Critically Appraising the Significance of the Oral Mycobiome

Abstract

Recent efforts to understand the oral microbiome have focused on its fungal component. Since fungi occupy a low proportion of the oral microbiome biomass, mycobiome studies rely on sequencing of internal transcribed spacer (ITS) amplicons. ITS-based studies usually detect hundreds of fungi in oral samples. Here, we review the oral mycobiome, critically appraising the significance of such large fungal diversity. When harsh lysis methods are used to extract DNA, 2 oral mycobiome community types (mycotypes) are evident, each dominated by only 1 genus, either Candida or Malassezia. The rest of the diversity in ITS surveys represents low-abundance fungi possibly acquired from the environment and ingested food. So far, Candida is the only genus demonstrated to reach a significant biomass in the oral cavity and clearly shown to be associated with a distinct oral ecology. Candida thrives in the presence of lower oral pH and is enriched in caries, with mechanistic studies in animal models suggesting it participates in the disease process by synergistically interacting with acidogenic bacteria. Candida serves as the main etiological agent of oral mucosal candidiasis, in which a Candida-bacteriome partnership plays a key role. The function of other potential oral colonizers, such as lipid-dependent Malassezia, is still unclear, with further studies needed to establish whether Malassezia are metabolically active oral commensals. Low-abundance oral mycobiome members acquired from the environment may be viable in the oral cavity, and although they may not play a significant role in microbiome communities, they could serve as opportunistic pathogens in immunocompromised hosts. We suggest that further work is needed to ascertain the significance of oral mycobiome members beyond Candida. ITS-based surveys should be complemented with other methods to determine the in situ biomass and metabolic state of fungi thought to play a role in the oral environment.

Introduction

Advances in DNA sequencing have facilitated the study of human microbiome communities under states of equilibrium with the host (health) or as modified by environmental and host-driven disruptions and associated with disease (dysbiosis). While most oral microbiome surveys have focused on bacteria, there is a recent emergence of studies on oral fungal communities—the oral mycobiome (see studies in the Table). These studies usually report hundreds of fungal taxa, suggesting a highly diverse mycobiome that could play a role in the oral microbiome by participating in the collective functions of the community and interacting with the adjacent mucosal tissues and teeth. Therefore, fungi have been suggested as understudied components of the oral microbiome with essential roles in health and disease. The reporting of a large collection of fungal DNA signatures in oral samples, however, has not been complemented by studies that demonstrate their functionality.

Table.

Most Commonly Reported Fungal Genera in Oral Mycobiome Studies.

Taxon	No. of Studies Reporting Taxon	No. of Studies from Previous Column That Sequenced ITS-1	Type of Oral Samples in Which Taxon Was Detected (See Coding Below)	Age Group of Subjects in Which Taxon Was Detected	Studies Reporting Taxon (See Coding Below)
Candida	20	14	SA, OR, MS, SUP, SUB	Adults, children	a to f, h to u
Cladosporium/Davidiella	15	10	SA, OR, MS, SUP	Adults, children	a, c, d, f, h to r, t
Saccharomyces	15	9	SA, OR, MS, SUP, SUB	Adults, children	b, c, d, f, h to k, m to o, r to u
Penicillium/Talaromyces	14	10	SA, OR, MS, SUP, SUB	Adults, children	a to d, f, g, h to k, m, n, q, u
Malassezia	13	8	SA, OR, MS, SUP, SUB	Adults, children	a to c, e, f, h, j to m, q to s
Alternaria/Lewia	12	7	SA, OR, MS, SUP	Adults, children	c, f, h, i, j, m, n, p to s, u
Pichia/Cyberlindnera	12	11	SA, OR, MS, SUP	Adults, children	a, e, f, i to l, n, p, q, t, u
Aspergillus/Emericella/Eurotium	11	9	SA, OR, MS, SUP	Adults, children	a, f, i to k, m, n, p, q, s, u
Cryptococcus/Cystofilobasidium	10	7	SA, OR, MS, SUP	Adults, children	a, c, f, h to k, n, q, t
Aureobasidium	8	6	SA, OR, MS, SUP, SUB	Adults, children	a, b, c, f, h, i, n, q
Rhodotorula	7	6	SA, OR, MS, SUP, SUB	Adults, children	b, h, j, k, n, p, q
Trichosporon	7	6	SA, OR, SUP	Adults, children	e, i, k to n, q
Debaromyces	6	4	SA, OR, MS, SUP, SUB	Adults, children	b, h, j, k, p, t
Wallemia	6	6	SA, OR, SUP	Adults, children	e, i, j, l, p, q
Clavispora	5	5	SA, OR	Adults	i to k, n, o
Meyerozima	5	3	OR, MS, SUP	Adults, children	c, k, l, q, r
Mycosphaerella	5	4	SA, OR, SUP	Adults, children	e, f, m, p, q
Phaeosphaeria	5	4	SA, OR	Adults	f, i, j, m, n
Unclassified Saccharomycetales	5	3	SA, MS, SUP	Adults, children	a, h, i, j, m
Trichoderma	5	3	SA, OR, SUP	Adults, children	c, f, k, m, q
Epicoccum	4	4	SA, OR	Adults	f, i, n, q
Toxicocladosporium	4	3	SA, OR, MS, SUP	Adults, children	h, j, k, p

Twenty-one original research articles evaluating the oral mycobiome were included (a, Abusleme et al. 2018; b, Annavajhala et al. 2020; c, Baraniya et al. 2020; d, Cui et al. 2015; e, de Jesus et al. 2020; f, Dupuy et al. 2014; g, El Jurdi et al. 2019; h, Fechney et al. 2019; i, Ghannoum et al. 2010; j, Hong et al. 2020; k, Ieda et al. 2014; l, Imabayashi et al. 2016; m, Li et al. 2019; n, Mukherjee et al. 2014; o, Mukherjee et al. 2018; p, O’Connell et al. 2020; q, Peters et al. 2017; r, Robinson et al. 2020; s, Shelburne et al. 2015; t, Ward et al. 2018; u, Watkins et al. 2016). Fungi were considered in the tally if they appeared in the main figures and tables of the manuscript.

ITS-1, internal transcribed spacer 1; MS, mucosal swab; OR, oral rinse; SA, saliva; SUB, subgingival plaque; SUP, supragingival plaque.

Here, we critically review oral mycobiome studies arguing that caution should be exercised when interpreting mycobiome sequencing surveys, especially those based on internal transcribed spacer (ITS) amplicons. We present evidence supporting the argument that until now, only a few fungi have been demonstrated to be true oral colonizers, that is, microorganisms capable of dividing and having an active metabolism in the oral cavity. Most fungal DNA signatures reported in mycobiome surveys possibly originate from transient colonizers present in ingested food or in the environment. We discuss (a) the ecological and clinical roles of fungi so far demonstrated to be significant colonizers, (b) the potential for fungi reported as common and abundant in molecular studies to constitute functional mycobiome members, and (c) the possible significance of the nonresident component of the oral mycobiome. We then discuss the need to complement ITS-based surveys with other methods to determine the in situ metabolic activity and biomass of fungal cells and to explore whether fungi have the potential to contribute in a significant manner to oral health or disease.

Oral Mycobiome Composition

In the past decade, more than 20 original research articles (see Table) have explored the composition of the mycobiome in oral samples, including rinses, saliva, mucosal swabs, and dental plaque collections. All these studies relied on amplification and sequencing of the ITS region, which is located between the 18S and 28S ribosomal RNA (rRNA) genes and includes ITS-1, the 5.8S gene, and the ITS-2 region. Most studies sequenced ITS-1, as it is the most rapidly evolving region, while 7 of 21 oral studies relied on ITS-2, which is a region evolving at moderate to rapid speed but that still harbors enough variability to allow taxonomic discrimination of a great number of fungi (Hershkovitz and Lewis 1996; Nilsson et al. 2008). ITS, however, has some limitations. For instance, for the genus Candida, which is the largest medically important genus comprising ~200 species, ITS is unable to discriminate among certain species (e.g., albicans vs. africana) due to high degree of homology (Brandt and Lockhart 2012).

To examine the taxa that comprise the oral mycobiome, we compared studies conducted to date creating a tally of fungi reported in the main tables and graphs of each manuscript, which usually include the most prevalent and abundant taxa and those fungi enriched under the conditions evaluated. This analysis showed a total of 193 genus-level taxa as significant components of the oral mycobiome. The total number of genera detected in these studies is much higher since only taxa reported in the main body of the manuscripts were included in the count. Moreover, the number of ITS-detected species would be much higher, but due to methodological limitations, the number of species represented in the reported genera is still unknown (Diaz et al. 2017). The number of taxa comprising the oral mycobiome, as suggested by ITS-based studies, seems therefore comparable to that in the oral bacteriome (Dewhirst et al. 2010). Are we thus missing knowledge on a component of the oral microbiome that could be playing a role as significant as that of bacteria?

When analyzing the tally used to construct the Table, it was striking to observe that out of 193 genera that appeared as significant components of the oral mycobiome in the 21 studies reviewed, the genus Candida was the only one reported in most studies, and just 22 genera appeared in at least 4 studies. In total, 116 genera were reported in only 1 study, highlighting the small overlap among different ITS surveys. Although some of the differences in reported fungi could be attributed to sample processing and specific population characteristics, a much higher degree of overlap among studies is to be expected, especially at a genus level, if a diverse core mycobiome existed in the human oral cavity.

Lack of overlap among mycobiome studies could be related to the low biomass at which most oral fungi are present, which decreases their detection probability. Measurement of total salivary fungal load using molecular methods in 410 elderly subjects has showed that the communities of subjects with the highest loads were those dominated by Candida, while those in which Candida was in low proportion had a low load (Zakaria et al. 2017). Cultivation studies agree with these results, detecting fungal concentrations in the range of 0 to 400 colony-forming units (CFU) per milliliter (of a 10-mL mouth rinse) in immunocompetent subjects free of mucosal disease (Monteiro-da-Silva et al. 2014). Candida, however, was the only genus seen at concentrations higher than 5 CFU/mL, with other yeast such as Rhodotorula and molds such as Aspergillus, Cladosporium, and Penicillium showing high prevalence but very low abundance (less than 5 CFU/mL). It appears, then, that Candida is the most abundant genus in oral samples, while other fungi are present in many subjects but in very low abundance.

Low-load fungi reported via ITS amplification may also represent environmentally acquired transient occupants. Even prevalent fungi common among oral mycobiome studies (shown in the Table) are frequently found in indoor and outdoor environments and in foods such as legumes, vegetables, nuts, cheese, meat products, and bread and, therefore, can be continuously acquired from the environment (Tong et al. 2017; Auchtung et al. 2018). The low biomass at which most ITS-reported components of the oral mycobiome are present perhaps suggests that the oral environment is not suitable for the growth of these fungi. Some fungi may be limited by their optimal growth temperature, which for species of Penicillium, Cladosporium, and Alternaria has been shown to be below 26°C (Sautour et al. 2001). In the oral cavity, temperature is maintained between 32°C and 37°C (Barclay et al. 2005). A cultivation study that compared growth of fungi from oral samples when incubated at 25°C or 37°C highlights that that the optimal growth temperature for oral mycobiome taxa is closer to room than body temperature, with all samples analyzed yielding molds and most yielding yeasts at 25°C, while less than half were positive for molds or yeasts at 37°C (Monteiro-da-Silva et al. 2014). Environmentally acquired fungi could therefore constitute a viable but metabolically inactive “nonresident” component of the oral mycobiome. Other mycobiome members such as Saccharomyces, Aspergillus, and Candida have the potential to replicate in the oral environment as they grow at a wide range of temperatures, including 37°C (Rhodes 2006; Munna et al. 2015; Drott et al. 2019). The growth of these fungi, however, may be limited by factors such as competition for nutrients with bacteria and by the antifungal capacity of the host (Bartels and Blechman 1964; Vylkova et al. 2007; Puri and Edgerton 2014). Knowledge on which fungi are able to replicate in the oral environment and participate in a significant manner in the metabolism of oral communities is important to avoid meaningless interrogations of mycobiome data sets.

Candida and Malassezia Are the Most Abundant Oral Mycobiome Components

If harsh lysis protocols, including zirconia or steel beads to disrupt fungal cell walls, are used prior to DNA extraction, the ITS-based mycobiome of saliva and mucosal samples is found to be dominated by 2 genera: Candida and Malassezia (Abusleme et al. 2018; Hong et al. 2020). These 2 genera, however, seem to have an exclusive relationship with samples dominated by either one or the other. In a recent study, we showed these 2 community types (mycotypes) are associated with specific clinical and bacteriome characteristics (Hong et al. 2020). The less diverse Candida mycotype was associated with a less diverse bacteriome and with acidogenic and aciduric bacteria such as Lactobacillus and Propionibacterium, while the more diverse Malassezia-dominated communities were associated with a more diverse bacteriome enriched with inflammophilic species from the genera Fusobacterium, Porphyromonas, Prevotella, and Leptotrichia, among others. Mycotypes also correlated with specific clinical characteristics, with the Candida-dominated communities appearing more frequently in subjects who smoked, used a removable dental prosthesis, had active caries, had a higher plaque index, or were taking steroids. These results suggested that lack of oral hygiene, dietary carbohydrate availability (associated with caries), and impairment of host surveillance (by steroids and smoking) promote oral growth of Candida. On the other hand, Malassezia, which rely on exogenously acquired lipids for metabolic activities, are able to live in assacharolytic environments such as those also preferred by inflammophilic bacteria. Malassezia may acquire lipids from oral fluids and the host diet through its lipases (Larsson et al. 1996; Wu, Zhao, et al. 2015). The characteristics of these 2 mycobiome community types are summarized in the Figure.

Figure.

Main components of the oral mycobiome and their significance. Two community types (mycotypes) have been detected in saliva (Hong et al. 2020) and are represented in the figure by the red and green circles. Either mycotype is numerically dominated by Candida or Malassezia. The less diverse Candida mycotype is associated with a bacteriome of lower diversity enriched for aciduric species. The more diverse Malassezia mycotype is enriched for other fungi present in low proportions. The Malassezia mycotype is associated with a more diverse bacteriome enriched for inflammophilic species. Factors hypothesized to drive selection of mycotypes are depicted in the top portion of the figure. Smoking, intake of corticosteroids, a diet rich in carbohydrates, and wearing a removable prosthesis favor a Candida mycotype, while a nonimpaired immune system, a diet low in carbohydrates and high in lipids, and good oral hygiene favor a Malassezia mycotype. While evidence exists for some of these associations, further studies are needed to understand some of these correlations (e.g., the relationship of Malassezia and dietary lipids). The bottom of the figure shows conditions in which mycotypes may be able to serve as predictors. Carrying the Candida mycotype may increase risk of oral candidiasis (Diaz et al. 2019) and may predispose to increased caries (Abusleme et al. 2018; Xiao et al. 2018). The size of the circles depicting mycotypes represents hypothesized differences in the total fungal load in each of these communities.

Malassezia are the most abundant commensals of human skin (Findley et al. 2013). The same species dominating skin communities are also the most numerous in the oral cavity (Hong et al. 2020). To evaluate the viability and load of oral Malassezia, we have attempted to cultivate them from samples that were also sequenced (Hong et al. 2020). However, only a small proportion of samples yielded Malassezia and at very low abundance. The most abundant species of Malassezia (Malassezia restricta and Malassezia globosa) in the ITS results did not appear by cultivation. The low-abundance Malassezia sympodialis was the only Malassezia isolated. It is therefore still unclear if Malassezia is present at a significant load in the oral cavity in those subjects with a Malassezia mycotype and whether Malassezia plays an active role in the oral microbiome metabolic activities. It has been recently reported in in vitro assays that spent medium from M. globosa, which is found in dental plaque of caries-free individuals, inhibits growth of Streptococcus mutans, the main etiological agent of caries (Baraniya et al. 2020). It is therefore possible that if M. globosa is present in the oral cavity in a viable state and at a significant load, it could have a role in the maintenance of health. Further work, however, is needed to understand the functional role of oral Malassezia.

The mycotype classification, which discriminates between subjects with high and low Candida load, may have diagnostic utility. The proportion of Candida in saliva is among an array of predictive factors that identify subjects at risk of developing oral candidiasis during cancer chemotherapy (Diaz et al. 2019). The presence of a Candida mycotype may also be related to caries risk. Irrespective of whether Candida directly participates in the etiology of caries (see discussion below), detection of a Candida mycotype may serve as an indicator of caries risk. The utility of the mycotype classification as a diagnostic biomarker needs further study.

Role of the Oral Mycobiome in Mucosal Disease

Despite the plethora of mycobiome surveys, Candida still remains the only fungal genus that unequivocally contributes to the etiology of most common oral mucosal infections. As opportunistic pathogens, Candida species are equipped with an array of virulence factors that can directly contribute to mucosal barrier breach in susceptible hosts. Candida, however, interact closely with the bacteriome to induce pathology.

To better understand global microbiome changes associated with oral mucosal disease, we conducted the first prospective longitudinal study of oral mycobiome and bacteriome changes in chemotherapy-treated cancer patients who developed oropharyngeal candidiasis (Diaz et al. 2019). In this cohort, oropharyngeal candidiasis development was not associated with mycobiome structure shifts but was the result of increased Candida load, with Candida albicans and Candida dubliniensis being the most abundant species during active oral infection. Bacterial analyses conducted in parallel revealed that mucosal disease was associated with a trend for increased oral bacterial burdens. Moreover, a lower bacterial diversity at baseline and abundance of aciduric bacteria significantly increased the risk for infection. Our findings thus suggest that a Candida mycotype combined with increased abundance of aciduric bacteria may be one of the risk factors underlying susceptibility to oropharyngeal candidiasis. Similarly, in humans with hyper-IgE inflammatory syndrome who develop oropharyngeal candidiasis due to defective STAT3/Th17 immunity, aciduric streptococci Streptococcus oralis and S. mutans have been identified as the top abundant bacterial species enriched during active fungal infection (Abusleme et al. 2018).

Although strong positive associations have been demonstrated between certain bacteriome taxa and oropharyngeal candidiasis in human oral microbiome surveys, the questions of whether bacterial colonizers influence C. albicans virulence or whether changes in bacterial diversity are a consequence of fungal-induced mucosal inflammation need to be addressed in experimental models. Mouse studies from our group focusing on the interactions of C. albicans with a single oral streptococcal species (S. oralis) revealed increased fungal virulence, with both fungal- and host-dependent mechanisms involved in pathogenic synergy (Xu et al. 2014, 2016, 2017). On the fungal side, S. oralis promoted efg1-mediated filamentous growth and increased efg1-dependent C. albicans als1 gene and protein expression on the surface of hyphae, enhancing interspecies coaggregation and oral mucosal biofilms (Xu et al. 2017). On the host side, we found an amplified mucosal inflammatory response to the 2 organisms together and synergistic activation of μ-calpain, a proteolytic enzyme that targets E-cadherin and occludin from epithelial junctions, resulting in enhanced barrier breach (Xu et al. 2014, 2016). These studies provided evidence to support the hypothesis that oral bacteriome shifts resulting in increased abundance of aciduric species such asS. oralis can aggravate mucosal fungal infection. However, these studies are still limited by the fact that other mucosal microbiome members that potentially contribute to the disease phenotype were not considered. More recent studies in a mouse model of cancer chemotherapy treatment provided experimental evidence for the role of indigenous bacterial mucosal communities in Candida pathogenesis. Antibiotic depletion of indigenous bacteria during fungal infection attenuated oral mucosal E-cadherin degradation and C. albicans mucosal breach without affecting fungal burdens, indicating that bacterial community changes indeed contribute to pathogenesis (Bertolini et al. 2019). In summary, both human and animal studies support the central tenet that the oral mycobiome, mainly Candida, contributes to mucosal disease in close interaction with the oral bacteriome. Therefore, Candida-bacterial interactions could represent novel targets for the development of therapeutic strategies against oral candidiasis.

Role of the Oral Mycobiome in Dental Caries

The role of the oral mycobiome in caries has been the focus of recent research. Several mycobiome studies have explored fungal community diversity in relation to caries in children (Fechney et al. 2019; Baraniya et al. 2020; de Jesus et al. 2020; O’Connell et al. 2020). Studies that compared the oral mycobiome between caries-free and caries-affected children with mixed dentition reported differences in abundance of certain fungi according to caries status. While Fechney et al. (2019) reported that a few genera and species were associated with health and no specific taxa were associated with caries, Baraniya et al. (2020) found that advanced caries was associated with increased prevalence and abundance of C. albicans, while M. globosa was enriched in caries-free children. A positive correlation between the abundance of C. albicans and that of the caries-associated bacteria S. mutans and Scardovia wiggsiae was also found (Baraniya et al. 2020). Although these studies present discordant results, it should be considered that only pooled plaque was analyzed, instead of lesion-specific material, and their findings are qualified by a small sample size.

More definitive analyses have been conducted in children with early childhood caries (ECC). In a comparison of the pooled plaque mycobiome of 40 children with ECC to the same number of caries-free children, it was found that the genus Candida was enriched in ECC (de Jesus et al. 2020). At a species level, however, it was observed that it was Candida dubliniensis, and not C. albicans, that dominated the mycobiome of children with caries. This study also reported differences in the mycobiome and its relationship to caries according to sex, with caries-free females showing an enrichment of Malassezia restricta. In a site-specific analysis of the mycobiome associated with ECC, caries-free children were compared to those with lesions in enamel or dentin, also discriminating between healthy surfaces, sites with enamel caries, and dentin lesions (O’Connell et al. 2020). This study showed a trend for decreased mycobiome diversity as caries severity increased and found C. albicans and C. dubliniensis were positively correlated with caries. Interestingly, C. albicans was only associated with severe disease, while C. dubliniensis increased steadily as caries severity also increased.

Overall, these studies point to an association of C. albicans with advanced caries, which is consistent with a meta-analysis that evaluated the association of C. albicans and ECC, finding that children with detectable C. albicans were 5 times more likely to have ECC than children without C. albicans (Xiao et al. 2018). Mechanistic studies in animal models provide evidence that C. albicans could have a pathogenic role in caries, as its co-inoculation with S. mutans promotes development of rampant carious lesions when rodents are also given a high-sucrose diet (Falsetta et al. 2014). The potential effect on this interaction of indigenous dental plaque microbiome members, enriched or depleted by a high-sucrose diet, has not been considered in this model. In support of this notion, we recently reported that a high-sucrose diet in a murine candidiasis model is associated with a significant reduction in indigenous enterococci, which are the dominant members of the murine bacterial microbiota in candidiasis lesions (Bertolini et al. 2019; Souza et al. 2020).

An emerging species associated with caries in 2 independent ECC cohorts is C. dubliniensis (de Jesus et al. 2020; O’Connell et al. 2020). This species, which is closely related to C. albicans but less pathogenic in mucosal disease models, has mostly been associated with mucosal disease in human immunodeficiency virus (HIV) cohorts (Sullivan et al. 1995) but in a recent cultivation study has been shown to be increased in children with caries (Al-Ahmad et al. 2016). ITS amplicon-based sequencing studies increasingly identify this species as a core member of the oral mycobiota, with higher abundance in children (Imabayashi et al. 2016; Zakaria et al. 2017; Diaz et al. 2019; de Jesus et al. 2020; O’Connell et al. 2020). Whether C. dubliniensis plays a contributory role in caries pathogenesis has yet to been examined in animal models. A still unanswered question regarding the role of Candida in caries is whether it contributes to disease initiation as suggested by animal models or merely thrives in the conditions locally induced by acidogenic bacterial species. In cross-sectional studies, Candida certainly appears enriched in certain populations that develop caries, but only longitudinal studies can answer whether the level of Candida in a healthy site constitutes a risk factor for lesion development. Another recently addressed question regarding the role of Candida in caries is whether Candida occupies a proportion of the biomass in lesion sites that is significant enough to contribute to biofilm development of cariogenic bacteria. Microscopic in situ evaluations of intact clinical biofilm samples from subjects with caries suggest this is the case, with images showing fungal-bacterial corncobs in which streptococci arrange around C. albicans hyphae (Dige and Nyvad 2019; Kim and Koo 2020). Interestingly, these studies also provide functional in situ evidence that the relationship of S. mutans and Candida is mediated by extracellular polymers, more specifically glucans produced by functional glucosyltransferases, bound to the C. albicans surface, as suggested in previous models (Falsetta et al. 2014; Kim and Koo 2020).

Some ITS-based investigations have also suggested that certain fungi associated with caries-free children, such as Debaromyces, Rhodotorula, or Malassezia, could antagonize cariogenic bacteria (Baraniya et al. 2020; O’Connell et al. 2020). In situ demonstrations that these fungi are a significant part of biomass in health and further mechanistic studies are needed. In summary, Candida are enriched in caries, with mycobiome studies suggesting C. albicans is enriched in advanced lesions, while C. dubliniensis could be playing a role earlier in the disease process. In a similar manner, as seen in oral candidiasis, Candida and bacteria may synergistically interact in caries lesions. Longitudinal clinical studies, including interventional trials using antifungals, would provide further evidence strengthening the role of fungi in caries initiation and progression.

Role of Fungi in Periodontal Disease

Fungi have been detected in subgingival plaque, but their role in these communities is still unclear. Cultivation studies have reported increased detection of yeasts, mostly Candida, in individuals with periodontitis (Canabarro et al. 2013). Candida, however, has not appeared as enriched in periodontitis in molecular studies. The only evaluation of the subgingival mycobiome using ITS sequencing was performed in HIV-positive subjects on antiretroviral therapy (Annavajhala et al. 2020). This study found periodontitis was associated with lower mycobiome diversity and changes in relative abundance of only 2 taxa (reduced Saccharomyces cerevisiae and increased proportions of a Filobasidiales species) compared to individuals with no/mild disease. A metagenomic evaluation of subgingival plaque has shown C. albicans is enriched in periodontal health (Dabdoub et al. 2016). The subgingival presence of Candida, however, has been hypothesized to contribute to the process of periodontal dysbiosis by facilitating biofilm formation by bacterial plaque constituents such as Fusobacterium nucleatum and Porphyromonas gingivalis (Wu, Cen, et al. 2015; Sztukowska et al. 2018) or by allowing bacterial tissue invasion (Tamai et al. 2011).

Significance of Nonresident Colonizers

While fungi present in extremely low abundance are not likely to influence in a significant manner the metabolic activities of oral microbiome communities, they could still play a role modulating immune responses or becoming opportunistic pathogens under conditions of impaired host surveillance. In rare instances involving severe immunosuppression, Cryptococcus spp. and filamentous fungi (Aspergillus spp.) have been reported to cause oral mucosal lesions and disseminated infection to extraoral sites (Schwartz and Thiel 1997; Iatta et al. 2009). A documented case shows that acquisition of Mucur velutinosus in the oral cavity of an individual during treatment for acute myelogenous leukemia later resulted in development of invasive mucormycosis (Shelburne et al. 2015). Therefore, given a susceptible host, the low abundance oral fungal biome acquired from environmental sources could serve as an infection reservoir for systemic mycoses.

Future Directions

This review highlights the need to collect evidence on the metabolic state and load of fungi, other than Candida, in oral mycobiome communities. As high-throughput DNA sequencing becomes more efficient and affordable, it may be possible to evaluate the functional mycobiome biomass after deep sequencing of oral transcriptomes, which can reveal which taxa are in a metabolically functional state. Metabolome analyses of tissue or saliva samples may also be appropriate in this regard. Prior to performing those studies, it is imperative to ascertain that efficient protocols for lysis of fungal cell walls are implemented to avoid underrepresentation of difficult to lyse taxa such as Malassezia. Cultivation approaches and targeted polymerase chain reaction methods could also help quantify the abundance of specific mycobiome components and reveal their metabolic state. In situ investigations using different microscopic techniques are also required to assess in clinical samples the biomass and the specific architectural arrangements and interactions of fungi thought to be clinically important with other microbiome members. So far, the only mycobiome member shown to be associated with certain oral environments, enriched in immunosuppressed hosts and clearly associated with oral disease, is Candida. In both oral candidiasis and caries, Candida is seen to closely interact with bacteria in what appears to be a synergistic partnership. Whether other fungi play a role in the oral microbiome will be revealed in the next years as the field moves beyond ITS-based surveys into functional mycobiome studies.

Author Contributions

P.I. Diaz, A. Dongari-Bagtzoglou, contributed to conception and design, drafted and critically revised the manuscript. All authors gave final approval and agree to be accountable for all aspects of the work.