm formation on titanium surfaces. Braz Oral Res . 2016 ; 30 : doi: 10.1590/1807-3107BOR-2016 . 53. Akers KS , Cardile AP , Wenke JC , Murray CK . Biofilm formation by clinical isolates and its relevance to clinical infections . Adv Exp Med Biol . 2015 ; 830 : 1 – 28 . Google Scholar CrossRef Search ADS PubMed 54. Rudney JD , Chen R , Lenton P et al. A reproducible oral microcosm biofilm model for testing dental materials . J Appl Microbiol . 2012 ; 113 : 1540 – 1553 . Google Scholar CrossRef Search ADS PubMed 55. Jenkinson HF , Lamont RJ . Oral microbial communities in sickness and in health . Trends Microbiol . 2005 ; 13 : 589 – 595 . Google Scholar CrossRef Search ADS PubMed 56. Oh S , Go GW , Mylonakis E , Kim Y . The bacterial signalling molecule indole attenuates the virulence of the fungal pathogen Candida albicans . J Appl Microbiol . 2012 ; 113 : 622 – 628 . Google Scholar CrossRef Search ADS PubMed 57. Krause J , Geginat G , Tammer I . Prostaglandin E2 from Candida albicans stimulates the growth of Staphylococcus aureus in mixed biofilms . PLoS ONE . 2015 ; 10 : e0135404 . Google Scholar CrossRef Search ADS PubMed 58. Nobbs AH , Lamont RJ , Jenkinson HF . Streptococcus adherence and colonization . Microbiol Mol Biol Rev . 2009 ; 73 : 407 – 450 . Google Scholar CrossRef Search ADS PubMed 59. Murciano C , Moyes DL , Runglall M et al. Evaluation of the role of Candida albicans aAgglutinin-like sequence (Als) proteins in human oral epithelial cell interactions . PLoS ONE . 2012 ; 7 : e33362 . Google Scholar CrossRef Search ADS PubMed 60. Periasamy S , Kolenbrander PE . Mutualistic biofilm communities develop with Porphyromonas gingivalis and initial, early, and late colonizers of enamel . J Bacteriol . 2009 ; 191 : 6804 – 6811 . Google Scholar CrossRef Search ADS PubMed 61. Ammann TW , Belibasakis GN , Thurnheer T . Impact of early colonizers on in vitro subgingival biofilm formation . PLoS ONE . 2013 ; 8 : e83090 . Google Scholar CrossRef Search ADS PubMed 62. Cavalcanti IMG , Ricomini Filho AP , Lucena-Ferreira SC et al. Salivary pellicle composition and multispecies biofilm developed on titanium nitrided by cold plasma . Arch Oral Biol . 2014 ; 59 : 695 – 703 . Google Scholar CrossRef Search ADS PubMed 63. van de Sande FH , Azevedo MS , Lund RG , Huysmans MCDNJM , Cenci MS . An in vitro biofilm model for enamel demineralization and antimicrobial dose-response studies . Biofouling . 2011 ; 27 : 1057 – 1063 . Google Scholar CrossRef Search ADS PubMed 64. Matsubara VH , Wang Y , Bandara HMHN , Mayer MPA , Samaranayake LP . Probiotic lactobacilli inhibit early stages of Candida albicans biofilm development by reducing their growth, cell adhesion, and filamentation . Appl Microbiol Biotechnol . 2016 ; 100 : 6415 – 6426 . Google Scholar CrossRef Search ADS PubMed 65. Jiao Y , Cody GD , Harding AK et al. Characterization of extracellular polymeric substances from acidophilic microbial biofilms . Appl Environ Microbiol . 2010 ; 76 : 2916 – 2922 . Google Scholar CrossRef Search ADS PubMed 66. Mitchell KF , Zarnowski R , Sanchez H et al. Community participation in biofilm matrix assembly and function . Proc Natl Acad Sci U S A . 2015 ; 112 : 4092 – 4097 . Google Scholar CrossRef Search ADS PubMed 67. Sandai D , Tabana YM , Ouweini AE , Ayodeji IO . Resistance of Candida albicans biofilms to drugs and the host immune system . Jundishapur J Microbiol . 2016 ; 9 : e3738 . Google Scholar CrossRef Search ADS 68. Bhattacharyya S , Gupta P , Banerjee G , Jain A , Singh M . Inhibition of biofilm formation and lipase in Candida albicans by culture filtrate of Staphylococcus epidermidis in vitro . Int J Appl Basic Med Res . 2014 ; 4 : S27 – 30 . Google Scholar CrossRef Search ADS PubMed 69. Junka AF , Szymczyk P , Smutnicka D et al. Microbial biofilms are able to destroy hydroxyapatite in the absence of host immunity in vitro . J Oral Maxillofac Surg . 2015 ; 73 : 451 – 464 . Google Scholar CrossRef Search ADS PubMed 70. Willems HM , Kos K , Jabra-Rizk MA , Krom BP . Candida albicans in oral biofilms could prevent caries . Pathog Dis . 2016 ; 74 : ftw039 . Google Scholar CrossRef Search ADS PubMed 71. Hannan S , Ready D , Jasni AS , Rogers M , Pratten J , Roberts AP . Transfer of antibiotic resistance by transformation with eDNA within oral biofilms . FEMS Immunol Med Microbiol . 2010 ; 59 : 345 – 349 . Google Scholar CrossRef Search ADS PubMed 72. Jeon J-G , Pandit S , Xiao J et al. Influences of trans-trans farnesol, a membrane-targeting sesquiterpenoid, on Streptococcus mutans physiology and survival within mixed-species oral biofilms . Int J Oral Sci . 2011 ; 3 : 98 – 106 . Google Scholar CrossRef Search ADS PubMed 73. Jenssen H , Hamill P , Hancock REW . Peptide antimicrobial agents . Clin Microbiol Rev . 2006 ; 19 : 491 – 511 . Google Scholar CrossRef Search ADS PubMed 74. Sherry L , Lappin G , O’Donnell LE et al. Viable compositional analysis of an eleven species oral polymicrobial biofilm . Front Microbiol . 2016 ; 7 : 912 . Google Scholar CrossRef Search ADS PubMed 75. Reese S , Guggenheim B . A novel TEM contrasting technique for extracellular polysaccharides in in vitro biofilms . Microsc Res Tech . 2007 ; 70 : 816 – 822 . Google Scholar CrossRef Search ADS PubMed 76. Xu H , Sobue T , Bertolini M , Thompson A , Dongari-Bagtzoglou A . Streptococcus oralis and Candida albicans synergistically activate μ-Calpain to degrade E-cadherin from oral epithelial junctions . J Infect Dis . 2016 ; 214 : 925 – 934 . Google Scholar CrossRef Search ADS PubMed 77. El-Azizi M , Farag N , Khardori N . Antifungal activity of amphotericin B and voriconazole against the biofilms and biofilm-dispersed cells of Candida albicans employing a newly developed in vitro pharmacokinetic model . Ann Clin Microbiol Antimicrob . 2015 ; 14 : 21 . Google Scholar CrossRef Search ADS PubMed 78. Yassin SA , German MJ , Rolland SL , Rickard AH , Jakubovics NS . Inhibition of multispecies biofilms by a fluoride-releasing dental prosthesis copolymer . J Dent . 2016 ; 48 : 62 – 70 . Google Scholar CrossRef Search ADS PubMed 79. Schlecht LM , Peters BM , Krom BP et al. Systemic Staphylococcus aureus infection mediated by Candida albicans hyphal invasion of mucosal tissue . Microbiol Read Engl . 2015 ; 161 : 168 – 181 . Google Scholar CrossRef Search ADS 80. Montelongo-Jauregui D , Srinivasan A , Ramasubramanian AK , Lopez-Ribot JL . An in vitro model for oral mixed biofilms of Candida albicans and Streptococcus gordonii in synthetic saliva . Front Microbiol . 2016 ; 7 : 686 . Google Scholar CrossRef Search ADS PubMed 81. Kim Y-S , Kang S-M , Lee E-S , Lee JH , Kim B-R , Kim B-I . Ecological changes in oral microcosm biofilm during maturation. J Biomed Opt . 2016 ; 21 : 101409 – 101409 . Google Scholar CrossRef Search ADS PubMed 82. Fernandez y Mostajo M , Exterkate RAM , Buijs MJ , Crielaard W , Zaura E . Effect of mouthwashes on the composition and metabolic activity of oral biofilms grown in vitro . Clin Oral Investig . 2016 ; 21 : 1221 – 1230 . Google Scholar CrossRef Search ADS PubMed 83. Paster BJ , Olsen I , Aas JA , Dewhirst FE . The breadth of bacterial diversity in the human periodontal pocket and other oral sites . Periodontol 2000 . 2006 ; 42 : 80 – 87 . Google Scholar CrossRef Search ADS PubMed 84. Arzmi MH , Dashper S , Catmull D , Cirillo N , Reynolds EC , McCullough M . Coaggregation of Candida albicans, Actinomyces naeslundii and Streptococcus mutans is Candida albicans strain dependent . FEMS Yeast Res . 2015 ; 15 : fov038 . Google Scholar CrossRef Search ADS PubMed 85. Sobue T , Diaz P , Xu H , Bertolini M , Dongari-Bagtzoglou A . Experimental models of C. albicans-Streptococcal co-infection . Methods Mol Biol Clifton NJ . 2016 ; 1356 : 137 – 152 . Google Scholar CrossRef Search ADS 86. Pereira-Cenci T , Deng DM , Kraneveld EA et al. The effect of Streptococcus mutans and Candida glabrata on Candida albicans biofilms formed on different surfaces . Arch Oral Biol . 2008 ; 53 : 755 – 764 . Google Scholar CrossRef Search ADS PubMed 87. Thein ZM , Samaranayake YH , Samaranayake LP . Effect of oral bacteria on growth and survival of Candida albicans biofilms . Arch Oral Biol . 2006 ; 51 : 672 – 680 . Google Scholar CrossRef Search ADS PubMed 88. Bamford CV , d’Mello A , Nobbs AH , Dutton LC , Vickerman MM , Jenkinson HF . Streptococcus gordonii modulates Candida albicans biofilm formation through intergeneric communication . Infect Immun . 2009 ; 77 : 3696 – 3704 . Google Scholar CrossRef Search ADS PubMed 89. van Leeuwen PT , van der Peet JM , Bikker FJ et al. Interspecies interactions between Clostridium difficile and Candida albicans . mSphere . 2016 ; 1 : e00187 – 16 . Google Scholar CrossRef Search ADS PubMed 90. Ramírez Granillo A , Canales MGM , Espíndola MES , Martínez Rivera MA , de Lucio VMB , Tovar AVR . Antibiosis interaction of Staphylococccus aureus on Aspergillus fumigatus assessed in vitro by mixed biofilm formation . BMC Microbiol . 2015 ; 15 : 33 . Google Scholar CrossRef Search ADS PubMed 91. Exterkate RA , Crielaard W , Ten Cate JM. Different response to amine fluoride by Streptococcus mutans and polymicrobial biofilms in a novel high-throughput active attachment model . Caries Res . 2010 ; 44 : 372 – 379 . Google Scholar CrossRef Search ADS PubMed 92. Krishnamurthy A , Kyd J . The roles of epithelial cell contact, respiratory bacterial interactions and phosphorylcholine in promoting biofilm formation by Streptococcus pneumoniae and nontypeable Haemophilus influenzae . Microbes Infect . 2014 ; 16 : 640 – 647 . Google Scholar CrossRef Search ADS PubMed 93. Townsend EM , Sherry L , Rajendran R et al. Development and characterisation of a novel three-dimensional inter-kingdom wound biofilm model . Biofouling . 2016 ; 32 : 1259 – 1270 . Google Scholar CrossRef Search ADS PubMed 94. Sampaio FC , Pereira M , do SV , Dias CS , Costa VCO , Conde NCO , Buzalaf MAR . In vitro antimicrobial activity of Caesalpinia ferrea Martius fruits against oral pathogens . J Ethnopharmacol . 2009 ; 124 : 289 – 294 . Google Scholar CrossRef Search ADS PubMed 95. Ammann TW , Gmür R , Thurnheer T . Advancement of the 10-species subgingival Zurich biofilm model by examining different nutritional conditions and defining the structure of the in vitro biofilms . BMC Microbiol . 2012 ; 12 : 227 . Google Scholar CrossRef Search ADS PubMed 96. Kolenbrander PE. Multispecies communities: interspecies interactions influence growth on saliva as sole nutritional source . Int J Oral Sci . 2011 ; 3 : 49 – 54 . Google Scholar CrossRef Search ADS PubMed 97. Thurnheer T , Belibasakis GN , Bostanci N . Colonisation of gingival epithelia by subgingival biofilms in vitro: role of “red complex” bacteria . Arch Oral Biol . 2014 ; 59 : 977 – 986 . Google Scholar CrossRef Search ADS PubMed 98. Shen Y , Stojicic S , Haapasalo M . Bacterial viability in starved and revitalized biofilms: comparison of viability staining and direct culture . J Endod . 2010 ; 36 : 1820 – 1823 . Google Scholar CrossRef Search ADS PubMed 99. Eick S , Markauskaite G , Nietzsche S , Laugisch O , Salvi GE , Sculean A . Effect of photoactivated disinfection with a light-emitting diode on bacterial species and biofilms associated with periodontitis and peri-implantitis. Photodiagnosis Photodyn Ther . 2013 ; 10 : 156 – 167 . Google Scholar CrossRef Search ADS PubMed 100. Filoche SK , Soma KJ , Sissons CH . Caries-related plaque microcosm biofilms developed in microplates . Oral Microbiol Immunol . 2007 ; 22 : 73 – 79 . Google Scholar CrossRef Search ADS PubMed 101. Mistry KS , Sanghvi Z , Parmar G , Shah S , Pushpalatha K . Antibacterial efficacy of Azadirachta indica, Mimusops elengi and 2% CHX on multispecies dentinal biofilm . J Conserv Dent . 2015 ; 18 : 461 – 466 . Google Scholar CrossRef Search ADS PubMed 102. Extremina CI , Costa L , Aguiar AI , Peixe L , Fonseca AP . Optimization of processing conditions for the quantification of enterococci biofilms using microtitre-plates. J Microbiol Methods . 2011 ; 84 : 167 – 173 . Google Scholar CrossRef Search ADS PubMed 103. Cavalcanti YW , Morse DJ , da Silva WJ et al. Virulence and pathogenicity of Candida albicans is enhanced in biofilms containing oral bacteria . Biofouling . 2015 ; 31 : 27 – 38 . Google Scholar CrossRef Search ADS PubMed 104. Standar K , Kreikemeyer B , Redanz S , Munter WL , Laue M , Podbielski A . Setup of an in vitro test system for basic studies on biofilm behavior of mixed-species cultures with dental and periodontal pathogens . PLoS ONE . 2010 ; 5 : e13135 . Google Scholar CrossRef Search ADS PubMed 105. Schlafer S , Raarup MK , Wejse PL et al. Osteopontin reduces biofilm formation in a multi-species model of dental biofilm . PLoS ONE . 2012 ; 7 : e41534 . Google Scholar CrossRef Search ADS PubMed 106. Chávez de Paz LE . Development of a multispecies biofilm community by four root canal bacteria . J Endod. 2012 ; 38 : 318 – 323 . Google Scholar CrossRef Search ADS PubMed 107. Luppens SBI , Kara D , Bandounas L et al. Effect of Veillonella parvula on the antimicrobial resistance and gene expression of Streptococcus mutans grown in a dual-species biofilm . Oral Microbiol Immunol . 2008 ; 23 : 183 – 189 . Google Scholar CrossRef Search ADS PubMed 108. Cao Y-Y , Cao Y-B , Xu Z et al. cDNA microarray analysis of differential gene expression in Candida albicans biofilm exposed to farnesol . Antimicrob Agents Chemother . 2005 ; 49 : 584 – 589 . Google Scholar CrossRef Search ADS PubMed © The Author(s) 2017. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Medical Mycology Oxford University Press

Oral fungal-bacterial biofilm models in vitro: a review

Medical Mycology , Volume 56 (6) – Aug 1, 2018

Loading next page...
 
/lp/oxford-university-press/oral-fungal-bacterial-biofilm-models-in-vitro-a-review-JiEUPL1agR

References (111)

Publisher
Oxford University Press
Copyright
© The Author(s) 2017. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology.
ISSN
1369-3786
eISSN
1460-2709
DOI
10.1093/mmy/myx111
pmid
29228383
Publisher site
See Article on Publisher Site

Abstract

Abstract Inclusion of fungi as commensals in oral biofilm is an important innovation in oral biology, and this work aimed to review the literature on the available biofilm and related disease in vitro models. Actually, thousands of bacterial and around one hundred of fungal phylotypes can colonize the oral cavity. Taxonomic profiling combined with functional expression analysis has revealed that Candida albicans, Streptococcus mutans and prominent periodontopathogens are not always present or numerically important in candidiasis, caries, or periodontitis lesions. However, C. albicans combined with Streptococcus spp. co-increase their virulence in invasive candidiasis, early childhood caries or peri-implantitis. As Candida species and many other fungi are also members of oral microcosms in healthy individuals, mixed fungal-bacterial biofilm models are increasingly valuable investigative tools, and new fungal-bacterial species combinations need to be investigated. Here we review the key points and current methods for culturing in vitro mixed fungal-bacterial models of oral biofilms. According to ecosystem under study (health, candidiasis, caries, periodontitis), protocol design will select microbial strains, biofilm support (polystyrene plate, cell culture, denture, tooth, implant), pre-treatment support (human or artificial saliva) and culture conditions. Growing mixed fungal-bacterial biofilm models in vitro is a difficult challenge. But reproducible models are needed, because oral hygiene products, food and beverage, medication, licit and illicit drugs can influence oral ecosystems. So, even though most oral fungi and bacteria are not cultivable, in vitro microbiological models should still be instrumental in adapting oral care products, dietary products and care protocols to patients at higher risk of oral diseases. Microbial biofilm models combined with oral epithelial cell cultures could also aid in understanding the inflammatory reaction. biofilms, Candida, co-culture, oral ecosystem, Streptococcus Introduction Inclusion of fungi as commensals in oral biofilm is an important innovation in oral biology, and this work aimed to review the literature on the available biofilm and related disease in vitro models. Actually, metagenomics and proteomics screening have revealed that thousands of bacterial and around one hundred of fungal phylotypes can colonise the oral cavity.1 Microbial communities colonising the mouth grow in a biofilm with a protective extracellular matrix. Specific biofilms colonise soft and hard oral surfaces, but all microbiome members are not biofilm formers. In addition to bacteria and fungi, they also contain archaea, parasites, and viruses infecting oral epithelia or vectored via saliva and respiratory secretions.2–7 In oral health, biofilms modulate the host immune system, which in turn tolerates them.8 Commensal bacteria and fungi and their polymeric and hydrated matrix constitute a first-line defense against pathogenic microorganisms. Any breakdown favors local infections (gingivitis and periodontitis, dental caries and endodontic infections, oral candidiasis, mucositis, peri-implantitis) as well as aspiration pneumonia and blood-borne infections (infectious endocarditis, deep abscesses).9–12 Oral infections are often complicated by oral pain and tooth loss, increasing the risk of anorexia and malnutrition.13 Evidence points to an association between oral infections, the resulting inflammation, and systemic diseases such as diabetes mellitus, rheumatoid arthritis, neurodegenerative diseases (Alzheimer's disease), atherosclerosis, cardiovascular disease, and stroke.12,14,15 Recent research has underlined the role of inter-kingdom microbial synergies or antagonisms in biofilms in health and oral diseases.16 For instance, oral Actinomyces and Lactobacillus spp. can inhibit Candida albicans biofilm formation.16,17 Conversely, C. albicans combined with Streptococcus spp. can co-increase their virulence in invasive candidiasis, early childhood caries or peri-implantitis.18–22 In vivo, the dental plaque typically presents a high microbial density, with approximately 1011 cells/g (wet weight). Approximately 700 oral bacterial species could be isolated and grown in vitro, and any given individual generally harbors 100 or more cultivable bacterial strains in its mouth.23 However, screening of various oral ecosystems has revealed that more than 19,000 noncultivable bacterial phylotypes could also colonise the human oral cavity.24–27 Similarly, more than 100 fungal species have been identified in the oral microbiome, most of them noncultivable.1,3,28 Animal models have been developed using mice, rats, and worms to mimic the human oral microcosm, but human oral ecosystems are different from animal ecosystems.17,19,29–31 In order to get closer to in vivo conditions, some biofilm models are grown with human epithelial cell cultures.18,32 In the model described by Ramage et al., the biofilm actually grows at a 0.5 mm distance from an epithelial monolayer.33 It is very likely that many noncultivable species play a role in health and disease.1,10,23,34 Among cultivable species, taxonomic profiling combined with functional expression analysis has recently revealed that C. albicans, Streptococcus mutans, and prominent periodontopathogens were not always present or numerically important in candidiasis, caries or periodontitis lesions.3,5,10,19,28 Thus, metagenomics and proteomics screening are driving new trends in oral disease prevention, diagnosis and treatment efficacy control based on individual follow-up.8,26,35–37 Future therapies will aim to replace cariogenic and periodontopathogenic microbiomes with the initial healthy microbiome of each subject.6,8,26,35 However, there is a need for reproducible in vitro models of mixed fungal and bacterial biofilms whenever it is necessary to compare different growth conditions or inhibitory substances at different concentrations.38–44 Multispecies culture with both fungal and bacterial strains is difficult because, in contrast to oral fungus Candida, most oral bacteria are slow-growing, nutritionally fastidious, and oxygen-sensitive. Oxygen is very important for Candida growth as well. Anaerobically, it grows yet very slowly, and it is out-competed by many bacterial species.45–47 In mixed biofilm models, strains can have two origins: some biofilms are grown with a defined consortium of bacteria and fungi, while others, called microcosms, are grown from saliva samples from donors. Co-cultures in defined consortiums are reproducible and easier to study because all the strains are known.48,49 In contrast, saliva sampling is closer to real-world conditions in terms of number and respective proportions of microbial species. One important advantage of using saliva is that the species and strains in the inoculum are better adapted to each other. Strain differences are probably important in dictating compatibility between species and kingdoms. However, saliva sampling suffers a lack of reproducibility due to the different saliva donors used.30,50–52 Moreover, only a limited number of bacterial and fungal species can be grown in vitro, and biofilms composition always differs from the initial inoculum.53,54 Choice of microbial strains or inoculum is pivotal. Co-culture conditions are critical too, as aerobic fungi are grown with strict anaerobic bacteria, hyphal formation is often required, and overgrowth of any one species is to be avoided. Here we review the key points and current methods for designing and culturing oral multispecies biofilms in vitro. Search criteria References in English were identified through PubMed and Science Direct searches for articles published since Jan 1, 2005. At first, the search terms used were “bacteria OR bacterial OR Streptococcus OR anaerobic bacteria” AND “fungi OR Candida” AND “biofilms OR models OR in vitro techniques.” Then second, search terms used were “dental caries OR periodontitis OR gingivitis OR abscess OR peri-implantitis OR denture stomatitis OR candidiasis OR mouth diseases OR oral health” AND “biofilms OR models OR in vitro techniques.” Finally, we retained only the studies with an in vitro model of bacterial and fungal cultures, in connection with the oral health or the diseases of the mouth. Stages of oral biofilm development In vivo and in vitro, biofilm development follows five stages: (1) adhesion to hard or soft tissues (adhesins and extracellular polysaccharides); (2) growth (microbial co-adhesion and co-aggregation, matrix formation); (3) maturation characterised by metabolic and genetic microbial exchanges, growth control by quorum-sensing molecules (auto-inducers) and antimicrobial peptides (bacteriocins); (4) tissue invasion/destruction (toxic metabolites and enzymes); and 5) surface detachment (enzymes). 21,35,55–57 Early biofilms Early microbial colonisers specifically adhere to cellular and to salivary receptors, such as mucins, proline-rich protein, statherin, salivary agglutinin (gp-340) and α-amylase.58,59 Organic salivary compounds are adsorbed to epithelial surfaces or hard surfaces (enamel, dentin, calculus, and restorative or prosthetic materials), and then provide static receptors for early colonizers.60–62 The salivary film coating dental enamel is called acquired salivary pellicle.62 The initial adhesion stage of oral bacteria lasts only a few seconds and is reversible. There are both nonspecific surface forces and recognition between microbial adhesins and their receptors. Hydrodynamic forces create either repulsive or attractive nonspecific connections via low-energy interactions (electrostatic, steric, hydrophobic, Van de Waals). Next step, the irreversible adhesion process is slower. Its duration depends on the microbial strains, its population density and the duration of its exponential growth phase.63,64 The taxonomic bacterial profile of early dental plaque, based on genomic data, has been recently detailed.23 A critical step is the choice of biofilm support (polystyrene plate, cell culture, acrylic resin for denture, tooth hydroxyapatite crystals, complete bovine or human teeth, titanium implant…) and pretreatment support (human or artificial saliva), adapted to early colonizers. Biofilm growth and maturation can take hours or days depending on the microbial species and environmental conditions involved, and, importantly, the frequency of nutrient replenishment. Bacteria and fungi multiply, colonise the support, and form aggregates (or microcolonies) that become confluent. The production of extracellular polymers varies according to microbial communities, local environment, and the biofilm maturity.65,66 Mature biofilms Mature biofilms are aggregates of microorganisms growing within an extracellular matrix. In vivo, Candida and streptococci form corn-cob-like structures.24 The matrix contains microbial metabolites, dead microbial and host cells (desquamated epithelial cells), other host components (mainly fibronectin, laminin, collagen and salivary constituents), food nutrients (sugars), and possibly also drugs. The matrix is well hydrated and crossed by channels conveying oxygen, nutrients and metabolites.31,67 Maturation of mixed biofilms depends on oxygen availability and metabolic interactions. End-chain products may be nutrients of different fungal and bacterial species, which may be either partners or competitors.68Streptococcus oralis combined with C. albicans synergistically increases both biofilm formation and virulence factor expression.18 In early-childhood caries, the presence of C. albicans and sucrose (but not glucose) synergistically increases S. mutans virulence, resulting in rapid onset of extensive caries lesions. C. albicans can tolerate acids in dental caries lesions.19,20,69 However, in a recent report, Willems and coworkers showed that while growth of S. muttons is increased by presence of Candida, and lactate production is also increased, the environmental pH increases to above the critical pH and Ca2+ release from hydroxyapatite disks is inhibited by the presence of Candida.70 Microorganisms are able to perceive various environmental parameters, either abiotic, such as physical-chemical signals (pH, osmolality, temperature), or biotic, such as signaling proteins.19,26,35,71–73 When signalling molecules reach a sufficient concentration, they can communicate and coordinate the formation of biofilm or the synthesis of virulence factors.71–73In vivo, quorum sensing coordinates the evolution from colonization stage to acute infection stage. In vitro, farnesol promotes the formation of Staphylococcus aureus biofilm at low levels (0.5–5 nM) and inhibits S. aureus growth at higher concentration (180 μM).57 In addition, some microbial species can produce antimicrobial peptides, although their direct microbe-killing effect is prevented in physiological conditions where they probably act as immunomodulators.73 In vitro, some studies aim to inhibit biofilm formation or to assay pre-formed biofilms, leading to models of early21,31,64 and mature biofilms,74 with corresponding culture duration, sugar pulses in caries models,75 sequential addition of strains, and progressive anaerobic conditions in peri-implantitis models.49 Late stages The invasion and destruction of soft tissues is mediated by microbial diffusible enzymes, such as lipases, proteases, nucleases and ureases. Enzymatic dissolution of mucosal barriers can also take place by activation of host enzymes triggered by C. albicans, for example, calpain.76 The destruction and invasion of hard dental tissues that results in tooth cavities starts with localised acid decalcification of the hydroxyapatite crystals constituting enamel and dentin, followed by an enzymatic lysis of organic structures.20 In vivo, the healthy adult's biofilm is bathed by a saliva flow of approximately 0.35 ml/min, which provides nutrients, moistens the mucus membranes, eliminates part of the bacteria and buffers the pH resulting in remineralization of the tooth surface.32,50 The biofilm's thickness is mechanically controlled by salivary flow, tongue and jaw movements, and by chewing solid food. The turnover of epithelial cells and the immune system (in saliva and epithelia) protect soft mucosal surfaces. In contrast, there is no cell turnover on the hard surfaces of teeth, calculus, dentures, and dental biomaterials.18,25 The late stage of biofilm development is the detachment of microbial cells or aggregates, which then colonise saliva and other supports. Detachment depends on support, microbial community, nutrient availability, hydrodynamics and physical-chemical conditions of the environment. Microbial aggregate detachment is facilitated by the lysis of extracellular polymers, such as by the production of dextranases by S. mutans and glucanases by C. albicans.65,77In vitro, sophisticated flux systems enable continuous renewal of the culture medium.18,30,51,78 Choice of microbial strains Consortium models Current consortium models contain cultivable species representative of ecosystems colonising oral mucosa, teeth, dentures, and peri-implantitis pockets. They can combine fungal and bacterial reference strains, wild-type strains or mutant strains. The number of species ranges from two31,69,79,80 to 11 strains.74 To our knowledge, neither archaea, viruses nor parasites have been used in multispecies bacterial biofilm models. Examples of consortium models combining fungal and bacterial strains are listed in Tables 1 and 2. Table 1. Oral candidiasis models. References Authors Model Microbial strains Supports Culture apparatus Main objective Assessment methods 18 Diaz 2012 Oral candidiasis Candida albicans Streptococcus gordonii Streptococcus oralis Streptococcus sanguinis Reconstructed oral and pharyngeal epithelium Flux system To demonstrate a synergistic interaction between commensal oral streptococci and Candida albicans Histology (FISH) CLSM RT-qPCR 21 Park 2014 Oral candidiasis Candida albicans Escherichia coli Proteus vulgaris Pseudomonas aeruginosa Staphylococcus aureus Streptococcus pyogenes Streptococcus salivarius Polystyrene plate 96-well plate To examine the influence of bacterial presence on biofilm formation of Candida albicans XTT cell viability assay SEM RT-qPCR 79 Schlecht 2015 Oral candidiasis Candida albicans Staphylococcus aureus Plastic Permanox slides 6-well plate To analyse how Staphylococcus aureus infection is mediated by Candida albicans hyphal invasion of mucosal tissue Phase-contrast microscopy Fluorescence microscopy CLSM 64 Matsubara 2016 Oral candidiasis Candida albicans Lactobacillus acidophilus Lactobacillus casei Lactobacillus rhamnosus Polystyrene plate 96-well plate To evaluate the inhibitory effects of the probiotic Lactobacillus species on different phases of Candida albicans biofilm development cfu count XTT cell viability assay SEM CLSM 32 Bertolini 2015 Xerostomia Candida albicans (and mutant strains) Streptococcus oralis Reconstructed oral epithelium Tissue-culture plate (not detailed) To analyse the structural and virulence characteristics of Candida-streptococcal mixed-biofilm models on reconstructed oral epithelium, in different conditions of moisture and nutrient availability CLSM Histology (FISH) 50 de Moraes 2012 Denture stomatitis Saliva microcosm Acrylic resin, soft denture liner 24-well plate To assess the antimicrobial activity of a triazine derivative added into denture liners cfu count 31 Barbosa 2016 Denture stomatitis Candida albicans Streptococcus mutans Polystyrene plate Acrylic resin 24-well plate To study the effects of Streptococcus mutans on biofilm formation and morphology of Candida albicans pH measure cfu count Bright-field microscopy: crystal violet and hyphae quantification SEM 74 Sherry 2016 Denture stomatitis Candida albicans Aggregatibacter actinomycetemcomitans Actinomyces naeslundii Fusobacterium nucleatum Fusobacterium nucleatum spp. vincentii Porphyromonas gingivalis Prevotella intermedia Streptococcus intermedius Streptococcus oralis Streptococcus mitis Veillonella dispar Acrylic resin 24-well plate To assess the activity of three protocols for cleaning removable dentures cfu count SEM CLSM Live-Dead PCR RT-qPCR 103 Cavalcanti 2015 Denture stomatitis Candida albicans Streptococcus mutans Streptococcus sanguinis Actinomyces viscosus Actinomyces odontolyticus Acrylic coupons Reconstructed human oral epithelium 24-well plate To examine the effect of a bacterial component on the virulence and pathogenicity of Candida biofilms and to assess epithelial cell responses to the different biofilm compositions Histology (FISH) CLSM RT-qPCR 78 Yassin 2016 Denture stomatitis Candida albicans Lactobacillus casei Streptococcus mutans Acrylic resin Flux system To assess the antimicrobial properties of a fluoride-releasing denture resin pH measure SEM CLSM RT-qPCR References Authors Model Microbial strains Supports Culture apparatus Main objective Assessment methods 18 Diaz 2012 Oral candidiasis Candida albicans Streptococcus gordonii Streptococcus oralis Streptococcus sanguinis Reconstructed oral and pharyngeal epithelium Flux system To demonstrate a synergistic interaction between commensal oral streptococci and Candida albicans Histology (FISH) CLSM RT-qPCR 21 Park 2014 Oral candidiasis Candida albicans Escherichia coli Proteus vulgaris Pseudomonas aeruginosa Staphylococcus aureus Streptococcus pyogenes Streptococcus salivarius Polystyrene plate 96-well plate To examine the influence of bacterial presence on biofilm formation of Candida albicans XTT cell viability assay SEM RT-qPCR 79 Schlecht 2015 Oral candidiasis Candida albicans Staphylococcus aureus Plastic Permanox slides 6-well plate To analyse how Staphylococcus aureus infection is mediated by Candida albicans hyphal invasion of mucosal tissue Phase-contrast microscopy Fluorescence microscopy CLSM 64 Matsubara 2016 Oral candidiasis Candida albicans Lactobacillus acidophilus Lactobacillus casei Lactobacillus rhamnosus Polystyrene plate 96-well plate To evaluate the inhibitory effects of the probiotic Lactobacillus species on different phases of Candida albicans biofilm development cfu count XTT cell viability assay SEM CLSM 32 Bertolini 2015 Xerostomia Candida albicans (and mutant strains) Streptococcus oralis Reconstructed oral epithelium Tissue-culture plate (not detailed) To analyse the structural and virulence characteristics of Candida-streptococcal mixed-biofilm models on reconstructed oral epithelium, in different conditions of moisture and nutrient availability CLSM Histology (FISH) 50 de Moraes 2012 Denture stomatitis Saliva microcosm Acrylic resin, soft denture liner 24-well plate To assess the antimicrobial activity of a triazine derivative added into denture liners cfu count 31 Barbosa 2016 Denture stomatitis Candida albicans Streptococcus mutans Polystyrene plate Acrylic resin 24-well plate To study the effects of Streptococcus mutans on biofilm formation and morphology of Candida albicans pH measure cfu count Bright-field microscopy: crystal violet and hyphae quantification SEM 74 Sherry 2016 Denture stomatitis Candida albicans Aggregatibacter actinomycetemcomitans Actinomyces naeslundii Fusobacterium nucleatum Fusobacterium nucleatum spp. vincentii Porphyromonas gingivalis Prevotella intermedia Streptococcus intermedius Streptococcus oralis Streptococcus mitis Veillonella dispar Acrylic resin 24-well plate To assess the activity of three protocols for cleaning removable dentures cfu count SEM CLSM Live-Dead PCR RT-qPCR 103 Cavalcanti 2015 Denture stomatitis Candida albicans Streptococcus mutans Streptococcus sanguinis Actinomyces viscosus Actinomyces odontolyticus Acrylic coupons Reconstructed human oral epithelium 24-well plate To examine the effect of a bacterial component on the virulence and pathogenicity of Candida biofilms and to assess epithelial cell responses to the different biofilm compositions Histology (FISH) CLSM RT-qPCR 78 Yassin 2016 Denture stomatitis Candida albicans Lactobacillus casei Streptococcus mutans Acrylic resin Flux system To assess the antimicrobial properties of a fluoride-releasing denture resin pH measure SEM CLSM RT-qPCR cfu, colony-forming unit; CLSM, confocal laser scanning electron microscopy; FISH, fluorescence in situ hybridisation; (RT)-Qpcr, quantitative (reverse transcription) polymerase chain reaction; SEM, scanning electron microscopy; XTT, (2,3-Bis-(2-Methoxy-4-Nitro-5-Sulfophenyl)-2H-Tetrazolium-5-Carboxanilide). View Large Table 1. Oral candidiasis models. References Authors Model Microbial strains Supports Culture apparatus Main objective Assessment methods 18 Diaz 2012 Oral candidiasis Candida albicans Streptococcus gordonii Streptococcus oralis Streptococcus sanguinis Reconstructed oral and pharyngeal epithelium Flux system To demonstrate a synergistic interaction between commensal oral streptococci and Candida albicans Histology (FISH) CLSM RT-qPCR 21 Park 2014 Oral candidiasis Candida albicans Escherichia coli Proteus vulgaris Pseudomonas aeruginosa Staphylococcus aureus Streptococcus pyogenes Streptococcus salivarius Polystyrene plate 96-well plate To examine the influence of bacterial presence on biofilm formation of Candida albicans XTT cell viability assay SEM RT-qPCR 79 Schlecht 2015 Oral candidiasis Candida albicans Staphylococcus aureus Plastic Permanox slides 6-well plate To analyse how Staphylococcus aureus infection is mediated by Candida albicans hyphal invasion of mucosal tissue Phase-contrast microscopy Fluorescence microscopy CLSM 64 Matsubara 2016 Oral candidiasis Candida albicans Lactobacillus acidophilus Lactobacillus casei Lactobacillus rhamnosus Polystyrene plate 96-well plate To evaluate the inhibitory effects of the probiotic Lactobacillus species on different phases of Candida albicans biofilm development cfu count XTT cell viability assay SEM CLSM 32 Bertolini 2015 Xerostomia Candida albicans (and mutant strains) Streptococcus oralis Reconstructed oral epithelium Tissue-culture plate (not detailed) To analyse the structural and virulence characteristics of Candida-streptococcal mixed-biofilm models on reconstructed oral epithelium, in different conditions of moisture and nutrient availability CLSM Histology (FISH) 50 de Moraes 2012 Denture stomatitis Saliva microcosm Acrylic resin, soft denture liner 24-well plate To assess the antimicrobial activity of a triazine derivative added into denture liners cfu count 31 Barbosa 2016 Denture stomatitis Candida albicans Streptococcus mutans Polystyrene plate Acrylic resin 24-well plate To study the effects of Streptococcus mutans on biofilm formation and morphology of Candida albicans pH measure cfu count Bright-field microscopy: crystal violet and hyphae quantification SEM 74 Sherry 2016 Denture stomatitis Candida albicans Aggregatibacter actinomycetemcomitans Actinomyces naeslundii Fusobacterium nucleatum Fusobacterium nucleatum spp. vincentii Porphyromonas gingivalis Prevotella intermedia Streptococcus intermedius Streptococcus oralis Streptococcus mitis Veillonella dispar Acrylic resin 24-well plate To assess the activity of three protocols for cleaning removable dentures cfu count SEM CLSM Live-Dead PCR RT-qPCR 103 Cavalcanti 2015 Denture stomatitis Candida albicans Streptococcus mutans Streptococcus sanguinis Actinomyces viscosus Actinomyces odontolyticus Acrylic coupons Reconstructed human oral epithelium 24-well plate To examine the effect of a bacterial component on the virulence and pathogenicity of Candida biofilms and to assess epithelial cell responses to the different biofilm compositions Histology (FISH) CLSM RT-qPCR 78 Yassin 2016 Denture stomatitis Candida albicans Lactobacillus casei Streptococcus mutans Acrylic resin Flux system To assess the antimicrobial properties of a fluoride-releasing denture resin pH measure SEM CLSM RT-qPCR References Authors Model Microbial strains Supports Culture apparatus Main objective Assessment methods 18 Diaz 2012 Oral candidiasis Candida albicans Streptococcus gordonii Streptococcus oralis Streptococcus sanguinis Reconstructed oral and pharyngeal epithelium Flux system To demonstrate a synergistic interaction between commensal oral streptococci and Candida albicans Histology (FISH) CLSM RT-qPCR 21 Park 2014 Oral candidiasis Candida albicans Escherichia coli Proteus vulgaris Pseudomonas aeruginosa Staphylococcus aureus Streptococcus pyogenes Streptococcus salivarius Polystyrene plate 96-well plate To examine the influence of bacterial presence on biofilm formation of Candida albicans XTT cell viability assay SEM RT-qPCR 79 Schlecht 2015 Oral candidiasis Candida albicans Staphylococcus aureus Plastic Permanox slides 6-well plate To analyse how Staphylococcus aureus infection is mediated by Candida albicans hyphal invasion of mucosal tissue Phase-contrast microscopy Fluorescence microscopy CLSM 64 Matsubara 2016 Oral candidiasis Candida albicans Lactobacillus acidophilus Lactobacillus casei Lactobacillus rhamnosus Polystyrene plate 96-well plate To evaluate the inhibitory effects of the probiotic Lactobacillus species on different phases of Candida albicans biofilm development cfu count XTT cell viability assay SEM CLSM 32 Bertolini 2015 Xerostomia Candida albicans (and mutant strains) Streptococcus oralis Reconstructed oral epithelium Tissue-culture plate (not detailed) To analyse the structural and virulence characteristics of Candida-streptococcal mixed-biofilm models on reconstructed oral epithelium, in different conditions of moisture and nutrient availability CLSM Histology (FISH) 50 de Moraes 2012 Denture stomatitis Saliva microcosm Acrylic resin, soft denture liner 24-well plate To assess the antimicrobial activity of a triazine derivative added into denture liners cfu count 31 Barbosa 2016 Denture stomatitis Candida albicans Streptococcus mutans Polystyrene plate Acrylic resin 24-well plate To study the effects of Streptococcus mutans on biofilm formation and morphology of Candida albicans pH measure cfu count Bright-field microscopy: crystal violet and hyphae quantification SEM 74 Sherry 2016 Denture stomatitis Candida albicans Aggregatibacter actinomycetemcomitans Actinomyces naeslundii Fusobacterium nucleatum Fusobacterium nucleatum spp. vincentii Porphyromonas gingivalis Prevotella intermedia Streptococcus intermedius Streptococcus oralis Streptococcus mitis Veillonella dispar Acrylic resin 24-well plate To assess the activity of three protocols for cleaning removable dentures cfu count SEM CLSM Live-Dead PCR RT-qPCR 103 Cavalcanti 2015 Denture stomatitis Candida albicans Streptococcus mutans Streptococcus sanguinis Actinomyces viscosus Actinomyces odontolyticus Acrylic coupons Reconstructed human oral epithelium 24-well plate To examine the effect of a bacterial component on the virulence and pathogenicity of Candida biofilms and to assess epithelial cell responses to the different biofilm compositions Histology (FISH) CLSM RT-qPCR 78 Yassin 2016 Denture stomatitis Candida albicans Lactobacillus casei Streptococcus mutans Acrylic resin Flux system To assess the antimicrobial properties of a fluoride-releasing denture resin pH measure SEM CLSM RT-qPCR cfu, colony-forming unit; CLSM, confocal laser scanning electron microscopy; FISH, fluorescence in situ hybridisation; (RT)-Qpcr, quantitative (reverse transcription) polymerase chain reaction; SEM, scanning electron microscopy; XTT, (2,3-Bis-(2-Methoxy-4-Nitro-5-Sulfophenyl)-2H-Tetrazolium-5-Carboxanilide). View Large Table 2. Caries and periodontitis models. Referen-ces Authors Model Microbial strains Supports Culture apparatus Main objective Assessment methods 75 Reese 2007 Dental plaque Candida albicans Actinomyces naeslundii Fusobacterium nucleatum Streptococcus oralis Streptococcus sobrinus Veillonella dispar Bovine enamel 24-well plate To observe the biofilm matrix in the dental plaque, before and after glucose-sucrose feeding TEM 86 Pereira-Cenci 2008 Dental plaque Candida albicans Candida glabrata Streptococcus mutans Hydroxyapatite Acrylic resin Soft denture liner 24-well plate To investigate how oral bacteria modulate the development and characteristics of Candida biofilms cfu count SEM CLSM 94 Sampaio 2009 Dental plaque Candida albicans Lactobacillus casei Streptococcus mutans Streptococcus oralis Streptococcus salivarius Bovine enamel Propylene tubes To assess the antimicrobial activity of a plant extract cfu count 80 Montelongo-Jauregui 2016 Dental plaque Candida albicans Streptococcus gordonii Polystyrene plate 96-well plate To assess the activity of antifungal and antibacterial drugs Bright-field microscopy: crystal violet Fluorescent plate reader: Presto Blue™ SEM CLSM 20 Falsetta 2014 Dental caries Candida albicans Streptococcus mutans (and mutant strains) Hydroxyapatite 24-well plate To study synergy (virulence) of Streptococcus mutans and Candida albicans in dental caries biofilm pH measure cfu count CLSM RT-qPCR 51 Koopman 2015 Dental caries Saliva microcosm Glass coverslips Multistation artificial mouth: drip system To assay the pH-raising effect of arginine pH measure RT-qPCR 101 Mistry 2015 Endodontic infection Candida albicans Enterococcus faecalis Staphylococcus aureus Streptococcus mutans Human dentin Eppendorf vials To assess the antibacterial activity of two plant extracts Cfu SEM 49 Thurnheer 2016 Periodontitis Candida albicans Actinomyces oris Campylobacter rectus Fusobacterium nucleatum Porphyromonas gingivalis Prevotella intermedia Streptococcus anginosus Streptococcus oralis Streptococcus mutans Tannerella forsythia Treponema denticola Veillonella dispar Hydroxyapatite 24-well plate To investigate changes in biofilm composition and structure during the shift from a ‘supragingival’ aerobic profile to a ‘subgingival’ anaerobic profile CLSM RT-qPCR 62 Cavalcanti 2014 Peri-implantitis Candida albicans Actinomyces naeslundii Fusobacterium nucleatum Streptococcus mutans Streptococcus oralis Veillonella dispar Titanium 24-well plate To assess surface properties of titanium disks nitrided by cold plasma Cfu SEM CLSM RT-qPCR 30 Sousa 2016 Peri-implantitis Saliva microcosm Titanium Flux system in vitro and secondly rabbit in vivo To assess three disinfection protocols with an experimental model for contamination of titanium surfaces cfu count Combined in vitro/in vivo animal model 69 Junka 2015 Bone destruction Candida albicans Streptococcus mutans Hydroxyapatite 24-well plate To assess bone hydroxyapatite destruction pH measure cfu count SEM CLSM Referen-ces Authors Model Microbial strains Supports Culture apparatus Main objective Assessment methods 75 Reese 2007 Dental plaque Candida albicans Actinomyces naeslundii Fusobacterium nucleatum Streptococcus oralis Streptococcus sobrinus Veillonella dispar Bovine enamel 24-well plate To observe the biofilm matrix in the dental plaque, before and after glucose-sucrose feeding TEM 86 Pereira-Cenci 2008 Dental plaque Candida albicans Candida glabrata Streptococcus mutans Hydroxyapatite Acrylic resin Soft denture liner 24-well plate To investigate how oral bacteria modulate the development and characteristics of Candida biofilms cfu count SEM CLSM 94 Sampaio 2009 Dental plaque Candida albicans Lactobacillus casei Streptococcus mutans Streptococcus oralis Streptococcus salivarius Bovine enamel Propylene tubes To assess the antimicrobial activity of a plant extract cfu count 80 Montelongo-Jauregui 2016 Dental plaque Candida albicans Streptococcus gordonii Polystyrene plate 96-well plate To assess the activity of antifungal and antibacterial drugs Bright-field microscopy: crystal violet Fluorescent plate reader: Presto Blue™ SEM CLSM 20 Falsetta 2014 Dental caries Candida albicans Streptococcus mutans (and mutant strains) Hydroxyapatite 24-well plate To study synergy (virulence) of Streptococcus mutans and Candida albicans in dental caries biofilm pH measure cfu count CLSM RT-qPCR 51 Koopman 2015 Dental caries Saliva microcosm Glass coverslips Multistation artificial mouth: drip system To assay the pH-raising effect of arginine pH measure RT-qPCR 101 Mistry 2015 Endodontic infection Candida albicans Enterococcus faecalis Staphylococcus aureus Streptococcus mutans Human dentin Eppendorf vials To assess the antibacterial activity of two plant extracts Cfu SEM 49 Thurnheer 2016 Periodontitis Candida albicans Actinomyces oris Campylobacter rectus Fusobacterium nucleatum Porphyromonas gingivalis Prevotella intermedia Streptococcus anginosus Streptococcus oralis Streptococcus mutans Tannerella forsythia Treponema denticola Veillonella dispar Hydroxyapatite 24-well plate To investigate changes in biofilm composition and structure during the shift from a ‘supragingival’ aerobic profile to a ‘subgingival’ anaerobic profile CLSM RT-qPCR 62 Cavalcanti 2014 Peri-implantitis Candida albicans Actinomyces naeslundii Fusobacterium nucleatum Streptococcus mutans Streptococcus oralis Veillonella dispar Titanium 24-well plate To assess surface properties of titanium disks nitrided by cold plasma Cfu SEM CLSM RT-qPCR 30 Sousa 2016 Peri-implantitis Saliva microcosm Titanium Flux system in vitro and secondly rabbit in vivo To assess three disinfection protocols with an experimental model for contamination of titanium surfaces cfu count Combined in vitro/in vivo animal model 69 Junka 2015 Bone destruction Candida albicans Streptococcus mutans Hydroxyapatite 24-well plate To assess bone hydroxyapatite destruction pH measure cfu count SEM CLSM cfu, colony forming unit; CLSM, confocal laser scanning electron microscopy; HIV, human immunodeficiency virus; RT-qPCR, quantitative reverse transcription polymerase chain reaction; SEM: scanning electron microscopy; TEM: transmission electron microscopy. View Large Table 2. Caries and periodontitis models. Referen-ces Authors Model Microbial strains Supports Culture apparatus Main objective Assessment methods 75 Reese 2007 Dental plaque Candida albicans Actinomyces naeslundii Fusobacterium nucleatum Streptococcus oralis Streptococcus sobrinus Veillonella dispar Bovine enamel 24-well plate To observe the biofilm matrix in the dental plaque, before and after glucose-sucrose feeding TEM 86 Pereira-Cenci 2008 Dental plaque Candida albicans Candida glabrata Streptococcus mutans Hydroxyapatite Acrylic resin Soft denture liner 24-well plate To investigate how oral bacteria modulate the development and characteristics of Candida biofilms cfu count SEM CLSM 94 Sampaio 2009 Dental plaque Candida albicans Lactobacillus casei Streptococcus mutans Streptococcus oralis Streptococcus salivarius Bovine enamel Propylene tubes To assess the antimicrobial activity of a plant extract cfu count 80 Montelongo-Jauregui 2016 Dental plaque Candida albicans Streptococcus gordonii Polystyrene plate 96-well plate To assess the activity of antifungal and antibacterial drugs Bright-field microscopy: crystal violet Fluorescent plate reader: Presto Blue™ SEM CLSM 20 Falsetta 2014 Dental caries Candida albicans Streptococcus mutans (and mutant strains) Hydroxyapatite 24-well plate To study synergy (virulence) of Streptococcus mutans and Candida albicans in dental caries biofilm pH measure cfu count CLSM RT-qPCR 51 Koopman 2015 Dental caries Saliva microcosm Glass coverslips Multistation artificial mouth: drip system To assay the pH-raising effect of arginine pH measure RT-qPCR 101 Mistry 2015 Endodontic infection Candida albicans Enterococcus faecalis Staphylococcus aureus Streptococcus mutans Human dentin Eppendorf vials To assess the antibacterial activity of two plant extracts Cfu SEM 49 Thurnheer 2016 Periodontitis Candida albicans Actinomyces oris Campylobacter rectus Fusobacterium nucleatum Porphyromonas gingivalis Prevotella intermedia Streptococcus anginosus Streptococcus oralis Streptococcus mutans Tannerella forsythia Treponema denticola Veillonella dispar Hydroxyapatite 24-well plate To investigate changes in biofilm composition and structure during the shift from a ‘supragingival’ aerobic profile to a ‘subgingival’ anaerobic profile CLSM RT-qPCR 62 Cavalcanti 2014 Peri-implantitis Candida albicans Actinomyces naeslundii Fusobacterium nucleatum Streptococcus mutans Streptococcus oralis Veillonella dispar Titanium 24-well plate To assess surface properties of titanium disks nitrided by cold plasma Cfu SEM CLSM RT-qPCR 30 Sousa 2016 Peri-implantitis Saliva microcosm Titanium Flux system in vitro and secondly rabbit in vivo To assess three disinfection protocols with an experimental model for contamination of titanium surfaces cfu count Combined in vitro/in vivo animal model 69 Junka 2015 Bone destruction Candida albicans Streptococcus mutans Hydroxyapatite 24-well plate To assess bone hydroxyapatite destruction pH measure cfu count SEM CLSM Referen-ces Authors Model Microbial strains Supports Culture apparatus Main objective Assessment methods 75 Reese 2007 Dental plaque Candida albicans Actinomyces naeslundii Fusobacterium nucleatum Streptococcus oralis Streptococcus sobrinus Veillonella dispar Bovine enamel 24-well plate To observe the biofilm matrix in the dental plaque, before and after glucose-sucrose feeding TEM 86 Pereira-Cenci 2008 Dental plaque Candida albicans Candida glabrata Streptococcus mutans Hydroxyapatite Acrylic resin Soft denture liner 24-well plate To investigate how oral bacteria modulate the development and characteristics of Candida biofilms cfu count SEM CLSM 94 Sampaio 2009 Dental plaque Candida albicans Lactobacillus casei Streptococcus mutans Streptococcus oralis Streptococcus salivarius Bovine enamel Propylene tubes To assess the antimicrobial activity of a plant extract cfu count 80 Montelongo-Jauregui 2016 Dental plaque Candida albicans Streptococcus gordonii Polystyrene plate 96-well plate To assess the activity of antifungal and antibacterial drugs Bright-field microscopy: crystal violet Fluorescent plate reader: Presto Blue™ SEM CLSM 20 Falsetta 2014 Dental caries Candida albicans Streptococcus mutans (and mutant strains) Hydroxyapatite 24-well plate To study synergy (virulence) of Streptococcus mutans and Candida albicans in dental caries biofilm pH measure cfu count CLSM RT-qPCR 51 Koopman 2015 Dental caries Saliva microcosm Glass coverslips Multistation artificial mouth: drip system To assay the pH-raising effect of arginine pH measure RT-qPCR 101 Mistry 2015 Endodontic infection Candida albicans Enterococcus faecalis Staphylococcus aureus Streptococcus mutans Human dentin Eppendorf vials To assess the antibacterial activity of two plant extracts Cfu SEM 49 Thurnheer 2016 Periodontitis Candida albicans Actinomyces oris Campylobacter rectus Fusobacterium nucleatum Porphyromonas gingivalis Prevotella intermedia Streptococcus anginosus Streptococcus oralis Streptococcus mutans Tannerella forsythia Treponema denticola Veillonella dispar Hydroxyapatite 24-well plate To investigate changes in biofilm composition and structure during the shift from a ‘supragingival’ aerobic profile to a ‘subgingival’ anaerobic profile CLSM RT-qPCR 62 Cavalcanti 2014 Peri-implantitis Candida albicans Actinomyces naeslundii Fusobacterium nucleatum Streptococcus mutans Streptococcus oralis Veillonella dispar Titanium 24-well plate To assess surface properties of titanium disks nitrided by cold plasma Cfu SEM CLSM RT-qPCR 30 Sousa 2016 Peri-implantitis Saliva microcosm Titanium Flux system in vitro and secondly rabbit in vivo To assess three disinfection protocols with an experimental model for contamination of titanium surfaces cfu count Combined in vitro/in vivo animal model 69 Junka 2015 Bone destruction Candida albicans Streptococcus mutans Hydroxyapatite 24-well plate To assess bone hydroxyapatite destruction pH measure cfu count SEM CLSM cfu, colony forming unit; CLSM, confocal laser scanning electron microscopy; HIV, human immunodeficiency virus; RT-qPCR, quantitative reverse transcription polymerase chain reaction; SEM: scanning electron microscopy; TEM: transmission electron microscopy. View Large Choice of bacterial strains The choice of bacterial strains commonly selected for caries and periodontitis models warrants update. Peterson et al. (2014) used microarrays and high-throughput sequencing to investigate biofilm physiology and microbial interactions in dental caries27 and demonstrated that taxonomic profile was not predictive of dental caries. In particular, S. mutans was not always prominent or present in caries ecosystems. Conversely, functional analysis based on RNA expression was more informative. Different bacterial species were shown to display similarities in gene expression patterns, and functional redundancy was common. Extensive listings of cultivable and noncultivable oral bacteria have recently been published. New prominent species have been identified in samples of healthy saliva,51,81,82 dental plaque,23,27,81,82 healthy gingiva,27 and periodontitis.27,81,83 The salivary flora is more similar to the tongue flora than to dental plaque.27 It is impossible to achieve the dynamics of the oral cavity using predefined combinations of laboratory or wild-type strains, but the choice of strains must also take into account co-occurrence and co-exclusion patterns in oral communities.10 For a single species, biofilm formation can be also strain-dependant.84 Even in simplified in vitro models, the source of inoculum may influence the model used. For instance, different results are anticipated when wild-type strains obtained from healthy young adults, healthy elderly people or specifically diseased individuals will be used instead of laboratory strains. Choice of fungal strains Knowledge on oral fungi is more limited, but several prominent genera have been identified in oral saliva (Table 3). To date, C. albicans is the most common species used in oral consortium studies as it is the most amenable to isolation, identification, and culture.6 Bertolini et al. (2015) also used three strains of mutant C. albicans.32,85 Pereira-Cenci et al. (2008) described a co-culture model with C. albicans and Candida glabrate,86 while Chew et al. (2015) recently developed a model of vulvovaginal candidiasis containing C. glabrata combined with two probiotic lactobacilli, Lactobacillus router and Lactobacillus rhamnosus.22 Furthermore, Pichia species have been identified in oral rinse samples of patients infected with human immunodeficiency virus (HIV), and this species could be antagonist to Candida, Aspergillus and Fusarium species.3 Table 3. Consensus members in the oral mycobiome. References Authors Oral ecosystem Main genera Prominent Candida species 1 Ghannoum 2010 20 oral rinse samples Frequency in the 20 subjects: Candida (75%) Cladosporium (65%) Aureobasidium (50%) Saccharomycetales (50%) Non-cultivable categories (36%) Aspergilllus (35%) Fusarium (30%) Cryptococcus (20%). Frequency of Candida species in the 20 subjects: Candida albicans (40%) Candida paraphilics (15%) Candida tropicalis (15%) Candida khmerensis (5%) Candida metapsilosis (5%) 28 Dupuy 2014 6 saliva samples Frequency of genera occurring in at least 50% of the six subjects: Malassezia (38%) Epicoccum (33%) Candida/Pichia (10%) Fusarium/Gibberella (4%) Cladosporium/Davidiella (3%) Alternaria/Lewia (2%) Aspergilllus/Emericella/Eurotium (2%) Cryptococcus/Filobasidiella (1%) Candida albicans Candida utilis/Pichia jadinii 3 Mukherjee 2014 24 oral rinse samples Frequency in the 12 subjects of each group: 12 HIV-noninfected patients: Candida (58%) Pichia (33%) Fusarium (33%) Cladosporium Penicillium 12 HIV-infected patients: Candida (92%) Epicoccum (33%) Alternaria (25%) Penicillium Trichosporon Main species identified in the Candida genus: Candida albicans (58%) Candida dublinensis (17%) Candida intermedia Candida albicans (83%) Candida dublinensis (17%) Candida sake References Authors Oral ecosystem Main genera Prominent Candida species 1 Ghannoum 2010 20 oral rinse samples Frequency in the 20 subjects: Candida (75%) Cladosporium (65%) Aureobasidium (50%) Saccharomycetales (50%) Non-cultivable categories (36%) Aspergilllus (35%) Fusarium (30%) Cryptococcus (20%). Frequency of Candida species in the 20 subjects: Candida albicans (40%) Candida paraphilics (15%) Candida tropicalis (15%) Candida khmerensis (5%) Candida metapsilosis (5%) 28 Dupuy 2014 6 saliva samples Frequency of genera occurring in at least 50% of the six subjects: Malassezia (38%) Epicoccum (33%) Candida/Pichia (10%) Fusarium/Gibberella (4%) Cladosporium/Davidiella (3%) Alternaria/Lewia (2%) Aspergilllus/Emericella/Eurotium (2%) Cryptococcus/Filobasidiella (1%) Candida albicans Candida utilis/Pichia jadinii 3 Mukherjee 2014 24 oral rinse samples Frequency in the 12 subjects of each group: 12 HIV-noninfected patients: Candida (58%) Pichia (33%) Fusarium (33%) Cladosporium Penicillium 12 HIV-infected patients: Candida (92%) Epicoccum (33%) Alternaria (25%) Penicillium Trichosporon Main species identified in the Candida genus: Candida albicans (58%) Candida dublinensis (17%) Candida intermedia Candida albicans (83%) Candida dublinensis (17%) Candida sake View Large Table 3. Consensus members in the oral mycobiome. References Authors Oral ecosystem Main genera Prominent Candida species 1 Ghannoum 2010 20 oral rinse samples Frequency in the 20 subjects: Candida (75%) Cladosporium (65%) Aureobasidium (50%) Saccharomycetales (50%) Non-cultivable categories (36%) Aspergilllus (35%) Fusarium (30%) Cryptococcus (20%). Frequency of Candida species in the 20 subjects: Candida albicans (40%) Candida paraphilics (15%) Candida tropicalis (15%) Candida khmerensis (5%) Candida metapsilosis (5%) 28 Dupuy 2014 6 saliva samples Frequency of genera occurring in at least 50% of the six subjects: Malassezia (38%) Epicoccum (33%) Candida/Pichia (10%) Fusarium/Gibberella (4%) Cladosporium/Davidiella (3%) Alternaria/Lewia (2%) Aspergilllus/Emericella/Eurotium (2%) Cryptococcus/Filobasidiella (1%) Candida albicans Candida utilis/Pichia jadinii 3 Mukherjee 2014 24 oral rinse samples Frequency in the 12 subjects of each group: 12 HIV-noninfected patients: Candida (58%) Pichia (33%) Fusarium (33%) Cladosporium Penicillium 12 HIV-infected patients: Candida (92%) Epicoccum (33%) Alternaria (25%) Penicillium Trichosporon Main species identified in the Candida genus: Candida albicans (58%) Candida dublinensis (17%) Candida intermedia Candida albicans (83%) Candida dublinensis (17%) Candida sake References Authors Oral ecosystem Main genera Prominent Candida species 1 Ghannoum 2010 20 oral rinse samples Frequency in the 20 subjects: Candida (75%) Cladosporium (65%) Aureobasidium (50%) Saccharomycetales (50%) Non-cultivable categories (36%) Aspergilllus (35%) Fusarium (30%) Cryptococcus (20%). Frequency of Candida species in the 20 subjects: Candida albicans (40%) Candida paraphilics (15%) Candida tropicalis (15%) Candida khmerensis (5%) Candida metapsilosis (5%) 28 Dupuy 2014 6 saliva samples Frequency of genera occurring in at least 50% of the six subjects: Malassezia (38%) Epicoccum (33%) Candida/Pichia (10%) Fusarium/Gibberella (4%) Cladosporium/Davidiella (3%) Alternaria/Lewia (2%) Aspergilllus/Emericella/Eurotium (2%) Cryptococcus/Filobasidiella (1%) Candida albicans Candida utilis/Pichia jadinii 3 Mukherjee 2014 24 oral rinse samples Frequency in the 12 subjects of each group: 12 HIV-noninfected patients: Candida (58%) Pichia (33%) Fusarium (33%) Cladosporium Penicillium 12 HIV-infected patients: Candida (92%) Epicoccum (33%) Alternaria (25%) Penicillium Trichosporon Main species identified in the Candida genus: Candida albicans (58%) Candida dublinensis (17%) Candida intermedia Candida albicans (83%) Candida dublinensis (17%) Candida sake View Large In vitro antagonisms between fungal and bacterial strains According to the data currently available, in vitro, some bacterial species decrease C. albicans biofilm formation and viability within biofilms, whereas C. albicans increases both bacterial growth and biofilm formation. For instance, C. albicans growth is inhibited by Escherichia coli, Pseudomonas aeruginosa, Proteus vulgaris, Staphylococcus aureus, Streptococcus pyogenes, Prevotella nigrescens, Porphyromonas gingivalis, and Streptococcus salivarius. Motile Gram-negative species display a greater inhibitory effect than Gram-positive species.21 The presence of Streptococcus intermedius seemed to have no effect.87 In contrast, both S. mutans20,86 and S. gorodki88 enhanced C. albicans hyphal development and biofilm formation, partially via the modulation of its signalling pathways. Conversely, C. albicans promoted the growth of S. aureus57 and the growth of Clostridium perfringens and Bacteroides fragilis, which are two strict anaerobes.2 This article published by Fox et al. (2014) showed that preformed Candida biofilms allowed cultivation of strict anaerobes. A more recent article published by van Leeuwen et al. (2016) showed that co-culturing does a similar thing.89 Furthermore, C. albicans synergistically promoted S. gordonii, S. mutans, S. oralis, S. sanguinis, and biofilm formation.18,76 The presence of sucrose greatly increased this synergistic effect of C. albicans and oral streptococci on biofilm formation.69 Similarly, Ramírez Granillo et al. (2015) developed a mixed Aspergillus fumigatus and S. aureus biofilm. Independently of bacterial concentration, they observed a low abundance of A. fumigatus biofilm production and abnormal fungal structures (hyphae and conidia).90 Microcosm models In microcosm models, the inoculum is constituted of saliva or dental plaque collected from donors.30,50,51 Rudney et al. (2012) recommended pooling saliva or plaque samples from multiple donors and then freezing aliquots in order to reproduce experiments.54 The resulting in vitro microcosms are much more diverse than consortia, but microcosms are difficult to characterise. Ex vivo and in vitro, culture-independent methodologies are expensive as they involve metagenomics data combined with sophisticated software analysis. Note too that many bacterial species, mostly fastidious anaerobic bacteria, are lost when the taxonomic profile of the microcosm is compared to the initial sample collected in vivo.54 There are few examples of oral microcosm models (Table 1 and 2). De Moraes et al. (2012) validated a model in 24-well tissue cultures plates.50 A commonly used model is the AAA-model first described by Exterkate et al. (2010).91 More recently, Koopman et al. (2015) and Sousa et al. (2016) developed microcosm models in a sophisticated flux system apparatus.30,51 Zijnge et al. (2010) showed that F. nucleateum and yeasts form aggregates in vivo.24 Krom et al. (2014) hypothesized that fungi could act as bridging organisms in analogy to Fusobacterium.10 Rudney et al. (2015) validated a reproducible oral microcosm model for experiments using plaque with sucrose pulsing, but taxonomic profiles were limited to bacteria and did not include fungal investigation.23 Culture conditions Biofilm support Solid supports and cell culture supports Biofilms need to grow onto a support, which can either be a solid material or an air-liquid surface (abiotic) or a cell culture (biotic). Oral biofilms are usually developed onto solid supports such as bovine enamel blocks, human enamel disks, human dentine, hydroxyapatite disks, polystyrene microtiter plates, methacrylate resin disks, soft denture liners, glass coverslips, and titanium disks (Table 1 and 2). Diaz et al. (2012) successfully developed fungal-bacterial biofilms onto reconstructed non-keratinised epithelium, using oral cells (OKF6/Tert-2) and oesophageal cells (EPC2) seeded on collagen type I-embedded fibroblasts (3T3 cells) (Table 2).18 Non-oral cell cultures have also been used, with either bacterial or fungal inoculum. For instance, Krishnamurthy & Kyd (2014) demonstrated that contact with lung epithelial cells (A549/CCL-185) increased the formation of a two-bacterial species biofilm compared to a no-contact model.92 Kavanaugh et al. (2014) worked with mucus-secreting cells derived from a human colorectal adenocarcinoma (HT29-MTX) and showed that mucins downregulated fungal genes involved in surface attachment (Als1, Als3) and suppressed surface adhesion of C. albicans.8 Recently, Townsend et al. (2016) developed a polymicrobial inter-kingdom in vitro wound biofilm model on complex hydrogel-based cellulose substrata to test commonly used topical treatments.93 Pre-treatment of the support Solid supports and epithelial cell cultures are frequently pre-incubated with human saliva to reproduce the salivary pellicle and provide receptor binding sites for bacteria.78,86,94 Diaz et al. (2012) used the culture medium of the biofilm (defined mucin medium) supplemented with saliva.18 The main limitation of using saliva is that its composition varies both intra- and inter-individually. To achieve an acceptably reproducible biofilm model, the saliva collection conditions must be standardised (no antibiotics during the previous weeks, hour of sampling, stimulated- or non-stimulated saliva, pooling various donors’ samples). The saliva is then filtered, aliquoted, and frozen, ready to perform all the experiments with the same saliva pool.62 The use of artificial saliva allows other authors to reproduce the model. Culture medium The culture medium used to grow the final multispecies mix is a key factor that influences the growth, stability, thickness, and composition of the biofilm.95 Consortium models: pre-culture of separate fungal and bacterial strains C. albicans can be pre-grown aerobically at 25°C, 35°C, or 37°C, with or without shaking. In different models, the starter culture broth is either a semi-defined medium with 18 mM glucose,86 Sabouraud Dextrose broth,94 yeast nitrogen base broth (YND) with 100 mM glucose,31 or yeast peptone dextrose medium (YPD).80 The final culture medium can contain the mix of every bacterial and fungal pre-culture broth, saliva, or fluid universal medium.96 In parallel, single bacterial species are grown separately in the most appropriate culture broth. There is no consensual medium. Pre-culture broth can be brain-heart infusion (BHI),18,69,94 BHI broth supplemented with 5% sucrose,31 ultrafiltered tryptone-yeast extract broth (UFTYE; 2.5% tryptone and 1.5% yeast extract, pH 7.0) with 1% glucose,20 Todd-Hewitt broth with 0.02% yeast extract media (THB + 0.02% YE),80 universal medium broth (thioglycolate medium supplemented with 67 mMl/l of Sorensen's solution (UM),94 or modified fluid universal medium (mFUM)97 with 0.3% glucose.75 Bacterial strains are pre-grown for 4 h to 18 h at 37°C, in aerobic, microaerophilic (5% CO2) or anaerobic (5% CO2, 95% N at 200 bar) conditions. Pre-cultures are grown to the exponential growth phase, and an aliquot is diluted in the final culture mix. The optimal inoculum concentration for bacteria and Candida strains generally ranges from 106 to 107 colony-forming units (cfu)/ml. The proportion of each species culture in the final mix must be carefully determined according to strains, support and culture conditions, to prevent any one species from overgrowing.35 Microcosm models In microcosm models, the culture medium must be directly adapted to fungal and bacterial strains. In a model of peri-implantitis, Sousa et al. (2016) developed a medium adapted to epithelial cell, fungal and bacterial growth. This unique medium mimicking the peri-implant sulcular fluid contained a tissue culture medium supplemented with horse serum, menadione, and haemin.30 Culture duration Protocols of current models are summarised in Table 1 and 2. Every protocol has its own unique design, with rinses and steps to promote Candida adhesion to the support, Candida filamentation, or biofilm formation, for instance.79 Culture duration ranges from 4 hours18 to 16 weeks.98 In studies designed to evaluate the efficacy of methods or compounds to eliminate bacteria in biofilm, the incubation time generally ranges from 2 to 4 days.94,99 Culture apparatus Batch cultures Batch culture models are grown in polystyrene plates, such as 96-well microtiter plates and 24-well tissue culture plates100 or more rarely in bottles (Fig. 1A). They are inexpensive and allow to test in parallel a large number of conditions.21,50 The biofilm is grown in the bottom of wells or onto solid samples placed into the wells (hydroxyapatite, resin or titanium disks, for instance). Extracted teeth specimens can be processed in Eppendorf tubes.101 In batch cultures, the culture is stopped at given times and submitted to rinses, new medium and/or sugar pulses or addition of antiseptics. Plates are well suited to the screening of several molecules at various concentrations or contact times. Mixed models contain facultative and/or obligate anaerobic bacteria as well as Candida sp. Some are grown in a microaerophilic atmosphere20,31,32,80 or an anaerobic atmosphere.62,74In vitro, agitation is important.102 Shaker speed usually ranges from 75 to 180 rpm.21,31,57 Figure 1. View largeDownload slide (A) Schematic representation of batch culture system: microtiter plate or bottle. (B) Schematic representation of flow cell system. This Figure is reproduced in color in the online version of Medical Mycology. Figure 1. View largeDownload slide (A) Schematic representation of batch culture system: microtiter plate or bottle. (B) Schematic representation of flow cell system. This Figure is reproduced in color in the online version of Medical Mycology. Flux systems Flux systems have been designed to continuously renew the culture medium, such as in a flow cell18,78 (Fig. 1B), an artificial mouth system,51 and a constant depth film fermentor.30 The model described by Diaz et al. was grown under aerobic conditions.18 Sousa et al. (2016) managed to create a peri-implantitis model in rabbits.30 The inoculum was a saliva microcosm, grown onto titanium discs. It was pumped by a peristaltic pump at the rate of 1 ml/min for 8 h. To simulate a subgingival environment, an anaerobic environment was progressively created as follows: day 0, aerobic atmosphere; days 1 to 9, microaerophilic environment (2% O2, 3% CO2, 95% N); days 10 to 30, anaerobic environment (5% CO2, 95% N at 200 bar). After a 30-day incubation period, the discs were surgically positioned onto rabbit tibial bone. Qualitative and quantitative methods to assess in vitro biofilm models The benefits and limitations of each method are presented in Table 4. A flow chart is also available (Fig. 2) to choose the suitable method. Figure 2. View largeDownload slide Flow chart to choose the suitable method. Figure 2. View largeDownload slide Flow chart to choose the suitable method. Table 4. Benefits and limitations of each method presented. Benefits Limitations Solid supports • Simplicity • Less close to the reality • Reproducibility • Moderate cost • No specific equipment required Biofilm support • Large number of samples at the same time Cell culture supports • Allow to take into account partially the immune response of the host • Difficulty of development • Not adapted for a large number of samples • Moderate cost Human saliva • To reproduce the salivary pellicle and provide receptor binding sites for bacteria • Composition varies both intra- and inter-individually Pre-treatment of the support • Closer to reality • To achieve an acceptable reproducibility of a biofilm model, the saliva collection conditions must be standardized (no antibiotics during the previous weeks, hour of sampling, pooling various donors’ samples) Artificial saliva • To reproduce the salivary pellicle and provide receptor binding sites for bacteria • Lack of potentially relevant molecules such as enzymes Batch cultures • Simplicity • Reproducibility • No renewal or renewal rough of the nutriments and oxygen • Moderate cost • No specific equipment required Culture apparatus • Large number of samples at the same time Flux systems • Reproducibility • Require scientific equipment • Moderate cost • Control of parameters (pH, flux, nutriments…) Numeration of viable cells (Colony Forming Units) • Simplicity • No consideration of viable but noncultivable bacteria • Reproducibility • Moderate cost • No specific equipment required Crystal violet staining • Simplicity • Moderate cost • Poor reproducibility • Inability to differentiate live or dead bacteria Qualitative and quantitative methods to assess in vitro biofilm models Routine laboratory methods Colorimetric XTT assay • Simplicity • Metabolic activity can be altered by a restricted access to nutrients and oxygen • Reproducibility • Limited to fungal cells • Moderate cost Fluorescence microscopy (i.e. LIVE/DEAD Baclight® method) • Simplicity • Limited applications and essentially for bacteria: (i.e: Gram stain kit, LIVE/DEAD viability kit…) • Multi-purpose method: a quantitative method for counting CFU/mL with a fluorescence microscope or a qualitative method to visualize biofilm colonization with a CSLM • Few fluorescent stains can be employed simultaneously • Less toxic than conventional assays Imaging Confocal scanning laser microscopy (CSLM) • Multi-purpose method: to estimate the biomass, to quantify extracellular polysaccharides and microbial cells • Only a semi-quantitative investigation • Few fluorescent stains can be employed simultaneously allowing the visualization of just a couple of components in the same image • Do not disrupt the biofilm Scanning electron microscopy (SEM) • High-resolution, • Samples preparation does not preserve the vitality of the biofilm • Three-dimensional images provide topographical, morphological and compositional information • Requires expensive scientific equipment RT-qPCR • Powerful and sensitive gene analysis techniques • Strict rules of sample preparation (No contaminants or PCR inhibitors; choice of the primers sequence …) • High costs • Difficulty of execution requiring expensive scientific equipment and skilled technical staff Genetic assays Fluorescence in situ hybridization (FISH) • Capacity to quantify nonculturable organisms • Cost • Inability to differentiate viable and nonviable cells • Impossibility to work on live material • Only qualitative method Microarrays • Allow the investigation of the biofilm physiology and microbial interactions • Powerful and sensitive gene analysis techniques • Strict rules of sample preparation • High costs • Difficulty of execution and expensive scientific equipment often lead to the use of platform of sequencing Benefits Limitations Solid supports • Simplicity • Less close to the reality • Reproducibility • Moderate cost • No specific equipment required Biofilm support • Large number of samples at the same time Cell culture supports • Allow to take into account partially the immune response of the host • Difficulty of development • Not adapted for a large number of samples • Moderate cost Human saliva • To reproduce the salivary pellicle and provide receptor binding sites for bacteria • Composition varies both intra- and inter-individually Pre-treatment of the support • Closer to reality • To achieve an acceptable reproducibility of a biofilm model, the saliva collection conditions must be standardized (no antibiotics during the previous weeks, hour of sampling, pooling various donors’ samples) Artificial saliva • To reproduce the salivary pellicle and provide receptor binding sites for bacteria • Lack of potentially relevant molecules such as enzymes Batch cultures • Simplicity • Reproducibility • No renewal or renewal rough of the nutriments and oxygen • Moderate cost • No specific equipment required Culture apparatus • Large number of samples at the same time Flux systems • Reproducibility • Require scientific equipment • Moderate cost • Control of parameters (pH, flux, nutriments…) Numeration of viable cells (Colony Forming Units) • Simplicity • No consideration of viable but noncultivable bacteria • Reproducibility • Moderate cost • No specific equipment required Crystal violet staining • Simplicity • Moderate cost • Poor reproducibility • Inability to differentiate live or dead bacteria Qualitative and quantitative methods to assess in vitro biofilm models Routine laboratory methods Colorimetric XTT assay • Simplicity • Metabolic activity can be altered by a restricted access to nutrients and oxygen • Reproducibility • Limited to fungal cells • Moderate cost Fluorescence microscopy (i.e. LIVE/DEAD Baclight® method) • Simplicity • Limited applications and essentially for bacteria: (i.e: Gram stain kit, LIVE/DEAD viability kit…) • Multi-purpose method: a quantitative method for counting CFU/mL with a fluorescence microscope or a qualitative method to visualize biofilm colonization with a CSLM • Few fluorescent stains can be employed simultaneously • Less toxic than conventional assays Imaging Confocal scanning laser microscopy (CSLM) • Multi-purpose method: to estimate the biomass, to quantify extracellular polysaccharides and microbial cells • Only a semi-quantitative investigation • Few fluorescent stains can be employed simultaneously allowing the visualization of just a couple of components in the same image • Do not disrupt the biofilm Scanning electron microscopy (SEM) • High-resolution, • Samples preparation does not preserve the vitality of the biofilm • Three-dimensional images provide topographical, morphological and compositional information • Requires expensive scientific equipment RT-qPCR • Powerful and sensitive gene analysis techniques • Strict rules of sample preparation (No contaminants or PCR inhibitors; choice of the primers sequence …) • High costs • Difficulty of execution requiring expensive scientific equipment and skilled technical staff Genetic assays Fluorescence in situ hybridization (FISH) • Capacity to quantify nonculturable organisms • Cost • Inability to differentiate viable and nonviable cells • Impossibility to work on live material • Only qualitative method Microarrays • Allow the investigation of the biofilm physiology and microbial interactions • Powerful and sensitive gene analysis techniques • Strict rules of sample preparation • High costs • Difficulty of execution and expensive scientific equipment often lead to the use of platform of sequencing View Large Table 4. Benefits and limitations of each method presented. Benefits Limitations Solid supports • Simplicity • Less close to the reality • Reproducibility • Moderate cost • No specific equipment required Biofilm support • Large number of samples at the same time Cell culture supports • Allow to take into account partially the immune response of the host • Difficulty of development • Not adapted for a large number of samples • Moderate cost Human saliva • To reproduce the salivary pellicle and provide receptor binding sites for bacteria • Composition varies both intra- and inter-individually Pre-treatment of the support • Closer to reality • To achieve an acceptable reproducibility of a biofilm model, the saliva collection conditions must be standardized (no antibiotics during the previous weeks, hour of sampling, pooling various donors’ samples) Artificial saliva • To reproduce the salivary pellicle and provide receptor binding sites for bacteria • Lack of potentially relevant molecules such as enzymes Batch cultures • Simplicity • Reproducibility • No renewal or renewal rough of the nutriments and oxygen • Moderate cost • No specific equipment required Culture apparatus • Large number of samples at the same time Flux systems • Reproducibility • Require scientific equipment • Moderate cost • Control of parameters (pH, flux, nutriments…) Numeration of viable cells (Colony Forming Units) • Simplicity • No consideration of viable but noncultivable bacteria • Reproducibility • Moderate cost • No specific equipment required Crystal violet staining • Simplicity • Moderate cost • Poor reproducibility • Inability to differentiate live or dead bacteria Qualitative and quantitative methods to assess in vitro biofilm models Routine laboratory methods Colorimetric XTT assay • Simplicity • Metabolic activity can be altered by a restricted access to nutrients and oxygen • Reproducibility • Limited to fungal cells • Moderate cost Fluorescence microscopy (i.e. LIVE/DEAD Baclight® method) • Simplicity • Limited applications and essentially for bacteria: (i.e: Gram stain kit, LIVE/DEAD viability kit…) • Multi-purpose method: a quantitative method for counting CFU/mL with a fluorescence microscope or a qualitative method to visualize biofilm colonization with a CSLM • Few fluorescent stains can be employed simultaneously • Less toxic than conventional assays Imaging Confocal scanning laser microscopy (CSLM) • Multi-purpose method: to estimate the biomass, to quantify extracellular polysaccharides and microbial cells • Only a semi-quantitative investigation • Few fluorescent stains can be employed simultaneously allowing the visualization of just a couple of components in the same image • Do not disrupt the biofilm Scanning electron microscopy (SEM) • High-resolution, • Samples preparation does not preserve the vitality of the biofilm • Three-dimensional images provide topographical, morphological and compositional information • Requires expensive scientific equipment RT-qPCR • Powerful and sensitive gene analysis techniques • Strict rules of sample preparation (No contaminants or PCR inhibitors; choice of the primers sequence …) • High costs • Difficulty of execution requiring expensive scientific equipment and skilled technical staff Genetic assays Fluorescence in situ hybridization (FISH) • Capacity to quantify nonculturable organisms • Cost • Inability to differentiate viable and nonviable cells • Impossibility to work on live material • Only qualitative method Microarrays • Allow the investigation of the biofilm physiology and microbial interactions • Powerful and sensitive gene analysis techniques • Strict rules of sample preparation • High costs • Difficulty of execution and expensive scientific equipment often lead to the use of platform of sequencing Benefits Limitations Solid supports • Simplicity • Less close to the reality • Reproducibility • Moderate cost • No specific equipment required Biofilm support • Large number of samples at the same time Cell culture supports • Allow to take into account partially the immune response of the host • Difficulty of development • Not adapted for a large number of samples • Moderate cost Human saliva • To reproduce the salivary pellicle and provide receptor binding sites for bacteria • Composition varies both intra- and inter-individually Pre-treatment of the support • Closer to reality • To achieve an acceptable reproducibility of a biofilm model, the saliva collection conditions must be standardized (no antibiotics during the previous weeks, hour of sampling, pooling various donors’ samples) Artificial saliva • To reproduce the salivary pellicle and provide receptor binding sites for bacteria • Lack of potentially relevant molecules such as enzymes Batch cultures • Simplicity • Reproducibility • No renewal or renewal rough of the nutriments and oxygen • Moderate cost • No specific equipment required Culture apparatus • Large number of samples at the same time Flux systems • Reproducibility • Require scientific equipment • Moderate cost • Control of parameters (pH, flux, nutriments…) Numeration of viable cells (Colony Forming Units) • Simplicity • No consideration of viable but noncultivable bacteria • Reproducibility • Moderate cost • No specific equipment required Crystal violet staining • Simplicity • Moderate cost • Poor reproducibility • Inability to differentiate live or dead bacteria Qualitative and quantitative methods to assess in vitro biofilm models Routine laboratory methods Colorimetric XTT assay • Simplicity • Metabolic activity can be altered by a restricted access to nutrients and oxygen • Reproducibility • Limited to fungal cells • Moderate cost Fluorescence microscopy (i.e. LIVE/DEAD Baclight® method) • Simplicity • Limited applications and essentially for bacteria: (i.e: Gram stain kit, LIVE/DEAD viability kit…) • Multi-purpose method: a quantitative method for counting CFU/mL with a fluorescence microscope or a qualitative method to visualize biofilm colonization with a CSLM • Few fluorescent stains can be employed simultaneously • Less toxic than conventional assays Imaging Confocal scanning laser microscopy (CSLM) • Multi-purpose method: to estimate the biomass, to quantify extracellular polysaccharides and microbial cells • Only a semi-quantitative investigation • Few fluorescent stains can be employed simultaneously allowing the visualization of just a couple of components in the same image • Do not disrupt the biofilm Scanning electron microscopy (SEM) • High-resolution, • Samples preparation does not preserve the vitality of the biofilm • Three-dimensional images provide topographical, morphological and compositional information • Requires expensive scientific equipment RT-qPCR • Powerful and sensitive gene analysis techniques • Strict rules of sample preparation (No contaminants or PCR inhibitors; choice of the primers sequence …) • High costs • Difficulty of execution requiring expensive scientific equipment and skilled technical staff Genetic assays Fluorescence in situ hybridization (FISH) • Capacity to quantify nonculturable organisms • Cost • Inability to differentiate viable and nonviable cells • Impossibility to work on live material • Only qualitative method Microarrays • Allow the investigation of the biofilm physiology and microbial interactions • Powerful and sensitive gene analysis techniques • Strict rules of sample preparation • High costs • Difficulty of execution and expensive scientific equipment often lead to the use of platform of sequencing View Large Routine laboratory methods Routine laboratory methods are a first step in observing consortium and microcosm models. C. albicans yeast-hyphal transition and fungal-bacterial interactions are observable directly in biofilms formed on glass coverslips and in the wells of polystyrene plates, with bright-field and phase-contrast microscopes.79 Light microscopy with x400 magnification is adapted to hyphae/yeast count.31 In dental plaque and caries models, pH evolution20,31,51,69,78 or metabolite releases51 are monitored in the culture medium bathing the biofilms. In-biofilm cell viability is commonly determined by scraping the microbial deposits formed onto solid supports followed by serial dilutions on appropriate agar plates for cfu count,30,31,50,62,64,69,74,86,94,101 but Falsetta et al. (2014) highlighted the limitations of cfu count data for C. albicans, as most hyphae are multicellular with a large biomass compared to yeasts yet like yeasts they form a single cfu.20 Alternatively, in-well biomass formation can be quantified with colorimetric methods needing a plate spectrophotometer.80 The XTT assay is based on the reduction of the tetrazolium salt of XTT in formazan by the succinate dehydrogenase system of the mitochondrial respiratory chain in fungal cells but not bacterial cells.21,64 The crystal violet assay dies in violet the total biomass, including fungal cells, bacterial cells and exopolysaccharides,31,80 but it quantifies both live and dead cells in the biofilm.74 Finally, routine histology are suitably adapted to analysing biofilms grown onto epithelial cell cultures.18,32,103 Imaging The LIVE/DEAD® Biofilm viability kit (BacLight, Invitrogen, Paisley, UK) method utilizes mixtures of green-fluorescent (SYTO9) and red-fluorescent (propidium iodide) nucleic acid stains for bacteria. The difference in stain penetration of bacterial and fungal cells allows making difference between healthy cells (green) and bacteria with damaged membranes (red). BacLight® can be used as a quantitative method or as a qualitative method.98,104 Figure 3A is an example of LIVE/DEAD® method, applied to a single species C. albicans biofilm. Figure 3. View largeDownload slide Comparison of different methods of imaging applied to a single species C. albicans biofilm. (A) Biofilm was stained using SYTO-9 (BacLight®) to stain live biofilm cells green and examined by fluorescence microscopy (x63); (B) Biofilm observed with SEM (Bar, 10 μm). (C) Biofilm 3D visualization after z-stack acquisition with CSLM (Bar, 50 μm) (Courtesy Pr. G. Ramage). This Figure is reproduced in color in the online version of Medical Mycology. Figure 3. View largeDownload slide Comparison of different methods of imaging applied to a single species C. albicans biofilm. (A) Biofilm was stained using SYTO-9 (BacLight®) to stain live biofilm cells green and examined by fluorescence microscopy (x63); (B) Biofilm observed with SEM (Bar, 10 μm). (C) Biofilm 3D visualization after z-stack acquisition with CSLM (Bar, 50 μm) (Courtesy Pr. G. Ramage). This Figure is reproduced in color in the online version of Medical Mycology. Scanning electron microscopy (SEM) is suitably adapted to observing fungal-bacterial organisation in a biofilm. It is also useful to observe the surface of the biofilm support before and after microbial colonisation, such as enamel acid attack in caries models.21,31,62,64,69,74,78,80,86,101 Figure 3B is an example of a C. albicans biofilm observed with SEM. Confocal laser scanning microscopy (CLSM) is suitably adapted to 2D and 3D analysis of the biofilm, combined with fluorescent staining of specific microbial cells and matrix components.18,20,32,64,69,74,78–80,103 Figure 3C is an example of CSLM method, applied to a single species C. albicans biofilm. After image reconstruction on appropriate software, it is possible to measure average thickness of the biofilm and to characterise its microbial species and their respective percentages in the biomass.49 Some fluorophore combinations are also adapted to identify and quantify live and dead cells in the biofilm.74 Genetic assays Quantitative reverse transcription polymerase chain reaction (RT-qPCR) is suitably adapted to characterising the taxonomic and functional profile of microcosm models based on selected genes,20,21,49,51,62,74,78 including cell viability74 and hyphal morphology18; however, RT-qPCR requires expensive equipment.54 Fluorescence in situ hybridization (FISH) is based on oligonucleotide probes labeled with fluorescent dyes. FISH can be used to determine the bacterial and fungal composition of a biofilm,105 to visualize spatial distribution in combination with confocal laser scanning microscopy (CLSM),106 or the colonization of gingival epithelia by subgingival biofilm.97 A microarray is a miniaturized solid support displaying a very large set of oligonucleotide probes, allowing the screening of >30.000 genes during a two-day protocol. For instance, microarrays allow the detection of variations in a gene sequence expression, the comparison of a bacterial genome expression at different times of growth or different culture conditions, or the comparison of two bacterial consortia grown in similar culture conditions.107 In a fungal biofilm model, Cao et al.108 used microarrays to show the influence of farnesol on C. albicans biofilm: some hyphal-formation-associated genes (including TUP1) were differentially expressed in farnesol-treated biofilms. Conclusion There is increasing interest in in vitro models of mixed fungal-bacterial biofilms designed to mimic various oral ecosystems. Protocol designs, culture broths and culture conditions are highly diverse, but the supports used to develop biofilms are relatively consensual, as is the choice of C. albicans and bacterial species in consortium models. S. mutans and oral streptococci are near-standard bacterial species in caries, periodontitis, and candidiasis models, but new bacterial and fungal combinations could be explored. Species selection could take into account genomic and proteomic results obtained in vivo, as recent studies have revealed new taxonomy profiles, unexpected quantitative compositions, and functional expressions in oral microcosms. This review also revealed a broad difference between culture apparatuses and assessment methods, ranging from microtiter plates to custom-made flux systems, and from cfu counts to CLSM and RT-qPCR. These sophisticated and expensive technologies may ultimately lead to therapies designed to clean up oral microcosms or improve oral health at molecular level. However, microtiter plate models still warrant attention, as they are well adapted to the development of new therapeutic agents. Many populations of patients still need basic oral hygiene education, first-line oral care, healthy diet, and medical help to reduce polymedication and combat addictions to alcohol, tobacco, and illicit drugs. In vivo, these conditions influence oral ecosystems, particularly in children and teenagers, in chronically ill, poly-medicated or malnourished patients, and in frail elderly people. In vitro assays with appropriate models could help improve oral care products, drug formulations, or the composition of foods, beverages, and oral nutritional supplements. Acknowledgements This work was supported by grants from the Association Française du Gougerot-Sjögren et des syndromes secs and the Fondation de l’Avenir (gAFGS-2013 and AP-RMA-2015-025). For manuscript revision, we thank Chetan S. Patil DDS, PhD, Periodontal Associate (LLC, Englewood, NJ, USA) and Clinical instructor (Columbia University College of Dental Medicine, NY, USA). The authors greatly acknowledge the CCMA (Centre Commun de Microscopie Appliquée, Université Côte d’Azur, Microscopy and Imaging platform Côte d’Azur, MICA) and its personnel. We also thank Pr. Gordon Ramage (Infection and Immunity Research Group, University of Glasgow, UK) for CLS micrograph. Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and the writing of the paper. References 1. Ghannoum MA , Jurevic RJ , Mukherjee PK et al. Characterization of the oral fungal microbiome (mycobiome) in healthy individuals . PLoS Pathog . 2010 ; 6 : e1000713 . Google Scholar CrossRef Search ADS PubMed 2. Fox EP , Cowley ES , Nobile CJ , Hartooni N , Newman DK , Johnson AD . Anaerobic bacteria grow within Candida albicans biofilms and induce biofilm formation in suspension cultures . Curr Biol . 2014 ; 24 : 2411 – 2416 . Google Scholar CrossRef Search ADS PubMed 3. Mukherjee PK , Chandra J , Retuerto M et al. Oral mycobiome analysis of HIV-infected patients: identification of Pichia as an antagonist of opportunistic fungi . PLoS Pathog . 2014 ; 10 : e1003996 . Google Scholar CrossRef Search ADS PubMed 4. Huynh HTT , Pignoly M , Nkamga VD , Drancourt M , Aboudharam G . The repertoire of archaea cultivated from severe periodontitis . PLoS ONE . 2015 ; 10 : e0121565 . Google Scholar CrossRef Search ADS PubMed 5. Diaz PI , Strausbaugh LD , Dongari-Bagtzoglou A . Fungal-bacterial interactions and their relevance to oral health: linking the clinic and the bench . Front Cell Infect Microbiol . 2014 ; 4 : 101 . Google Scholar CrossRef Search ADS PubMed 6. Xu H , Dongari-Bagtzoglou A . Shaping the oral mycobiota: interactions of opportunistic fungi with oral bacteria and the host . Curr Opin Microbiol . 2015 ; 26 : 65 – 70 . Google Scholar CrossRef Search ADS PubMed 7. Vincent-Bugnas S , Vitale S , Mouline CC et al. EBV infection is common in gingival epithelial cells of the periodontium and worsens during chronic periodontitis . PloS One . 2013 ; 8 : e80336 . Google Scholar CrossRef Search ADS PubMed 8. Kavanaugh NL , Zhang AQ , Nobile CJ , Johnson AD , Ribbeck K . Mucins suppress virulence traits of Candida albicans . mBio . 2014 ; 5 : e0 1911. Google Scholar CrossRef Search ADS 9. Ewan V , Perry JD , Mawson T et al. Detecting potential respiratory pathogens in the mouths of older people in hospital . Age Ageing . 2010 ; 39 : 122 – 125 . Google Scholar CrossRef Search ADS PubMed 10. Krom BP , Kidwai S , ten Cate JM . Candida and other fungal species: forgotten players of healthy oral microbiota . J Dent Res . 2014 ; 93 : 445 – 451 . Google Scholar CrossRef Search ADS PubMed 11. Ortega O , Sakwinska O , Combremont S et al. High prevalence of colonization of oral cavity by respiratory pathogens in frail older patients with oropharyngeal dysphagia . Neurogastroenterol Motil Off J Eur Gastrointest Motil Soc . 2015 ; 12 : 1804 – 1816 . Google Scholar CrossRef Search ADS 12. Scannapieco FA , Cantos A . Oral inflammation and infection, and chronic medical diseases: implications for the elderly . Periodontol 2000 . 2016 ; 72 : 153 – 175 . Google Scholar CrossRef Search ADS PubMed 13. Saarela RKT , Lindroos E , Soini H et al. Dentition, nutritional status and adequacy of dietary intake among older residents in assisted living facilities . Gerodontology . 2016 ; 33 : 225 – 232 . Google Scholar CrossRef Search ADS PubMed 14. Otomo-Corgel J , Pucher JJ , Rethman MP , Reynolds MA . State of the science: chronic periodontitis and systemic health . J Evid Based Dent Pract . 2012 ; 12 : 20 – 28 . Google Scholar CrossRef Search ADS PubMed 15. Gil-Montoya JA , Ferreira de Mello AL , Barrios R , Gonzalez-Moles MA , Bravo M . Oral health in the elderly patient and its impact on general well-being: a nonsystematic review . Clin Interv Aging . 2015 ; 10 : 461 – 467 . Google Scholar CrossRef Search ADS PubMed 16. Guo Y , Wei C , Liu C et al. Inhibitory effects of oral Actinomyces on the proliferation, virulence and biofilm formation of Candida albicans . Arch Oral Biol . 2015 ; 60 : 1368 – 1374 . Google Scholar CrossRef Search ADS PubMed 17. Vilela SFG , Barbosa JO , Rossoni RD et al. Lactobacillus acidophilus ATCC 4356 inhibits biofilm formation by C. albicans and attenuates the experimental candidiasis in Galleria mellonella. Virulence . 2015 ; 6 : 29 – 39 . Google Scholar CrossRef Search ADS PubMed 18. Diaz PI , Xie Z , Sobue T et al. Synergistic Interaction between Candida albicans and commensal oral streptococci in a novel in vitro mucosal model . Infect Immun . 2012 ; 80 : 620 – 632 . Google Scholar CrossRef Search ADS PubMed 19. Xu H , Sobue T , Thompson A et al. Streptococcal co-infection augments Candida pathogenicity by amplifying the mucosal inflammatory response . Cell Microbiol . 2014 ; 16 : 214 – 231 . Google Scholar CrossRef Search ADS PubMed 20. Falsetta ML , Klein MI , Colonne PM et al. Symbiotic relationship between Streptococcus mutans and Candida albicans synergizes virulence of plaque biofilms in vivo . Infect Immun . 2014 ; 82 : 1968 – 1981 . Google Scholar CrossRef Search ADS PubMed 21. Park SJ , Han K-H , Park JY , Choi SJ , Lee K-H . Influence of bacterial presence on biofilm formation of Candida albicans . Yonsei Med J . 2014 ; 55 : 449 – 458 . Google Scholar CrossRef Search ADS PubMed 22. Chew SY , Cheah YK , Seow HF , Sandai D , Than LTL . In vitro modulation of probiotic bacteria on the biofilm of Candida glabrata . Anaerobe . 2015 ; 34 : 132 – 138 . Google Scholar CrossRef Search ADS PubMed 23. Rudney JD , Jagtap PD , Reilly CS et al. Protein relative abundance patterns associated with sucrose-induced dysbiosis are conserved across taxonomically diverse oral microcosm biofilm models of dental caries . Microbiome . 2015 ; 3 : 69 . Google Scholar CrossRef Search ADS PubMed 24. Zijnge V , van Leeuwen MBM , Degener JE et al. Oral biofilm architecture on natural teeth . PloS One . 2010 ; 5 : e9321 . Google Scholar CrossRef Search ADS PubMed 25. Frias-Lopez J , Duran-Pinedo A . Effect of periodontal pathogens on the metatranscriptome of a healthy multispecies biofilm model . J Bacteriol . 2012 ; 194 : 2082 – 2095 . Google Scholar CrossRef Search ADS PubMed 26. Palmer RJ. Composition and development of oral bacterial communities . Periodontol 2000 . 2014 ; 64 : 20 – 39 . Google Scholar CrossRef Search ADS PubMed 27. Peterson SN , Meissner T , Su AI et al. Functional expression of dental plaque microbiota . Front Cell Infect Microbiol . 2014 ; 4 : 108 . Google Scholar CrossRef Search ADS PubMed 28. Dupuy AK , David MS , Li L et al. Redefining the human oral mycobiome with improved practices in amplicon-based taxonomy: discovery of Malassezia as a prominent commensal . PloS One . 2014 ; 9 : e90899 . Google Scholar CrossRef Search ADS PubMed 29. Vanhoecke BWA , De Ryck TRG , De boel K et al. Low-dose irradiation affects the functional behavior of oral microbiota in the context of mucositis . Exp Biol Med Maywood NJ . 2016 ; 241 : 60 – 70 . Google Scholar CrossRef Search ADS 30. Sousa V , Mardas N , Spratt D , Boniface D , Dard M , Donos N . Experimental models for contamination of titanium surfaces and disinfection protocols . Clin Oral Implants Res . 2016 ; 10 : 1233 – 1242 . Google Scholar CrossRef Search ADS 31. Barbosa JO , Rossoni RD , Vilela SFG et al. Streptococcus mutans can modulate biofilm formation and attenuate the virulence of Candida albicans . PloS One . 2016 ; 11 : e0150457 . Google Scholar CrossRef Search ADS PubMed 32. Bertolini MM , Xu H , Sobue T , Nobile CJ , Del Bel Cury AA , Dongari-Bagtzoglou A . Candida-streptococcal mucosal biofilms display distinct structural and virulence characteristics depending on growth conditions and hyphal morphotypes . Mol Oral Microbiol . 2015 ; 30 : 307 – 322 . Google Scholar CrossRef Search ADS PubMed 33. Ramage G , Lappin DF , Millhouse E et al. The epithelial cell response to health and disease associated oral biofilm models . J Periodontal Res . 2016 ; 52 : 325 – 333 . Google Scholar CrossRef Search ADS PubMed 34. Wade WG. Characterisation of the human oral microbiome . J Oral Biosci . 2013 ; 55 : 143 – 148 . Google Scholar CrossRef Search ADS 35. Kreth J , Merritt J , Qi F . Bacterial and host interactions of oral streptococci . DNA Cell Biol . 2009 ; 28 : 397 – 403 . Google Scholar CrossRef Search ADS PubMed 36. Jorth P , Turner KH , Gumus P , Nizam N , Buduneli N , Whiteley M . Metatranscriptomics of the human oral microbiome during health and disease . mBio . 2014 ; 5 : e01012 – 01014 . Google Scholar CrossRef Search ADS PubMed 37. Rams TE , Degener JE , van Winkelhoff AJ . Antibiotic resistance in human chronic periodontitis microbiota . J Periodontol. 2014 ; 85 : 160 – 169 . Google Scholar CrossRef Search ADS PubMed 38. Brändle N , Zehnder M , Weiger R , Waltimo T . Impact of growth conditions on susceptibility of five microbial species to alkaline stress . J Endod . 2008 ; 34 : 579 – 582 . Google Scholar CrossRef Search ADS PubMed 39. Yamazaki H , Ohshima T , Tsubota Y , Yamaguchi H , Jayawardena JA , Nishimura Y . Microbicidal activities of low frequency atmospheric pressure plasma jets on oral pathogens . Dent Mater J . 2011 ; 30 : 384 – 391 . Google Scholar CrossRef Search ADS PubMed 40. Ramalingam K , Amaechi BT , Ralph RH , Lee VA . Antimicrobial activity of nanoemulsion on cariogenic planktonic and biofilm organisms . Arch Oral Biol . 2012 ; 57 : 15 – 22 . Google Scholar CrossRef Search ADS PubMed 41. Muhammad OH , Chevalier M , Rocca J-P , Brulat-Bouchard N , Medioni E . Photodynamic therapy versus ultrasonic irrigation: Interaction with endodontic microbial biofilm, an ex vivo study . Photodiagnosis Photodyn Ther . 2014 ; 11 : 171 – 181 . Google Scholar CrossRef Search ADS PubMed 42. Salli KM , Ouwehand AC . The use of in vitro model systems to study dental biofilms associated with caries: a short review . J Oral Microbiol . 2015 ; 7 : 26149 . Google Scholar CrossRef Search ADS PubMed 43. Rahmani-Badi A , Sepehr S , Babaie-Naiej H . A combination of cis-2-decenoic acid and chlorhexidine removes dental plaque . Arch Oral Biol . 2015 ; 60 : 1655 – 1661 . Google Scholar CrossRef Search ADS PubMed 44. Soares GMS , Teles F , Starr JR et al. Effects of azithromycin, metronidazole, amoxicillin, andmetronidazole plus amoxicillin on an in vitro polymicrobial subgingival biofilm model . Antimicrob Agents Chemother . 2015 ; 59 : 2791 – 2798 . Google Scholar CrossRef Search ADS PubMed 45. Periasamy S , Kolenbrander PE . Aggregatibacter actinomycetemcomitans builds mutualistic biofilm communities with Fusobacterium nucleatum and Veillonella species in saliva . Infect Immun . 2009 ; 77 : 3542 – 3551 . Google Scholar CrossRef Search ADS PubMed 46. Li H , Zhang C , Liu P , Liu W , Gao Y , Sun S . In vitro interactions between fluconazole and minocycline against mixed cultures of Candida albicans and Staphylococcus aureus . J Microbiol Immunol Infect . 2015 ; 48 : 655 – 661 . Google Scholar CrossRef Search ADS PubMed 47. Shen Y , Stojicic S , Qian W , Olsen I , Haapasalo M . The synergistic antimicrobial effect by mechanical agitation and two chlorhexidine preparations on biofilm bacteria . J Endod . 2010 ; 36 : 100 – 104 . Google Scholar CrossRef Search ADS PubMed 48. Fröjd V , Chávez de Paz L , Andersson M , Wennerberg A , Davies JR , Svensäter G . In situ analysis of multispecies biofilm formation on customized titanium surfaces . Mol Oral Microbiol . 2011 ; 26 : 241 – 252 . Google Scholar CrossRef Search ADS PubMed 49. Thurnheer T , Bostanci N , Belibasakis GN . Microbial dynamics during conversion from supragingival to subgingival biofilms in an in vitro model . Mol Oral Microbiol . 2016 ; 31 : 125 – 135 . Google Scholar CrossRef Search ADS PubMed 50. de Moraes AP Barwaldt CK , Nunes TZ et al. Effect of triazine derivative added to denture materials on a microcosm biofilm model . J Biomed Mater Res B Appl Biomater . 2012 ; 100 : 1328 – 1333 . Google Scholar CrossRef Search ADS PubMed 51. Koopman JE , Röling WFM , Buijs MJ et al. Stability and resilience of oral microcosms toward acidification and Candida outgrowth by arginine supplementation . Microb Ecol . 2015 ; 69 : 422 – 433 . Google Scholar CrossRef Search ADS PubMed 52. Verardi G , Cenci MS , Maske TT , Webber B , Santos LR dos . Antiseptics and microcosm biofilm formation on titanium surfaces. Braz Oral Res . 2016 ; 30 : doi: 10.1590/1807-3107BOR-2016 . 53. Akers KS , Cardile AP , Wenke JC , Murray CK . Biofilm formation by clinical isolates and its relevance to clinical infections . Adv Exp Med Biol . 2015 ; 830 : 1 – 28 . Google Scholar CrossRef Search ADS PubMed 54. Rudney JD , Chen R , Lenton P et al. A reproducible oral microcosm biofilm model for testing dental materials . J Appl Microbiol . 2012 ; 113 : 1540 – 1553 . Google Scholar CrossRef Search ADS PubMed 55. Jenkinson HF , Lamont RJ . Oral microbial communities in sickness and in health . Trends Microbiol . 2005 ; 13 : 589 – 595 . Google Scholar CrossRef Search ADS PubMed 56. Oh S , Go GW , Mylonakis E , Kim Y . The bacterial signalling molecule indole attenuates the virulence of the fungal pathogen Candida albicans . J Appl Microbiol . 2012 ; 113 : 622 – 628 . Google Scholar CrossRef Search ADS PubMed 57. Krause J , Geginat G , Tammer I . Prostaglandin E2 from Candida albicans stimulates the growth of Staphylococcus aureus in mixed biofilms . PLoS ONE . 2015 ; 10 : e0135404 . Google Scholar CrossRef Search ADS PubMed 58. Nobbs AH , Lamont RJ , Jenkinson HF . Streptococcus adherence and colonization . Microbiol Mol Biol Rev . 2009 ; 73 : 407 – 450 . Google Scholar CrossRef Search ADS PubMed 59. Murciano C , Moyes DL , Runglall M et al. Evaluation of the role of Candida albicans aAgglutinin-like sequence (Als) proteins in human oral epithelial cell interactions . PLoS ONE . 2012 ; 7 : e33362 . Google Scholar CrossRef Search ADS PubMed 60. Periasamy S , Kolenbrander PE . Mutualistic biofilm communities develop with Porphyromonas gingivalis and initial, early, and late colonizers of enamel . J Bacteriol . 2009 ; 191 : 6804 – 6811 . Google Scholar CrossRef Search ADS PubMed 61. Ammann TW , Belibasakis GN , Thurnheer T . Impact of early colonizers on in vitro subgingival biofilm formation . PLoS ONE . 2013 ; 8 : e83090 . Google Scholar CrossRef Search ADS PubMed 62. Cavalcanti IMG , Ricomini Filho AP , Lucena-Ferreira SC et al. Salivary pellicle composition and multispecies biofilm developed on titanium nitrided by cold plasma . Arch Oral Biol . 2014 ; 59 : 695 – 703 . Google Scholar CrossRef Search ADS PubMed 63. van de Sande FH , Azevedo MS , Lund RG , Huysmans MCDNJM , Cenci MS . An in vitro biofilm model for enamel demineralization and antimicrobial dose-response studies . Biofouling . 2011 ; 27 : 1057 – 1063 . Google Scholar CrossRef Search ADS PubMed 64. Matsubara VH , Wang Y , Bandara HMHN , Mayer MPA , Samaranayake LP . Probiotic lactobacilli inhibit early stages of Candida albicans biofilm development by reducing their growth, cell adhesion, and filamentation . Appl Microbiol Biotechnol . 2016 ; 100 : 6415 – 6426 . Google Scholar CrossRef Search ADS PubMed 65. Jiao Y , Cody GD , Harding AK et al. Characterization of extracellular polymeric substances from acidophilic microbial biofilms . Appl Environ Microbiol . 2010 ; 76 : 2916 – 2922 . Google Scholar CrossRef Search ADS PubMed 66. Mitchell KF , Zarnowski R , Sanchez H et al. Community participation in biofilm matrix assembly and function . Proc Natl Acad Sci U S A . 2015 ; 112 : 4092 – 4097 . Google Scholar CrossRef Search ADS PubMed 67. Sandai D , Tabana YM , Ouweini AE , Ayodeji IO . Resistance of Candida albicans biofilms to drugs and the host immune system . Jundishapur J Microbiol . 2016 ; 9 : e3738 . Google Scholar CrossRef Search ADS 68. Bhattacharyya S , Gupta P , Banerjee G , Jain A , Singh M . Inhibition of biofilm formation and lipase in Candida albicans by culture filtrate of Staphylococcus epidermidis in vitro . Int J Appl Basic Med Res . 2014 ; 4 : S27 – 30 . Google Scholar CrossRef Search ADS PubMed 69. Junka AF , Szymczyk P , Smutnicka D et al. Microbial biofilms are able to destroy hydroxyapatite in the absence of host immunity in vitro . J Oral Maxillofac Surg . 2015 ; 73 : 451 – 464 . Google Scholar CrossRef Search ADS PubMed 70. Willems HM , Kos K , Jabra-Rizk MA , Krom BP . Candida albicans in oral biofilms could prevent caries . Pathog Dis . 2016 ; 74 : ftw039 . Google Scholar CrossRef Search ADS PubMed 71. Hannan S , Ready D , Jasni AS , Rogers M , Pratten J , Roberts AP . Transfer of antibiotic resistance by transformation with eDNA within oral biofilms . FEMS Immunol Med Microbiol . 2010 ; 59 : 345 – 349 . Google Scholar CrossRef Search ADS PubMed 72. Jeon J-G , Pandit S , Xiao J et al. Influences of trans-trans farnesol, a membrane-targeting sesquiterpenoid, on Streptococcus mutans physiology and survival within mixed-species oral biofilms . Int J Oral Sci . 2011 ; 3 : 98 – 106 . Google Scholar CrossRef Search ADS PubMed 73. Jenssen H , Hamill P , Hancock REW . Peptide antimicrobial agents . Clin Microbiol Rev . 2006 ; 19 : 491 – 511 . Google Scholar CrossRef Search ADS PubMed 74. Sherry L , Lappin G , O’Donnell LE et al. Viable compositional analysis of an eleven species oral polymicrobial biofilm . Front Microbiol . 2016 ; 7 : 912 . Google Scholar CrossRef Search ADS PubMed 75. Reese S , Guggenheim B . A novel TEM contrasting technique for extracellular polysaccharides in in vitro biofilms . Microsc Res Tech . 2007 ; 70 : 816 – 822 . Google Scholar CrossRef Search ADS PubMed 76. Xu H , Sobue T , Bertolini M , Thompson A , Dongari-Bagtzoglou A . Streptococcus oralis and Candida albicans synergistically activate μ-Calpain to degrade E-cadherin from oral epithelial junctions . J Infect Dis . 2016 ; 214 : 925 – 934 . Google Scholar CrossRef Search ADS PubMed 77. El-Azizi M , Farag N , Khardori N . Antifungal activity of amphotericin B and voriconazole against the biofilms and biofilm-dispersed cells of Candida albicans employing a newly developed in vitro pharmacokinetic model . Ann Clin Microbiol Antimicrob . 2015 ; 14 : 21 . Google Scholar CrossRef Search ADS PubMed 78. Yassin SA , German MJ , Rolland SL , Rickard AH , Jakubovics NS . Inhibition of multispecies biofilms by a fluoride-releasing dental prosthesis copolymer . J Dent . 2016 ; 48 : 62 – 70 . Google Scholar CrossRef Search ADS PubMed 79. Schlecht LM , Peters BM , Krom BP et al. Systemic Staphylococcus aureus infection mediated by Candida albicans hyphal invasion of mucosal tissue . Microbiol Read Engl . 2015 ; 161 : 168 – 181 . Google Scholar CrossRef Search ADS 80. Montelongo-Jauregui D , Srinivasan A , Ramasubramanian AK , Lopez-Ribot JL . An in vitro model for oral mixed biofilms of Candida albicans and Streptococcus gordonii in synthetic saliva . Front Microbiol . 2016 ; 7 : 686 . Google Scholar CrossRef Search ADS PubMed 81. Kim Y-S , Kang S-M , Lee E-S , Lee JH , Kim B-R , Kim B-I . Ecological changes in oral microcosm biofilm during maturation. J Biomed Opt . 2016 ; 21 : 101409 – 101409 . Google Scholar CrossRef Search ADS PubMed 82. Fernandez y Mostajo M , Exterkate RAM , Buijs MJ , Crielaard W , Zaura E . Effect of mouthwashes on the composition and metabolic activity of oral biofilms grown in vitro . Clin Oral Investig . 2016 ; 21 : 1221 – 1230 . Google Scholar CrossRef Search ADS PubMed 83. Paster BJ , Olsen I , Aas JA , Dewhirst FE . The breadth of bacterial diversity in the human periodontal pocket and other oral sites . Periodontol 2000 . 2006 ; 42 : 80 – 87 . Google Scholar CrossRef Search ADS PubMed 84. Arzmi MH , Dashper S , Catmull D , Cirillo N , Reynolds EC , McCullough M . Coaggregation of Candida albicans, Actinomyces naeslundii and Streptococcus mutans is Candida albicans strain dependent . FEMS Yeast Res . 2015 ; 15 : fov038 . Google Scholar CrossRef Search ADS PubMed 85. Sobue T , Diaz P , Xu H , Bertolini M , Dongari-Bagtzoglou A . Experimental models of C. albicans-Streptococcal co-infection . Methods Mol Biol Clifton NJ . 2016 ; 1356 : 137 – 152 . Google Scholar CrossRef Search ADS 86. Pereira-Cenci T , Deng DM , Kraneveld EA et al. The effect of Streptococcus mutans and Candida glabrata on Candida albicans biofilms formed on different surfaces . Arch Oral Biol . 2008 ; 53 : 755 – 764 . Google Scholar CrossRef Search ADS PubMed 87. Thein ZM , Samaranayake YH , Samaranayake LP . Effect of oral bacteria on growth and survival of Candida albicans biofilms . Arch Oral Biol . 2006 ; 51 : 672 – 680 . Google Scholar CrossRef Search ADS PubMed 88. Bamford CV , d’Mello A , Nobbs AH , Dutton LC , Vickerman MM , Jenkinson HF . Streptococcus gordonii modulates Candida albicans biofilm formation through intergeneric communication . Infect Immun . 2009 ; 77 : 3696 – 3704 . Google Scholar CrossRef Search ADS PubMed 89. van Leeuwen PT , van der Peet JM , Bikker FJ et al. Interspecies interactions between Clostridium difficile and Candida albicans . mSphere . 2016 ; 1 : e00187 – 16 . Google Scholar CrossRef Search ADS PubMed 90. Ramírez Granillo A , Canales MGM , Espíndola MES , Martínez Rivera MA , de Lucio VMB , Tovar AVR . Antibiosis interaction of Staphylococccus aureus on Aspergillus fumigatus assessed in vitro by mixed biofilm formation . BMC Microbiol . 2015 ; 15 : 33 . Google Scholar CrossRef Search ADS PubMed 91. Exterkate RA , Crielaard W , Ten Cate JM. Different response to amine fluoride by Streptococcus mutans and polymicrobial biofilms in a novel high-throughput active attachment model . Caries Res . 2010 ; 44 : 372 – 379 . Google Scholar CrossRef Search ADS PubMed 92. Krishnamurthy A , Kyd J . The roles of epithelial cell contact, respiratory bacterial interactions and phosphorylcholine in promoting biofilm formation by Streptococcus pneumoniae and nontypeable Haemophilus influenzae . Microbes Infect . 2014 ; 16 : 640 – 647 . Google Scholar CrossRef Search ADS PubMed 93. Townsend EM , Sherry L , Rajendran R et al. Development and characterisation of a novel three-dimensional inter-kingdom wound biofilm model . Biofouling . 2016 ; 32 : 1259 – 1270 . Google Scholar CrossRef Search ADS PubMed 94. Sampaio FC , Pereira M , do SV , Dias CS , Costa VCO , Conde NCO , Buzalaf MAR . In vitro antimicrobial activity of Caesalpinia ferrea Martius fruits against oral pathogens . J Ethnopharmacol . 2009 ; 124 : 289 – 294 . Google Scholar CrossRef Search ADS PubMed 95. Ammann TW , Gmür R , Thurnheer T . Advancement of the 10-species subgingival Zurich biofilm model by examining different nutritional conditions and defining the structure of the in vitro biofilms . BMC Microbiol . 2012 ; 12 : 227 . Google Scholar CrossRef Search ADS PubMed 96. Kolenbrander PE. Multispecies communities: interspecies interactions influence growth on saliva as sole nutritional source . Int J Oral Sci . 2011 ; 3 : 49 – 54 . Google Scholar CrossRef Search ADS PubMed 97. Thurnheer T , Belibasakis GN , Bostanci N . Colonisation of gingival epithelia by subgingival biofilms in vitro: role of “red complex” bacteria . Arch Oral Biol . 2014 ; 59 : 977 – 986 . Google Scholar CrossRef Search ADS PubMed 98. Shen Y , Stojicic S , Haapasalo M . Bacterial viability in starved and revitalized biofilms: comparison of viability staining and direct culture . J Endod . 2010 ; 36 : 1820 – 1823 . Google Scholar CrossRef Search ADS PubMed 99. Eick S , Markauskaite G , Nietzsche S , Laugisch O , Salvi GE , Sculean A . Effect of photoactivated disinfection with a light-emitting diode on bacterial species and biofilms associated with periodontitis and peri-implantitis. Photodiagnosis Photodyn Ther . 2013 ; 10 : 156 – 167 . Google Scholar CrossRef Search ADS PubMed 100. Filoche SK , Soma KJ , Sissons CH . Caries-related plaque microcosm biofilms developed in microplates . Oral Microbiol Immunol . 2007 ; 22 : 73 – 79 . Google Scholar CrossRef Search ADS PubMed 101. Mistry KS , Sanghvi Z , Parmar G , Shah S , Pushpalatha K . Antibacterial efficacy of Azadirachta indica, Mimusops elengi and 2% CHX on multispecies dentinal biofilm . J Conserv Dent . 2015 ; 18 : 461 – 466 . Google Scholar CrossRef Search ADS PubMed 102. Extremina CI , Costa L , Aguiar AI , Peixe L , Fonseca AP . Optimization of processing conditions for the quantification of enterococci biofilms using microtitre-plates. J Microbiol Methods . 2011 ; 84 : 167 – 173 . Google Scholar CrossRef Search ADS PubMed 103. Cavalcanti YW , Morse DJ , da Silva WJ et al. Virulence and pathogenicity of Candida albicans is enhanced in biofilms containing oral bacteria . Biofouling . 2015 ; 31 : 27 – 38 . Google Scholar CrossRef Search ADS PubMed 104. Standar K , Kreikemeyer B , Redanz S , Munter WL , Laue M , Podbielski A . Setup of an in vitro test system for basic studies on biofilm behavior of mixed-species cultures with dental and periodontal pathogens . PLoS ONE . 2010 ; 5 : e13135 . Google Scholar CrossRef Search ADS PubMed 105. Schlafer S , Raarup MK , Wejse PL et al. Osteopontin reduces biofilm formation in a multi-species model of dental biofilm . PLoS ONE . 2012 ; 7 : e41534 . Google Scholar CrossRef Search ADS PubMed 106. Chávez de Paz LE . Development of a multispecies biofilm community by four root canal bacteria . J Endod. 2012 ; 38 : 318 – 323 . Google Scholar CrossRef Search ADS PubMed 107. Luppens SBI , Kara D , Bandounas L et al. Effect of Veillonella parvula on the antimicrobial resistance and gene expression of Streptococcus mutans grown in a dual-species biofilm . Oral Microbiol Immunol . 2008 ; 23 : 183 – 189 . Google Scholar CrossRef Search ADS PubMed 108. Cao Y-Y , Cao Y-B , Xu Z et al. cDNA microarray analysis of differential gene expression in Candida albicans biofilm exposed to farnesol . Antimicrob Agents Chemother . 2005 ; 49 : 584 – 589 . Google Scholar CrossRef Search ADS PubMed © The Author(s) 2017. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices)

Journal

Medical MycologyOxford University Press

Published: Aug 1, 2018

There are no references for this article.