Investigating the oral microbiome in retrospective and prospective cases of prostate, colon, and breast cancer.

The human microbiome has been proposed as a potentially useful biomarker for several cancers. To examine this, we made use of salivary samples from the Atlantic Partnership for Tomorrow's Health (PATH) project and Alberta's Tomorrow Project (ATP). Sample selection was divided into both a retrospective and prospective case control design examining prostate, breast, and colon cancer. In total 89 retrospective and 260 prospective cancer cases were matched to non-cancer controls and saliva samples were sequenced using 16S rRNA gene sequencing. We found no significant differences in alpha diversity. All beta diversity measures were insignificant except for unweighted UniFrac profiles in retrospective breast cancer cases and weighted UniFrac, Bray-Curtis and Robust Atchinson's distances in colon cancer after testing with age and sex adjusted MiRKAT models. Differential abundance (DA) analysis showed several taxa that were associated with previous cancer in all three groupings. Only one genus (Clostridia UCG-014) in breast cancer and one ASV (Fusobacterium periodonticum) in colon cancer was identified by more than one DA tool. In prospective cases three ASVs were associated with colon cancer, one ASV with breast cancer, and one ASV with prostate cancer. Random Forest classification showed low levels of signal in both study designs in breast and prostate cancer. Contrastingly, colon cancer did show signal in our retrospective analysis (AUC: 0.737) and in one of two prospective cohorts (AUC: 0.717). Our results indicate that it is unlikely that reliable microbial oral biomarkers for breast and prostate cancer exist.. However, further research into the oral microbiome and colon cancer could be fruitful.

Exploring the microbiome of oral epithelial dysplasia as a predictor of malignant progression.

A growing body of research associates the oral microbiome and oral cancer. Well-characterized clinical samples with outcome data are required to establish relevant associations between the microbiota and disease. The objective of this study was to characterize the community variations and the functional implications of the microbiome in low-grade oral epithelial dysplasia (OED) using 16S rRNA gene sequencing from annotated archival swabs in progressing (P) and non-progressing (NP) OED. We characterised the microbial community in 90 OED samples - 30 swabs from low-grade OED that progressed to cancer (cases) and 60 swabs from low-grade OED that did not progress after a minimum of 5 years of follow up (matched control subjects). There were small but significant differences between P and NP samples in terms of alpha diversity as well as beta diversity in conjunction with other clinical factors such as age and smoking status for both taxa and functional predictions. Across all samples, the most abundant genus was Streptococcus, followed by Haemophilus, Rothia, and Neisseria. Taxa and predicted functions were identified that were significantly differentially abundant with progression status (all Ps and NPs), when samples were grouped broadly by the number of years between sampling and progression or in specific time to progression for Ps only. However, these differentially abundant features were typically present only at low abundances. For example, Campylobacter was present in slightly higher abundance in Ps (1.72%) than NPs (1.41%) and this difference was significant when Ps were grouped by time to progression. Furthermore, several of the significantly differentially abundant functions were linked to the Campylobacteraceae family in Ps and may justify further investigation. Larger cohort studies to further explore the microbiome as a potential biomarker of risk in OED are warranted.

The gastrointestinal antibiotic resistome in pediatric leukemia and lymphoma patients.

Introduction: Most children with leukemia and lymphoma experience febrile neutropenia. These are treated with empiric antibiotics that include ß-lactams and/or vancomycin. These are often administered for extended periods, and the effect on the resistome is unknown. Methods: We examined the impact of repeated courses and duration of antibiotic use on the resistome of 39 pediatric leukemia and lymphoma patients. Shotgun metagenome sequences from 127 stool samples of pediatric oncology patients were examined for abundance of antibiotic resistance genes (ARGs) in each sample. Abundances were grouped by repeated courses (no antibiotics, 1-2 courses, 3+ courses) and duration (no use, short duration, long and/or mixed durationg) of ß-lactams, vancomycin and "any antibiotic" use. We assessed changes in both taxonomic composition and prevalence of ARGs among these groups. Results: We found that Bacteroidetes taxa and ß-lactam resistance genes decreased, while opportunistic Firmicutes and Proteobacteria taxa, along with multidrug resistance genes, increased with repeated courses and/or duration of antibiotics. Efflux pump related genes predominated (92%) among the increased multidrug genes. While we found ß-lactam ARGs present in the resistome, the taxa that appear to contain them were kept in check by antibiotic treatment. Multidrug ARGs, mostly efflux pumps or regulators of efflux pump genes, were associated with opportunistic pathogens, and both increased in the resistome with repeated antibiotic use and/or increased duration. Conclusions: Given the strong association between opportunistic pathogens and multidrug-related efflux pumps, we suggest that drug efflux capacity might allow the opportunistic pathogens to persist or increase despite repeated courses and/or duration of antibiotics. While drug efflux is the most direct explanation, other mechanisms that enhance the ability of opportunistic pathogens to handle environmental stress, or other aspects of the treatment environment, could also contribute to their ability to flourish within the gut during treatment. Persistence of opportunistic pathogens in an already dysbiotic and weakened gastrointestinal tract could increase the likelihood of life-threatening blood borne infections. Of the 39 patients, 59% experienced at least one gastrointestinal or blood infection and 60% of bacteremia's were bacteria found in stool samples. Antimicrobial stewardship and appropriate use and duration of antibiotics could help reduce morbidity and mortality in this vulnerable population.

Antibiotic and antifungal use in pediatric leukemia and lymphoma patients are associated with increasing opportunistic pathogens and decreasing bacteria responsible for activities that enhance colonic defense.

Due to decreased immunity, both antibiotics and antifungals are regularly used in pediatric hematologic-cancer patients as a means to prevent severe infections and febrile neutropenia. The general effect of antibiotics on the human gut microbiome is profound, yielding decreased diversity and changes in community structure. However, the specific effect on pediatric oncology patients is not well-studied. The effect of antifungal use is even less understood, having been studied only in mouse models. Because the composition of the gut microbiome is associated with regulation of hematopoiesis, immune function and gastrointestinal integrity, changes within the patient gut can have implications for the clinical management of hematologic malignancies. The pediatric population is particularly challenging because the composition of the microbiome is age dependent, with some of the most pronounced changes occurring in the first three years of life. We investigated how antibiotic and antifungal use shapes the taxonomic composition of the stool microbiome in pediatric patients with leukemia and lymphoma, as inferred from both 16S rRNA and metagenome data. Associations with age, antibiotic use and antifungal use were investigated using multiple analysis methods. In addition, multivariable differential abundance was used to identify and assess specific taxa that were associated with multiple variables. Both antibiotics and antifungals were linked to a general decline in diversity in stool samples, which included a decrease in relative abundance in butyrate producers that play a critical role in host gut physiology (e.g., Faecalibacterium, Anaerostipes, Dorea, Blautia),. Furthermore, antifungal use was associated with a significant increase in relative abundance of opportunistic pathogens. Collectively, these findings have important implications for the treatment of leukemia and lymphoma patients. Butyrate is important for gastrointestinal integrity; it inhibits inflammation, reinforces colonic defense, mucosal immunity. and decreases oxidative stress. The routine use of broad-spectrum anti-infectives in pediatric oncology patients could simultaneously contribute to a decline in gastrointestinal integrity and colonic defense while promoting increases in opportunistic pathogens within the patient gut. Because the gut microbiome has been linked to both short-term clinical outcomes, and longer-lasting health effects, systematic characterization of the gut microbiome in pediatric patients during, and beyond, treatment is warranted.

Gut bacterial gene changes following pegaspargase treatment in pediatric patients with acute lymphoblastic leukemia.

Treatment of pediatric acute lymphoblastic leukemia (ALL) with pegaspargase exploits ALL cells dependency on asparagine. Pegaspargase depletes asparagine, consequentially affecting aspartate, glutamine and glutamate. The gut as a confounding source of these amino acids (AAs) and the role of gut microbiome metabolism of AAs has not been examined. We examined asparagine, aspartate, glutamine and glutamate in stool samples from patients over pegaspargase treatment. Microbial gene-products, which interact with these AAs were identified. Stool asparagine declined significantly, and 31 microbial genes changed over treatment. Changes were complex, and included genes involved in AA metabolism, nutrient sensing, and pathways increased in cancers. While we identified changes in a gene (iaaA) with limited asparaginase activity, it lacked significance after correction leaving open other mechanisms for asparagine decline, possibly including loss from gut to blood. Understanding pathways that change AA availability, including by microbes in the gut, could be useful in optimizing pegaspargase therapy.

An in vivo genome-wide shRNA screen identifies BCL6 as a targetable biomarker of paclitaxel resistance in breast cancer.

Paclitaxel is a common breast cancer drug; however, some tumors are resistant. The identification of biomarkers for paclitaxel resistance or sensitivity would enable the development of strategies to improve treatment efficacy. A genome-wide in vivo shRNA screen was performed on paclitaxel-treated mice with MDA-MB-231 tumors to identify genes associated with paclitaxel sensitivity or resistance. Gene expression of the top screen hits was associated with tumor response (resistance or sensitivity) among patients who received neoadjuvant chemotherapy containing paclitaxel. We focused our validation on screen hit B-cell lymphoma 6 (BCL6), which is a therapeutic target in cancer but for which no effects on drug response have been reported. Knockdown of BCL6 resulted in increased tumor regression in mice treated with paclitaxel. Similarly, inhibiting BCL6 using a small molecule inhibitor enhanced paclitaxel treatment efficacy both in vitro and in vivo in breast cancer models. Mechanism studies revealed that reduced BCL6 enhances the efficacy of paclitaxel by inducing sustained G1/S arrest, concurrent with increased apoptosis and expression of target gene cyclin-dependent kinase inhibitor 1A. In summary, the genome-wide shRNA knockdown screen has identified BCL6 as a potential targetable resistance biomarker of paclitaxel response in breast cancer.

Investigating the gut microbial community and genes in children with differing levels of change in serum asparaginase activity during pegaspargase treatment for acute lymphoblastic leukemia.

Asparaginase (ASNase) is an effective treatment of pediatric acute lymphoblastic leukemia (ALL). Changes in ASNase activity may lead to suboptimal treatment and poorer outcomes. The gut microbiome produces metabolites that could impact ASNase therapy, however, remains uninvestigated. We examined gut-microbial community and microbial-ASNase and asparagine synthetase (ASNS) genes using 16SrRNA and metagenomic sequence data from stool samples of pediatric ALL patients. Comparing ASNase activity between consecutive ASNase-doses, we found microbial communities differed between decreased- and increased-activity samples. Escherichia predominated in the decreased-activity community while Bacteroides and Streptococcus predominated in the increased-activity community. In addition microbial ASNS was significantly (p=.004) negatively correlated with change in serum ASNase activity. These preliminary findings suggest microbial communities prior to treatment could affect serum ASNase levels, although the mechanism is unknown. Replication in an independent cohort is needed, and future research on manipulation of these communities and genes could prove useful in optimizing ASNase therapy.

The human tumor microbiome is composed of tumor type-specific intracellular bacteria.

Bacteria were first detected in human tumors more than 100 years ago, but the characterization of the tumor microbiome has remained challenging because of its low biomass. We undertook a comprehensive analysis of the tumor microbiome, studying 1526 tumors and their adjacent normal tissues across seven cancer types, including breast, lung, ovary, pancreas, melanoma, bone, and brain tumors. We found that each tumor type has a distinct microbiome composition and that breast cancer has a particularly rich and diverse microbiome. The intratumor bacteria are mostly intracellular and are present in both cancer and immune cells. We also noted correlations between intratumor bacteria or their predicted functions with tumor types and subtypes, patients' smoking status, and the response to immunotherapy.

Bacterial Taxa and Functions Are Predictive of Sustained Remission Following Exclusive Enteral Nutrition in Pediatric Crohn's Disease.

BACKGROUND: The gut microbiome is extensively involved in induction of remission in pediatric Crohn's disease (CD) patients by exclusive enteral nutrition (EEN). In this follow-up study of pediatric CD patients undergoing treatment with EEN, we employ machine learning models trained on baseline gut microbiome data to distinguish patients who achieved and sustained remission (SR) from those who did not achieve remission nor relapse (non-SR) by 24 weeks. METHODS: A total of 139 fecal samples were obtained from 22 patients (8-15 years of age) for up to 96 weeks. Gut microbiome taxonomy was assessed by 16S rRNA gene sequencing, and functional capacity was assessed by metagenomic sequencing. We used standard metrics of diversity and taxonomy to quantify differences between SR and non-SR patients and to associate gut microbial shifts with fecal calprotectin (FCP), and disease severity as defined by weighted Pediatric Crohn's Disease Activity Index. We used microbial data sets in addition to clinical metadata in random forests (RFs) models to classify treatment response and predict FCP levels. RESULTS: Microbial diversity did not change after EEN, but species richness was lower in low-FCP samples (<250 µg/g). An RF model using microbial abundances, species richness, and Paris disease classification was the best at classifying treatment response (area under the curve [AUC] = 0.9). KEGG Pathways also significantly classified treatment response with the addition of the same clinical data (AUC = 0.8). Top features of the RF model are consistent with previously identified IBD taxa, such as Ruminococcaceae and Ruminococcus gnavus. CONCLUSIONS: Our machine learning approach is able to distinguish SR and non-SR samples using baseline microbiome and clinical data.

Infectious Complications Are Associated With Alterations in the Gut Microbiome in Pediatric Patients With Acute Lymphoblastic Leukemia.

Acute lymphoblastic leukemia is the most common pediatric cancer. Fortunately, survival rates exceed 90%, however, infectious complications remain a significant issue that can cause reductions in the quality of life and prognosis of patients. Recently, numerous studies have linked shifts in the gut microbiome composition to infection events in various hematological malignances including acute lymphoblastic leukemia (ALL). These studies have been limited to observing broad taxonomic changes using 16S rRNA gene profiling, while missing possible differences within microbial functions encoded by individual species. In this study we present the first combined 16S rRNA gene and metagenomic shotgun sequencing study on the gut microbiome of an independent pediatric ALL cohort during treatment. In this study we found distinctive differences in alpha diversity and beta diversity in samples from patients with infectious complications in the first 6 months of therapy. We were also able to find specific species and functional pathways that were significantly different in relative abundance between samples that came from patients with infectious complications. Finally, machine learning models based on patient metadata and bacterial species were able to classify samples with high accuracy (84.09%), with bacterial species being the most important classifying features. This study strengthens our understanding of the association between infection and pediatric acute lymphoblastic leukemia treatment and warrants further investigation in the future.

Enhancement of the gut barrier integrity by a microbial metabolite through the Nrf2 pathway.

The importance of gut microbiota in human health and pathophysiology is undisputable. Despite the abundance of metagenomics data, the functional dynamics of gut microbiota in human health and disease remain elusive. Urolithin A (UroA), a major microbial metabolite derived from polyphenolics of berries and pomegranate fruits displays anti-inflammatory, anti-oxidative, and anti-ageing activities. Here, we show that UroA and its potent synthetic analogue (UAS03) significantly enhance gut barrier function and inhibit unwarranted inflammation. We demonstrate that UroA and UAS03 exert their barrier functions through activation of aryl hydrocarbon receptor (AhR)- nuclear factor erythroid 2-related factor 2 (Nrf2)-dependent pathways to upregulate epithelial tight junction proteins. Importantly, treatment with these compounds attenuated colitis in pre-clinical models by remedying barrier dysfunction in addition to anti-inflammatory activities. Cumulatively, the results highlight how microbial metabolites provide two-pronged beneficial activities at gut epithelium by enhancing barrier functions and reducing inflammation to protect from colonic diseases.

Differences in adiposity and diet quality among individuals with inflammatory bowel disease in Eastern Canada.

The objective of the current study was to characterize the relationship between diet quality and body composition in participants living with IBD, specifically Crohn's disease (CD) or ulcerative colitis (UC), in Atlantic Canada. Participants from the Atlantic Partnership for Tomorrow's Health (PATH) study are residents of one of the four Atlantic Canada provinces. Participants who completed the dietary questionnaire and had body composition measured were included in the study (n = 12,462 without IBD, n = 111 CD, n = 119 UC). A greater number of participants with IBD reported having multiple chronic conditions compared to those without IBD. Those with UC had statistically higher body weight and body mass index (BMI) compared to those without IBD. Overall, significant positive correlations were observed between adiposity and servings of refined grains, and meats and alternatives such as eggs and fish, whereas negative correlations were observed with servings of vegetables, fruit, whole grains, and alternatives such as tofu, and nuts/seeds. Participants with IBD (both CD and UC) consumed more refined grains than those without IBD. Using logistic regression analysis, participants consuming more servings of vegetables and whole grains were less likely to have CD where as those consuming more serving of fruit and bean/legumes were less likely to have UC. In the Atlantic PATH cohort, which includes a region of the world with a high incidence of IBD, distinct differences in adiposity and diet quality were observed in individuals with specific types of IBD compared to those without. There is a need for collaborative efforts to address weight management and diet quality issues in those living with IBD in the Atlantic Canadian region.

Multi-omics differentially classify disease state and treatment outcome in pediatric Crohn's disease.

BACKGROUND: Crohn's disease (CD) has an unclear etiology, but there is growing evidence of a direct link with a dysbiotic microbiome. Many gut microbes have previously been associated with CD, but these have mainly been confounded with patients' ongoing treatments. Additionally, most analyses of CD patients' microbiomes have focused on microbes in stool samples, which yield different insights than profiling biopsy samples. RESULTS: We sequenced the 16S rRNA gene (16S) and carried out shotgun metagenomics (MGS) from the intestinal biopsies of 20 treatment-naïve CD and 20 control pediatric patients. We identified the abundances of microbial taxa and inferred functional categories within each dataset. We also identified known human genetic variants from the MGS data. We then used a machine learning approach to determine the classification accuracy when these datasets, collapsed to different hierarchical groupings, were used independently to classify patients by disease state and by CD patients' response to treatment. We found that 16S-identified microbes could classify patients with higher accuracy in both cases. Based on follow-ups with these patients, we identified which microbes and functions were best for predicting disease state and response to treatment, including several previously identified markers. By combining the top features from all significant models into a single model, we could compare the relative importance of these predictive features. We found that 16S-identified microbes are the best predictors of CD state whereas MGS-identified markers perform best for classifying treatment response. CONCLUSIONS: We demonstrate for the first time that useful predictors of CD treatment response can be produced from shotgun MGS sequencing of biopsy samples despite the complications related to large proportions of host DNA. The top predictive features that we identified in this study could be useful for building an improved classifier for CD and treatment response based on sufferers' microbiome in the future. The BISCUIT project is funded by a Clinical Academic Fellowship from the Chief Scientist Office (Scotland)-CAF/08/01.

Moderate Exercise Has Limited but Distinguishable Effects on the Mouse Microbiome.

The gut microbiome is known to have a complex yet vital relationship with host health. While both exercise and the gut microbiome have been shown to impact human health independently, the direct effects of moderate exercise on the intestinal microbiota remain unclear. In this study, we compared gut microbial diversity and changes in inflammatory markers associated with exercise over an 8-week period in mice that performed either voluntary exercise (VE) (n = 10) or moderate forced exercise (FE) (n = 11) and mice that did not perform any exercise (n = 21). VE mice, but not FE mice, had increased food intake and lean body mass compared to sedentary mice. The levels of inflammatory markers associated with exercise were similar for mice in all three groups. Traditional microbial profiles comparing operational taxonomic units (OTUs) in samples (P > 0.1) and multivariate analysis of beta diversity via Adonis testing (P > 0.1) did not identify significantly altered taxonomic profiles in the voluntary or forced exercise group compared to the sedentary controls. However, a random forests machine learning model, which takes into account the relationships between bacteria in a community, classified voluntary exercisers and nonexercisers with 97% accuracy at 8 weeks. The top bacteria used by the model allowed us to identify known taxa (Bacteroides, S24-7, and Lactobacillus) and novel taxa (Rikenellaceae and Lachnospiraceae) associated with exercise. Although aerobic exercise in mice did not result in significant changes of abundance in gut microbes or in host inflammatory response, more sophisticated computational methods could identify some microbial shifts. More study is needed on the effects of various exercise intensities and their impact on the gut microbiome. IMPORTANCE The bacteria that live in our gut have a complex yet vital relationship with our health. Environmental factors that influence the gut microbiome are of great interest, as recent research demonstrates that these microbes, mostly bacteria, are important for normal host physiology. Diseases such as obesity, diabetes, inflammatory bowel disease, and colon cancer have also been linked to shifts in the microbiome. Exercise is known to have beneficial effects on these diseases; however, much less is known about its direct impact on the gut microbiome. Our results illustrate that exercise has a moderate but measurable effect on gut microbial communities in mice. These methods can be used to provide important insight into other factors affecting the microbiome and our health.

The Impact of Exclusive Enteral Nutrition (EEN) on the Gut Microbiome in Crohn's Disease: A Review.

Crohn's disease (CD), a form of inflammatory bowel disease (IBD), is thought to arise from a complex interaction of genetics, the gut microbiome, and environmental factors, such as diet. There is clear evidence that dietary intervention is successful in the treatment of CD-exclusive enteral nutrition (EEN) is able to induce remission in up to 80% of CD patients. While the mechanism of action of EEN is not clear, EEN is known to cause profound changes in the gut microbiome. Understanding how EEN modifies the gut microbiome to induce remission could provide insight into CD etiopathogenesis and aid the development of microbiome-targeted interventions to guide ongoing dietary therapy to sustain remission. This review includes current literature on changes in composition and function of the gut microbiome associated with EEN treatment in CD patients.

Newly introduced genomic prophage islands are critical determinants of in vivo competitiveness in the Liverpool Epidemic Strain of Pseudomonas aeruginosa.

Pseudomonas aeruginosa isolates have a highly conserved core genome representing up to 90% of the total genomic sequence with additional variable accessory genes, many of which are found in genomic islands or islets. The identification of the Liverpool Epidemic Strain (LES) in a children's cystic fibrosis (CF) unit in 1996 and its subsequent observation in several centers in the United Kingdom challenged the previous widespread assumption that CF patients acquire only unique strains of P. aeruginosa from the environment. To learn about the forces that shaped the development of this important epidemic strain, the genome of the earliest archived LES isolate, LESB58, was sequenced. The sequence revealed the presence of many large genomic islands, including five prophage clusters, one defective (pyocin) prophage cluster, and five non-phage islands. To determine the role of these clusters, an unbiased signature tagged mutagenesis study was performed, followed by selection in the chronic rat lung infection model. Forty-seven mutants were identified by sequencing, including mutants in several genes known to be involved in Pseudomonas infection. Furthermore, genes from four prophage clusters and one genomic island were identified and in direct competition studies with the parent isolate; four were demonstrated to strongly impact on competitiveness in the chronic rat lung infection model. This strongly indicates that enhanced in vivo competitiveness is a major driver for maintenance and diversifying selection of these genomic prophage genes.