Integrated Analysis of Clinical and Microbiome Risk Factors Associated with the Development of Oral Candidiasis during Cancer Chemotherapy.
ABSTRACT: Oral candidiasis is a common side effect of cancer chemotherapy. To better understand predisposing factors, we followed forty-five subjects who received 5-fluorouracil- or doxorubicin-based treatment, during one chemotherapy cycle. Subjects were evaluated at baseline, prior to the first infusion, and at three additional visits within a two-week window. We assessed the demographic, medical and oral health parameters, neutrophil surveillance, and characterized the salivary bacteriome and mycobiome communities through amplicon high throughput sequencing. Twenty percent of all subjects developed oral candidiasis. Using multivariate statistics, we identified smoking, amount of dental plaque, low bacteriome and mycobiome alpha-diversity, and the proportions of specific bacterial and fungal taxa as baseline predictors of oral candidiasis development during the treatment cycle. All subjects who developed oral candidiasis had baseline microbiome communities dominated by Candida and enriched in aciduric bacteria. Longitudinally, oral candidiasis was associated with a decrease in salivary flow prior to lesion development, and occurred simultaneously or before oral mucositis. Candidiasis was also longitudinally associated with a decrease in peripheral neutrophils but increased the neutrophil killing capacity of Candida albicans. Oral candidiasis was not found to be associated with mycobiome structure shifts during the cycle but was the result of an increase in Candida load, with C. albicans and Candida dubliniensis being the most abundant species comprising the salivary mycobiome of the affected subjects. In conclusion, we identified a set of clinical and microbiome baseline factors associated with susceptibility to oral candidiasis, which might be useful tools in identifying at risk individuals, prior to chemotherapy.
Project description:Although the term "microbiome" refers to all microorganisms, the majority of microbiome studies focus on the bacteriome. Here, we characterize the oral mycobiome, including mycobiome-bacteriome interactions, in the setting of remission-induction chemotherapy (RIC) for acute myeloid leukemia (AML). Oral samples (n?=?299) were prospectively collected twice weekly from 39 AML patients during RIC until neutrophil recovery. Illumina MiSeq 16S rRNA gene (V4) and internal transcribed spacer 2 (ITS2) sequencing were used to determine bacterial and fungal diversity and community composition. Intrakingdom and interkingdom network connectivity at baseline (T1) and at midpoint (T3) and a later time point (T6) were assessed via SPIEC-EASI (sparse inverse covariance estimation for ecological association inference). In this exploratory study, mycobiome ?-diversity was not significantly associated with antibiotic or antifungal receipt. However, postchemotherapy mycobiome ?-diversity was lower in subjects receiving high-intensity chemotherapy. Additionally, greater decreases in Malassezia levels were seen over time among patients on high-intensity RIC compared to low-intensity RIC (P?=?0.003). A significantly higher relative abundance of Candida was found among patients who had infection (P?=?0.008), while a significantly higher relative abundance of Fusarium was found among patients who did not get an infection (P?=?0.03). Analyses of intrakingdom and interkingdom relationships at T1, T3, and T6 indicated that interkingdom connectivity increased over the course of IC as bacterial ?-diversity diminished. In (to our knowledge) the first longitudinal mycobiome study performed during AML RIC, we found that mycobiome-bacteriome interactions are highly dynamic. Our study data suggest that inclusion of mycobiome analysis in the design of microbiome studies may be necessary to optimally understand the ecological and functional role of microbial communities in clinical outcomes.IMPORTANCE This report highlights the importance of longitudinal, parallel characterization of oral fungi and bacteria in order to better elucidate the dynamic changes in microbial community structure and interkingdom functional interactions during the injury of chemotherapy and antibiotic exposure as well as the clinical consequences of these interrelated alterations.
Project description:Oral microbiota contribute to health and disease, and their disruption may influence the course of oral diseases. Here, we used pyrosequencing to characterize the oral bacteriome and mycobiome of 12 HIV-infected patients and matched 12 uninfected controls. The number of bacterial and fungal genera in individuals ranged between 8-14 and 1-9, among uninfected and HIV-infected participants, respectively. The core oral bacteriome (COB) comprised 14 genera, of which 13 were common between the two groups. In contrast, the core oral mycobiome (COM) differed between HIV-infected and uninfected individuals, with Candida being the predominant fungus in both groups. Among Candida species, C. albicans was the most common (58% in uninfected and 83% in HIV-infected participants). Furthermore, 15 and 12 bacteria-fungi pairs were correlated significantly within uninfected and HIV-infected groups, respectively. Increase in Candida colonization was associated with a concomitant decrease in the abundance of Pichia, suggesting antagonism. We found that Pichia spent medium (PSM) inhibited growth of Candida, Aspergillus and Fusarium. Moreover, Pichia cells and PSM inhibited Candida biofilms (P?=?.002 and .02, respectively, compared to untreated controls). The mechanism by which Pichia inhibited Candida involved nutrient limitation, and modulation of growth and virulence factors. Finally, in an experimental murine model of oral candidiasis, we demonstrated that mice treated with PSM exhibited significantly lower infection score (P?=?.011) and fungal burden (P?=?.04) compared to untreated mice. Moreover, tongues of PSM-treated mice had few hyphae and intact epithelium, while vehicle- and nystatin-treated mice exhibited extensive fungal invasion of tissue with epithelial disruption. These results showed that PSM was efficacious against oral candidiasis in vitro and in vivo. The inhibitory activity of PSM was associated with secretory protein/s. Our findings provide the first evidence of interaction among members of the oral mycobiota, and identifies a potential novel antifungal.
Project description:Background: Recent studies have reveled the presence of a complex fungal community (mycobiome) in the oral cavity. However, the role of oral mycobiome in dental caries and its interaction with caries-associated bacteria is not yet clear. Methods: Whole-mouth supragingival plaque samples from 30 children (6-10 years old) with no caries, early caries, or advanced caries were sequenced for internal transcribed spacer 2 (ITS-2). The mycobiome profiles were correlated with previously published bacteriome counterparts. Interaction among selected fungal and bacterial species was assessed by co-culture or spent media experiments. Results: Fungal load was extremely low. Candida, Malassezia, Cryptococcus, and Trichoderma spp. were the most prevalent/abundant taxa. Advanced caries was associated with significantly higher fungal load and prevalence/abundance of Candida albicans. Cryptococcus neoformans and Candida sake were significantly over-abundant in early caries, while Malassezia globosa was significantly enriched in caries-free subjects. C. albicans correlated with Streptococcus mutans and Scardovia wiggsiae among other caries-associated bacteria, while M. globosa inversely correlated with caries-associated bacteria. In-vitro, M. globosa demonstrated inhibitory properties against S. mutans. Conclusions: the results substantiate the potential role of the oral mycobiome, primarily Candida species, in dental caries. Inter-kingdom correlations and inhibition of S. mutans by M. globosa are worth further investigation.
Project description:BACKGROUND:Gastrointestinal mucosal injury (mucositis), commonly affecting the oral cavity, is a clinically significant yet incompletely understood complication of cancer chemotherapy. Although antineoplastic cytotoxicity constitutes the primary injury trigger, the interaction of oral microbial commensals with mucosal tissues could modify the response. It is not clear, however, whether chemotherapy and its associated treatments affect oral microbial communities disrupting the homeostatic balance between resident microorganisms and the adjacent mucosa and if such alterations are associated with mucositis. To gain knowledge on the pathophysiology of oral mucositis, 49 subjects receiving 5-fluorouracil (5-FU) or doxorubicin-based chemotherapy were evaluated longitudinally during one cycle, assessing clinical outcomes, bacterial and fungal oral microbiome changes, and epithelial transcriptome responses. As a control for microbiome stability, 30 non-cancer subjects were longitudinally assessed. Through complementary in vitro assays, we also evaluated the antibacterial potential of 5-FU on oral microorganisms and the interaction of commensals with oral epithelial tissues. RESULTS:Oral mucositis severity was associated with 5-FU, increased salivary flow, and higher oral granulocyte counts. The oral bacteriome was disrupted during chemotherapy and while antibiotic and acid inhibitor intake contributed to these changes, bacteriome disruptions were also correlated with antineoplastics and independently and strongly associated with oral mucositis severity. Mucositis-associated bacteriome shifts included depletion of common health-associated commensals from the genera Streptococcus, Actinomyces, Gemella, Granulicatella, and Veillonella and enrichment of Gram-negative bacteria such as Fusobacterium nucleatum and Prevotella oris. Shifts could not be explained by a direct antibacterial effect of 5-FU, but rather resembled the inflammation-associated dysbiotic shifts seen in other oral conditions. Epithelial transcriptional responses during chemotherapy included upregulation of genes involved in innate immunity and apoptosis. Using a multilayer epithelial construct, we show mucositis-associated dysbiotic shifts may contribute to aggravate mucosal damage since the mucositis-depleted Streptococcus salivarius was tolerated as a commensal, while the mucositis-enriched F. nucleatum displayed pro-inflammatory and pro-apoptotic capacity. CONCLUSIONS:Altogether, our work reveals that chemotherapy-induced oral mucositis is associated with bacterial dysbiosis and demonstrates the potential for dysbiotic shifts to aggravate antineoplastic-induced epithelial injury. These findings suggest that control of oral bacterial dysbiosis could represent a novel preventive approach to ameliorate oral mucositis.
Project description:The etiology of head and neck squamous cell carcinoma (HNSCC) is not fully understood. While risk factors such as positive human papilloma virus (HPV) status, smoking and tobacco use have been identified, they do not account for all cases of the disease. We aimed to characterize the bacteriome, mycobiome and mycobiome-bacteriome interactions of oral wash in HNSCC patients and to determine if they are distinct from those of the oral wash of matched non-HNSCC patients. Oral wash samples were collected from 46 individuals with HNSCC and 46 controls for microbiome analyses. We identified three fungal phyla and eleven bacterial phyla of which Ascomycota (fungi, 72%) and Firmicutes (bacteria, 39%) were the most dominant, respectively. A number of organisms were identified as being differentially abundant between oral wash samples from patients with HNSCC and oral wash samples from those without HNSCC. Of note, strains of Candida albicans and Rothia mucilaginosa were differentially abundant and Schizophyllum commune was depleted in those with HNSCC compared to oral wash from those without HNSCC. Our results suggest that the oral cavity of HNSCC patients harbors unique differences in the mycobiome, bacteriome, and microbiome interactions when compared to those of control patients.
Project description:We implemented a Systems Biology approach using Correlation Difference Probability Network (CDPN) analysis to provide insights into the statistically significant functional differences between HIV-infected patients and uninfected individuals. The analysis correlates bacterial microbiome ("bacteriome"), fungal microbiome ("mycobiome"), and metabolome data to model the underlying biological processes comprising the Human Oral Metabiome. CDPN highlights the taxa-metabolite-taxa differences between the cohorts that frequently capture quorum-sensing modifications that reflect communication disruptions in the dysbiotic HIV cohort. The results also highlight the significant role of cyclic mono and dipeptides as quorum-sensing (QS) mediators between oral bacteria and fungal genus. The developed CDPN approach allowed us to model the interactions of taxa and key metabolites, and hypothesize their possible contribution to the etiology of Oral Candidiasis (OC).
Project description:BACKGROUND:Microbial flora in several organs of HIV-infected individuals have been characterized; however, the palatine tonsil bacteriome and mycobiome and their relationship with each other remain unclear. Determining the palatine tonsil microbiome may provide a better understanding of the pathogenesis of oral and systemic complications in HIV-infected individuals. We conducted a cross-sectional study to characterize the palatine tonsil microbiome in HIV-infected individuals. RESULTS:Palatine tonsillar swabs were collected from 46 HIV-infected and 20 HIV-uninfected individuals. The bacteriome and mycobiome were analyzed by amplicon sequencing using Illumina MiSeq. The palatine tonsil bacteriome of the HIV-infected individuals differed from that of HIV-uninfected individuals in terms of the decreased relative abundances of the commensal genera Neisseria and Haemophilus. At the species level, the relative abundances and presence of Capnocytophaga ochracea, Neisseria cinerea, and Selenomonas noxia were higher in the HIV-infected group than those in the HIV-uninfected group. In contrast, fungal diversity and composition did not differ significantly between the two groups. Microbial intercorrelation analysis revealed that Candida and Neisseria were negatively correlated with each other in the HIV-infected group. HIV immune status did not influence the palatine tonsil microbiome in the HIV-infected individuals. CONCLUSIONS:HIV-infected individuals exhibit dysbiotic changes in their palatine tonsil bacteriome, independent of immunological status.
Project description:HIV-infected individuals constitute a population highly susceptible to opportunistic infections, particularly oral candidiasis caused by the most pathogenic human fungal species Candida albicans. Host-produced salivary antimicrobial peptides are considered to be an important part of the host innate immune system involved in protection of the oral cavity against colonization and infection by microbial species. Histatin-5 (Hst-5) specifically has exhibited potent anti-candidal properties in vitro. However, its importance in protecting the oral mucosa against candidal colonization and importantly, its contribution to the observed enhanced susceptibility of HIV-infected individuals to candidiasis has not been previously investigated. To that end, a novel immunoassay was used to demonstrate significant decrease in salivary Hst-5 levels in HIV+ individuals concomitant with enhanced candidal prevalence. Further, saliva's anti-candidal potency was found to be proportional to Hst-5 concentration and significantly compromised in HIV+ subjects compared to controls. The key role for Hst-5 was further confirmed upon exposure to the Hst-5-specific antibody where saliva's initial killing activity was substantially compromised. Combined, these findings identify Hst-5 as a key anti-candidal salivary component and demonstrate its decreased levels in HIV infection providing new insights into oral Innate immune defense mechanisms and the enhanced susceptibility of HIV+ individuals to oral candidiasis.
Project description:Squamous cell carcinoma of the oral (mobile) tongue (OMTC), a non-human papilloma virus-associated oral cancer, is rapidly increasing without clear etiology. Poor oral hygiene has been associated with oral cancers, suggesting that oral bacteriome (bacterial community) and mycobiome (fungal community) could play a role. While the bacteriome is increasingly recognized as an active participant in health, the role of the mycobiome has not been studied in OMTC. Tissue DNA was extracted from 39 paired tumor and adjacent normal tissues from patients with OMTC. Microbiome profiling, principal coordinate, and dissimilarity index analyses showed bacterial diversity and richness, and fungal richness, were significantly reduced in tumor tissue (TT) compared to their matched non-tumor tissues (NTT, P<0.006). Firmicutes was the most abundant bacterial phylum, which was significantly increased in TT compared to NTT (48% vs. 40%, respectively; P=0.004). Abundance of Bacteroidetes and Fusobacteria were significantly decreased in TT compared to matched NTT (P?0.003 for both). Abundance of 22 bacterial and 7 fungal genera was significantly different between the TT and NTT, including Streptococcus, which was the most abundant and significantly increased in the tumor group (34% vs. 22%, P<0.001). Abundance of fungal genus Aspergillus in TT correlated negatively with bacteria (Actinomyces, Prevotella, Streptococcus), but positively with Aggregatibacter. Patients with high T-stage disease had lower mean differences between TT and NTT compared with patients with low T-stage disease (0.07 vs. 0.21, P=0.04). Our results demonstrate differences in bacteriome and mycobiome between OMTC and their matched normal oral epithelium, and their association with T-stage.
Project description:Oropharyngeal candidiasis (OPC, thrush) is an opportunistic infection caused by the commensal fungus Candida albicans. An understanding of immunity to Candida has recently begun to unfold with the identification of fungal pattern-recognition receptors such as C-type lectin receptors, which trigger protective T-helper (Th)17 responses in the mucosa. Hyper-IgE syndrome (HIES/Job's syndrome) is a rare congenital immunodeficiency characterized by dominant-negative mutations in signal transducer and activator of transcription 3, which is downstream of the Th17-inductive cytokines interleukin (IL)-6 and IL-23, and hence patients with HIES exhibit dramatic Th17 deficits. HIES patients develop oral and mucocutaneous candidiasis, supporting a protective role for Th17 cells in immunity to OPC. However, the Th17-dependent mechanisms of antifungal immunity in OPC are still poorly defined. An often unappreciated aspect of oral immunity is saliva, which is rich in antimicrobial proteins (AMPs) and exerts direct antifungal activity. In this study, we show that HIES patients show significant impairment in salivary AMPs, including ?-defensin 2 and Histatins. This tightly correlates with reduced candidacidal activity of saliva and concomitantly elevated colonization with Candida. Moreover, IL-17 induces histatins in cultured salivary gland cells. This is the first demonstration that HIES is associated with defective salivary activity, and provides a mechanism for the severe susceptibility of these patients to OPC.