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 dy...

Summary High throughput sequencing of 16S ribosomal RNA gene amplicons is a cost-effective method for characterization of oral bacterial communities. However, before undertaking large-scale studies, it is necessary to understand the technique-associated limitations and intrinsic variability of the oral ecosystem. In this work we evaluated bias in species representation using an in vitro-assembled mock community of oral bacteria. We then characterized the bacterial communities in saliva and buccal mucosa of five healthy subjects to investigate the power of high throughput sequencing in revealing their diversity and biogeography patterns. Mock community analysis showed primer and DNA isolation biases and an overestimation of diversity that was reduced after eliminating singleton operational taxonomic units (OTUs). Sequencing of salivary and mucosal communities found a total of 455 OTUs (0.3% dissimilarity) with only 78 of these present in all subjects. We demonstrate that this variability was partly the result of incomplete richness coverage even at great sequencing depths, and so comparing communities by their structure was more effective than comparisons based solely on membership. With respect to oral biogeography, we found inter-subject variability in community structure was lower than site differences between salivary and mucosal communities within subjects. These differences were evident at very low sequencing depths and were mostly caused by the abundance of Streptococcus mitis and Gemella haemolysans in mucosa. In summary, we present an experimental and data analysis framework that will facilitate design and interpretation of pyrosequencing-based studies. Despite challenges associated with this technique, we demonstrate its power for evaluation of oral diversity and biogeography patterns.

Background Recurrent Aphthous Stomatitis (RAS) is a common and painful oral mucosal disease. Possible etiologies include genetics, vitamin deficiencies, trauma, immune dysfunction and stress. The goal of the present study was to examine the relationship between the occurrence, type, and magnitude of stressful events and the onset and duration of RAS episodes. Methods 160 subjects with a history of RAS completed a weekly phone survey for up to one year, providing data on the occurrence of RAS episodes and details of any stressful events they experienced during the previous week. During RAS episodes, subjects also completed daily paper diaries that recorded incidence and duration of the RAS episode. Stressful events were quantified using the validated Recent Life Changes Questionnaire (RLCQ), and were classified as mental or physical stressors. Results Stressful life events were significantly associated with the onset of RAS episodes (p<0.001), however not with duration of the RAS episodes. Experiencing a stressful life event increased the odds of an RAS episode by almost three times (OR=2.72; 95% CI 2.04-3.62). When controlled for each other, mental stressors had a larger effect (OR=3.46, 95% CI=2.54-4.72) than physical stressors (OR=1.44; 95% CI 1.04-1.99) on the occurrence of RAS episodes. RAS episodes did not occur more frequently or last longer with increasing stress severity. Conclusions In patients with a history of RAS, stressful events may mediate changes involved in initiation of new RAS episodes. Mental stressors are more strongly associated with RAS episodes than physical stressors.

In addition to disease diagnostics, there is a need for biomarkers to predict severity of cancer therapy side effects such as oral mucositis. Oral mucositis is an inflammatory lesion of oral mucosa caused by high dose chemotherapy and/or radiation that is especially prevalent during oral cancer treatment. We describe here a semiautomated, modular microfluidic immunoarray optimized for ultrasensitive detection of pro-inflammatory cytokines involved in pathobiology of oral mucositis. Our goal is methodology to identify risk of mucositis early in oral cancer treatment, before the onset of lesions. Biomarkers include tumor necrosis factor (TNF-α), interleukin-6 (IL-6), interleukin-1β (IL-1β), and C-reactive protein (CRP). Protein analytes were captured from serum in a capture chamber by 1 μm magnetic beads coated with antibodies and enzyme labels. Beads are then transported downstream to a detection chamber containing an 8-sensor array coated with glutathione-gold nanoparticles (GSH-AuNP) and a second set of antibodies to capture the beads with analyte proteins. In this first application of the immunoarray to 4-protein multiplexed measurements, ultralow detection limits of 10–40 fg mL−1 in 5 μL serum were achieved for simultaneous detection in 30 min. Mass detection limits were 2.5–10 zeptomol, as few as 1500 molecules. Accuracy and diagnostic utility of the arrays was demonstrated by correlation of levels of the 4 biomarker proteins in serum from head and neck cancer patients with results from standard ELISA. This approach may lead to rapid, low-cost estimates of projected risk for severity of oral mucositis in cancer patients to enable improved therapeutic management.