Integrated Analysis of Clinical and Microbiome Risk Factors Associated with the Development of Oral Candidiasis during Cancer Chemotherapy.

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

Chemotherapy-induced oral mucositis is associated with detrimental bacterial dysbiosis.

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

Further Defining Spectral Type "Y" and Exploring the Low-mass End of the Field Brown Dwarf Mass Function

We present the discovery of another seven Y dwarfs from the Wide-field Infrared Survey Explorer (WISE). Using these objects, as well as the first six WISE Y dwarf discoveries from Cushing et al., we further explore the transition between spectral types T and Y. We find that the T/Y boundary roughly coincides with the spot where the J-H colors of brown dwarfs, as predicted by models, turn back to the red. Moreover, we use preliminary trigonometric parallax measurements to show that the T/Y boundary may also correspond to the point at which the absolute H (1.6 um) and W2 (4.6 um) magnitudes plummet. We use these discoveries and their preliminary distances to place them in the larger context of the Solar Neighborhood. We present a table that updates the entire stellar and substellar constituency within 8 parsecs of the Sun, and we show that the current census has hydrogen-burning stars outnumbering brown dwarfs by roughly a factor of six. This factor will decrease with time as more brown dwarfs are identified within this volume, but unless there is a vast reservoir of cold brown dwarfs invisible to WISE, the final space density of brown dwarfs is still expected to fall well below that of stars. We also use these new Y dwarf discoveries, along with newly discovered T dwarfs from WISE, to investigate the field substellar mass function. We find that the overall space density of late-T and early-Y dwarfs matches that from simulations describing the mass function as a power law with slope -0.5 < alpha < 0.0; however, a power-law may provide a poor fit to the observed object counts as a function of spectral type because there are tantalizing hints that the number of brown dwarfs continues to rise from late-T to early-Y. More detailed monitoring and characterization of these Y dwarfs, along with dedicated searches aimed at identifying more examples, are certainly required.Comment: 91 pages, 15 figures, accepted for publication in The Astrophysical Journa

Fungal Communities in Oral Health and Disease

Fungal Communities in Oral Health and Disease Amanda K. Dupuy, PhD University of Connecticut, [2015] With advances in, and cost reduction of next generation sequencing technologies, assessing the presence of microbes in host niches in healthy and diseased states has become a more feasible task. However, these studies are often only limited to bacterial characterization, ignoring other important community members such as fungi, viruses, and protists. The research presented here begins to fill this gap by creating a roadmap for fungal community analysis. With fungal outbreaks on the rise, it is essential that the fungal kingdom is included in future microbiome analyses to gain a more comprehensive understanding of commensal species, how they maintain a healthy niche, and their impact on acquired diseases. One particularly at-risk group of immune compromised patients are those undergoing chemotherapy. Approximately 40% of such patients develop a debilitating side effect known as oral mucositis, which is complicated by severe pain, inability to eat and speak, and severe bacterial and fungal infections. The research in this dissertation focuses on three aims necessary for answering the question of how fungal genera are implicated in oral mucositis. First, we present a roadmap from sample processing to data analysis, describing challenges and solutions for characterizing fungal communities in any human-health related metagenomics study. Second, we address the healthy fungal mycobiome of saliva, providing evidence for new and existing members of the oral niche, while assessing the temporal variability in community composition in a healthy state. Third, we characterize the oral genera during chemotherapy in a longitudinal study of cancer patients, and document their changes during the progression and development of oral mucositis. Revealing and meeting the challenges associated with fungal metagenomic analysis by means of initial hand curation will pave way for development of new, much needed library preparation and bioinformatics tools. But above all, pinpointing community trends for susceptible subjects will ultimately provide unprecedented insight for implementation of prophylactic measures in cancer patients

Redefining the Human Oral Mycobiome with Improved Practices in Amplicon-based Taxonomy: Discovery of <i>Malassezia</i> as a Prominent Commensal

<div><p>Fungi are a large, complex group, increasingly recognized as emerging threats. Their roles as modifiers of health mandate accurate portrayals of fungal communities in humans. As an entry point into the airways and gastrointestinal tract, fungi in the mouth are relevant to several biocompartments. We have revised current practices in sequence-based taxonomy assignments and employed the improvements to address the question of the fungal genera present in the healthy human mouth. The human oral mycobiome was surveyed using massively parallel, high throughput sequencing of internal transcribed spacer 1 (ITS1) amplicons from saliva following robust extraction methods. Taxonomy was assigned by comparison to a curated reference dataset, followed by filtering with an empirically determined BLAST E-value match statistic (10⁻⁴²). Nomenclature corrections further refined results by conjoining redundant names for a single fungal genus. Following these curation steps, about two-thirds of the initially identified genera were eliminated. In comparison with the one similar metagenomic study and several earlier culture-based ones, our findings change the current conception of the oral mycobiome, especially with the discovery of the high prevalence and abundance of the genus <i>Malassezia</i>. Previously identified as an important pathogen of the skin, and recently reported as the predominant fungal genus at the nostril and backs of the head and ear, this is the first account of <i>Malassezia</i> in the human mouth. Findings from this study were in good agreement with others on the existence of many consensus members of the core mycobiome, and on unique patterns for individual subjects. This research offered a cautionary note about unconditional acceptance of lengthy lists of community members produced by automated assignments, provided a roadmap for enhancing the likely biological relevance of sequence-based fungal surveys, and built the foundation for understanding the role of fungi in health and disease of the oral cavity.</p></div

Quality Controls.

a<p>One region of an 8 gasket PTP was used for a positive control (C). Negatives, positives, and pilot samples (representing a subset of three subjects) were sequenced in one region on the same run, and pilot l (A) and negative control (B) sequences partitioned by MID.</p>b<p>QIIME restrictions: Minimum length  =  100 (after trimming forward primer and MID); maximum “N” = 1, maximum homocopolymer  = 10; maximum forward primer mismatch  = 2; maximum barcode mismatch  = 2.</p>c<p>Genus assignments (sequence counts): Unclassified fungi (6 sequences); Saccharomyces (3); Tumularia (3); Malassezia (2); Rhodotorula (2); Candida (1); Ceratobasidium (1); Galerina (1). One of the genera assignments (Ceratobasidium) was at a very weak E-value (3.9); all others were at very strong E-values (−85 to −177).</p>d<p>Includes <i>Candida</i>. All non-<i>Candida</i> genera are constituted by singleton sequences; 4 (<i>Scutellospora,Tomentella, Saccharomyces, Cryptococcus</i>) have very weak E-values (0.11–4.8); 1 (<i>Fusarium</i>) has a strong E-value (−119).</p

Increasingly Rigorous E-value Intervals Maximize Recovery of Authentic Fungal Assignments.

<p>Bars depict relative proportion of total sequence assignments in that interval that are accounted for by each color-coded category: low abundance representation (orange); plant-derived incorrect assignments (gray); occurrence only in that or a weaker E-value interval (yellow); assignments at class or order levels (blue), and genus assignments that are also included in other very low E-value assignments (green).</p

Frequency, abundance, and distribution of genera occurring in at least 50% of the six subjects.

<p>Genera ordered by frequency of occurrence, with normalized representation and sequence counts (columns 2, 3, 4). Heatmap depiction (columns 5–10) summarizes qualitative and quantitative distribution of genera in six individuals (50, 51, 52, 54, 56, 57) and depth of sequencing for each subject (row 2). Values within individual heatmap cells are the percentage representation within that subject.</p