Elucidating the roles of the mammary and gut microbiomes in breast cancer development

The mammary microbiome is a newly characterized bacterial niche that might offer biological insight into the development of breast cancer. Together with in-depth analysis of the gut microbiome in breast cancer, current evidence using next-generation sequencing and metabolic profiling suggests compositional and functional shifts in microbial consortia are associated with breast cancer. In this review, we discuss the fundamental studies that have progressed this important area of research, focusing on the roles of both the mammary tissue microbiome and the gut microbiome. From the literature, we identified the following major conclusions, (I) There are unique breast and gut microbial signatures (both compositional and functional) that are associated with breast cancer, (II) breast and gut microbiome compositional and breast functional dysbiosis represent potential early events of breast tumor development, (III) specific breast and gut microbes confer host immune responses that can combat breast tumor development and progression, and (IV) chemotherapies alter the microbiome and thus maintenance of a eubiotic microbiome may be key in breast cancer treatment. As the field expectantly advances, it is necessary for the role of the microbiome to continue to be elucidated using multi-omic approaches and translational animal models in order to improve predictive, preventive, and therapeutic strategies for breast cancer

Acute Beetroot Juice Ingestion Increases Nitric Oxide Bioavailability Without Changing Oral Microbial Composition in Healthy Young Women

Dietary nitrate supplementation can elicit beneficial health and exercise performance effects. Oral microbiota are critical for the metabolism of exogenously consumed nitrate; however, limited data are available on the influence of dietary nitrate ingestion on bacterial taxa and in women. PURPOSE: To investigate if acute dietary nitrate ingestion alters the oral microbiota in young healthy women compared to a nitrate-depleted placebo. METHODS: In a randomized double-blinded crossover design, fifteen recreationally active women (mean ± SD: age 20 ± 1 years; body mass 63 ± 10 kg; height 1.68 ± 0.1 m) participated in two conditions to ingest nitrate-rich beetroot juice (BR; 12 mmol of nitrate) and nitrate-depleted beetroot juice (PL, negligible nitrate), 2.5 hours prior to a resting blood draw and buccal swab sample. Plasma [nitrate] and [nitrite] were analyzed using gas phase chemiluminescence. Buccal swab samples were used for DNA extraction and isolation. DNA was amplified using polymerase chain reaction targeting the V3 - V4 region of the 16S rRNA gene. Following index PCR, amplicons were pooled and sequenced using the iSeq Illumina NGS sequencer. Reads were clustered into amplicon sequence variants and analyzed for alpha and beta diversity and relative abundance. RESULTS: BR increased plasma [nitrate] (PL: 52 ± 14 µM vs. BR: 629 ± 132 µM, P \u3c 0.001) and plasma [nitrite] (PL: 276 ± 286 nM vs. BR: 703 ± 391 nM, P \u3c 0.001). One sample had insufficient DNA and thus, a subset of samples was analyzed for oral microbial composition (n = 14). Alpha (i.e., species richness or evenness) and beta diversity was not different between PL and BR (P \u3e 0.05). The relative abundance of the phylum and genus were not influenced by BR (P \u3e 0.05). CONCLUSION: Acute nitrate ingestion did not improve or worsen the composition of global or lower taxonomic levels of bacteria in young recreationally active women. These data indicate that acute nitrate ingestion is an intervention to rapidly increase nitric oxide bioavailability in young recreationally active women, which is an effect that did not require changes to the oral microbial community. Further research is required to understand the impact of dosing regimen and population on oral bacterial taxa and the efficacy of nitrate on nitrate-induced effects

Composition and Functional Potential of the Human Mammary Microbiota Prior to and following Breast Tumor Diagnosis

Microbiota studies have reported changes in the microbial composition of the breast upon cancer development. However, results are inconsistent and limited to the later phases of cancer development (after diagnosis). We analyzed and compared the resident bacterial taxa of histologically normal breast tissue (healthy, H, n = 49) with those of tissues donated prior to (prediagnostic, PD, n = 15) and after (adjacent normal, AN, n = 49, and tumor, T, n = 46) breast cancer diagnosis (n total = 159). DNA was isolated from tissue samples and submitted for Illumina MiSeq paired-end sequencing of the V3-V4 region of the 16S gene. To infer bacterial function in breast cancer, we predicted the functional bacteriome from the 16S sequencing data using PICRUSt2. Bacterial compositional analysis revealed an intermediary taxonomic signature in the PD tissue relative to that of the H tissue, represented by shifts in Bacillaceae, Burkholderiaceae, Corynebacteriaceae, Streptococcaceae, and Staphylococcaceae. This compositional signature was enhanced in the AN and T tissues. We also identified significant metabolic reprogramming of the microbiota of the PD, AN, and T tissue compared with the H tissue. Further, preliminary correlation analysis between host transcriptome profiling and microbial taxa and genes in H and PD tissues identified altered associations between the human host and mammary microbiota in PD tissue compared with H tissue. These findings suggest that compositional shifts in bacterial abundance and metabolic reprogramming of the breast tissue microbiota are early events in breast cancer development that are potentially linked with cancer susceptibility

Co-Ingestion of Dietary Nitrate and Ascorbic Acid on Nitric Oxide Biomarkers and The Oral Microbiome in Sedentary Hispanic Women

Nitric oxide bioavailability increases following nitrate supplementation wherein oral microbiota facilitate the metabolism and absorption of nitrate. However, few studies have examined if co-ingestion of nitrate with antioxidants can further elevate nitric oxide bioavailability. Moreover, our understanding on how the oral microbiome responds to nitrate supplementation is limited, especially in women. PURPOSE: To examine the effects of ingesting dietary nitrate and ascorbic acid independently and concurrently on markers of nitric oxide bioavailability and oral microbiota species. METHODS: Twelve sedentary women of Hispanic descent (mean ± SD: age 20 ± 1 years; body mass 74 ± 15 kg; height 1.62 ± 0.09 m) consumed nitrate-rich beetroot juice (BR), nitrate-depleted beetroot juice (PL), ascorbic acid (AA), and crystal light (CRY) in four conditions: BR combined with AA (BR+AA); BR only (BR+CRY); AA only (PL+AA); and placebo-control (PL+CRY). Supplements were ingested 2.5 hours prior to a resting blood draw and buccal swab sample. Plasma [nitrate] and [nitrite] were analyzed using gas phase chemiluminescence. Buccal swab samples were used for DNA extraction and isolation. DNA was amplified using polymerase chain reaction (PCR) targeting the V3 - V4 region of the 16S rRNA gene. Following index PCR, amplicons were pooled and sequenced using the iSeq Illumina NGS sequencer. Reads were clustered into amplicon sequence variants and analyzed for alpha and beta diversity and relative abundance. RESULTS: BR increased plasma [nitrate] (BR+AA: 641 ± 252 vs. BR+CRY: 528 ± 307 vs. PL+AA: 35 ± 10 vs. PL+CRY: 35 ± 12 µM, P \u3c 0.001) and plasma [nitrite] (BR+AA: 710 ± 336 vs. BR+CRY: 578 ± 428 vs. PL+AA: 209 ± 88 vs. PL+CRY: 198 ± 82 nM, P \u3c 0.001) with no differences within BR and PL conditions. Alpha and beta diversity, and the relative abundance of higher and lower taxonomic levels were not significantly different between all conditions (P \u3e 0.05) CONCLUSION: Concurrent nitrate and AA supplementation did not elicit additional increases to nitric oxide compared to nitrate ingestion alone. Acute beetroot juice and ascorbic acid were ineffective at modulating oral microbial composition. Further research is required to understand the impact of supplementation regimen and population on the physiological effects of dietary nitrate

Associations between infant fungal and bacterial dysbiosis and childhood atopic wheeze in a nonindustrialized setting.

BACKGROUND: Asthma is the most prevalent chronic disease of childhood. Recently, we identified a critical window early in the life of both mice and Canadian infants during which gut microbial changes (dysbiosis) affect asthma development. Given geographic differences in human gut microbiota worldwide, we studied the effects of gut microbial dysbiosis on atopic wheeze in a population living in a distinct developing world environment. OBJECTIVE: We sought to determine whether microbial alterations in early infancy are associated with the development of atopic wheeze in a nonindustrialized setting. METHODS: We conducted a case-control study nested within a birth cohort from rural Ecuador in which we identified 27 children with atopic wheeze and 70 healthy control subjects at 5 years of age. We analyzed bacterial and eukaryotic gut microbiota in stool samples collected at 3 months of age using 16S and 18S sequencing. Bacterial metagenomes were predicted from 16S rRNA data by using Phylogenetic Investigation of Communities by Reconstruction of Unobserved States and categorized by function with Kyoto Encyclopedia of Genes and Genomes ontology. Concentrations of fecal short-chain fatty acids were determined by using gas chromatography. RESULTS: As previously observed in Canadian infants, microbial dysbiosis at 3 months of age was associated with later development of atopic wheeze. However, the dysbiosis in Ecuadorian babies involved different bacterial taxa, was more pronounced, and also involved several fungal taxa. Predicted metagenomic analysis emphasized significant dysbiosis-associated differences in genes involved in carbohydrate and taurine metabolism. Levels of the fecal short-chain fatty acids acetate and caproate were reduced and increased, respectively, in the 3-month stool samples of children who went on to have atopic wheeze. CONCLUSIONS: Our findings support the importance of fungal and bacterial microbiota during the first 100 days of life on the development of atopic wheeze and provide additional support for considering modulation of the gut microbiome as a primary asthma prevention strategy

Defining the role of the human intestinal microbiota in childhood asthma and atopic disease

Asthma is a chronic inflammatory atopic disease of the airways affecting one in ten children in Westernized countries. The microflora hypothesis of allergic disease proposes the intestinal microbiota as a potential mechanistic link connecting environmental exposures to changes in the developing immune system. Further, animal model studies allude to an early life critical window, during which the immune system is most vulnerable to compositional and functional changes in the intestinal microbiota. We conducted an epidemiological assessment of early life environmental factors associated with atopy and wheezing at age 1 year and preschool-age asthma in children enrolled in the Canadian Healthy Infant Longitudinal Development (CHILD) Study (n = 2,695). Here, we identified early life microflora hypothesis related variables (e.g. pre- and post-natal antibiotic exposure, gestational age, and birth mode) as risk and protective factors for asthma and atopic disease. Informed by this epidemiological assessment, we used 16S ribosomal RNA sequencing and quantitative polymerase chain reaction to analyze the 3-month and 1-year fecal microbiota of one-year-old CHILD Study subjects positive or negative for atopy and wheezing (n = 319) and among this same cohort, those who were diagnosed with preschool-age asthma or non-atopic non-wheezing controls (n = 76). The fecal microbiota of atopic wheezing subjects compared to controls showed decreases in the abundances of four gut bacterial genera, Faecalibacterium, Lachnospira, Rothia, and Veillonella, combined with a reduction in fecal acetate at the 3-month time point only. Further, we found shifts in the relative abundances of two bacterial taxa in the 3-month fecal microbiota of preschool-age asthmatic children compared to controls; Lachnospira remained decreased among asthmatic children and Clostridium neonatale was increased in asthmatics. Quartile analysis at 3-months revealed a negative association between the ratio of these two bacteria (Lachnospira/Clostridium neonatale) and asthma risk. Altogether, this research highlights environmental factors that may be contributing to gut bacterial alterations in subjects with asthma or atopic disease. Additionally, these microbial alterations were no longer present by 1-year of age, suggesting the first 100 days of life as the critical window during which taxa-specific gut bacterial dysbiosis is associated with asthma and atopic disease in humans.Science, Faculty ofMicrobiology and Immunology, Department ofGraduat

Asthma and the microbiome: defining the critical window in early life

Asthma is a chronic inflammatory immune disorder of the airways affecting one in ten children in westernized countries. The geographical disparity combined with a generational rise in prevalence, emphasizes that changing environmental exposures play a significant role in the etiology of this disease. The microflora hypothesis suggests that early life exposures are disrupting the composition of the microbiota and consequently, promoting immune dysregulation in the form of hypersensitivity disorders. Animal model research supports a role of the microbiota in asthma and atopic disease development. Further, these model systems have identified an early life critical window, during which gut microbial dysbiosis is most influential in promoting hypersensitivity disorders. Until recently this critical window had not been characterized in humans, but now studies suggest that the ideal time to use microbes as preventative treatments or diagnostics for asthma in humans is within the first 100 days of life. This review outlines the major mouse-model and human studies leading to characterization of the early life critical window, emphasizing studies analyzing the intestinal and airway microbiotas in asthma and atopic disease. This research has promising future implications regarding childhood immune health, as ultimately it may be possible to therapeutically administer specific microbes in early life to prevent the development of asthma in children.Medicine, Faculty ofScience, Faculty ofOther UBCMicrobiology and Immunology, Department ofPediatrics, Department ofReviewedFacult