Characterization of microbial communities in the mammary glands and the influence of this microbiota on health and disease

The collective microbes and their genomic activity within us, referred to as the human microbiome, is crucial in maintaining health, and disruptions in composition and activity have been associated with various diseases. Considering the importance of breast milk in neonatal development and the high rate of breast cancer in women, the objective of my thesis was to obtain a comprehensive analysis of the microbiota of the mammary glands and to examine the influence of these organisms on health and disease. Human milk is an important source of bacteria for the developing infant. While a variety of studies over the last 20 years have provided a greater appreciation for how diverse and variable the milk microbiota is, little is known about these bacterial communities. Many studies have emphasized the adverse effects of antibiotics on the infant microbiota, however the impact of drugs on the lactating mother has not been studied. In Chapter 2 we report the changes caused by chemotherapy (over a 4 month period) on the milk microbiota and metabolome of a woman undergoing treatment for Hodgkin’s lymphoma. In Chapter 3, we show from a study of 39 Canadian women that neither gestation, mode of delivery nor gender of the child explains the diversity of the milk microbiota. As milk is not sterile and given the nutrient-rich fatty composition of the breast and its exposure to the external environment via the nipple, we hypothesized that breast tissue has its own indigenous microbiota. In Chapter 4, we confirmed this in a study of breast tissue from 81 Canadian and Irish women, with the discovery of diverse bacterial communities dominated by the phylum Proteobacteria. To determine whether this tissue microbiota could play a role in breast cancer development, we compared, in Chapter 5, bacterial profiles in tissue between women with breast cancer and those who were disease free. We show that differences do exist and that women with cancer have higher numbers of bacteria with the ability to induce host DNA damage. This work forms the platform for further studies that examine how breast milk and tissue are first colonized and how these microbiotas can be manipulated to promote optimal maternal and infant health

Microbiome-liver crosstalk: A multihit therapeutic target for liver disease.

Liver disease has become a leading cause of death, particularly in the West, where it is attributed to more than two million deaths annually. The correlation between gut microbiota and liver disease is still not fully understood. However, it is well known that gut dysbiosis accompanied by a leaky gut causes an increase in lipopolysaccharides in circulation, which in turn evoke massive hepatic inflammation promoting liver cirrhosis. Microbial dysbiosis also leads to poor bile acid metabolism and low short-chain fatty acids, all of which exacerbate the inflammatory response of liver cells. Gut microbial homeostasis is maintained through intricate processes that ensure that commensal microbes adapt to the low oxygen potential of the gut and that they rapidly occupy all the intestinal niches, thus outcompeting any potential pathogens for available nutrients. The crosstalk between the gut microbiota and its metabolites also guarantee an intact gut barrier. These processes that protect against destabilization of gut microbes by potential entry of pathogenic bacteria are collectively called colonization resistance and are equally essential for liver health. In this review, we shall investigate how the mechanisms of colonization resistance influence the liver in health and disease and the microbial-liver crosstalk potential as therapeutic target areas

The microbiota of breast tissue and its association with breast cancer

In the United States, 1 in 8 women will be diagnosed with breast cancer in her lifetime. Along with genetics, the environmentcontributes to disease development, but what these exact environmental factors are remains unknown. We have previouslyshown that breast tissue is not sterile but contains a diverse population of bacteria. We thus believe that the host\u27s local microbiomecould be modulating the risk of breast cancer development. Using 16S rRNA amplicon sequencing, we show that bacterialprofiles differ between normal adjacent tissue from women with breast cancer and tissue from healthy controls. Women withbreast cancer had higher relative abundances of Bacillus, Enterobacteriaceae and Staphylococcus. Escherichia coli (a member ofthe Enterobacteriaceae family) and Staphylococcus epidermidis, isolated from breast cancer patients, were shown to induce DNAdouble-stranded breaks in HeLa cells using the histone-2AX (H2AX) phosphorylation (γ-H2AX) assay. We also found that microbialprofiles are similar between normal adjacent tissue and tissue sampled directly from the tumor. This study raises importantquestions as to what role the breast microbiome plays in disease development or progression and how we can manipulatethis for possible therapeutics or prevention

A Ratio of Spore to Viable Organisms: A Case Study of the JPL-SAF Cleanroom

Spacecraft surfaces that are destined to land on potential life-harboring celestial bodies are required to be rigorously cleaned and continuously monitored for spore bioburden as a proxy for spacecraft cleanliness. The NASA standard assay (NSA), used for spacecraft bioburden estimates, specifically measures spores that are cultivable, aerobic, resistant to heat shock, and grow at 30 C in a nutrient-rich medium. Since the vast majority of microorganisms cannot be cultivated using the NSA, it is necessary to utilize state-of-the art molecular techniques to better understand the presence of all viable microorganisms, not just those measured with the NSA. In this study, the nutrient-deprived low biomass cleanrooms, where spacecraft are assembled, were used as a surrogate for spacecraft surfaces to measure the ratio of NSA spores in relation to the total viable microorganism population in order to make comparisons with the 2006 Space Studies Board (SSB) estimate of 1 spore per approximately 50,000 viable organisms. Ninety-eight surface wipe samples were collected from the Spacecraft Assembly Facility (SAF) cleanroom at the Jet Propulsion Laboratory (JPL) over a 6-month period. The samples were processed and analyzed using classical microbiology along with molecular methodology. Traditional microbiology plating methods were used to determine the cultivable bacterial, fungal, and spore populations. Molecular assays were used to determine the total organisms (TO, dead and live) and the viable organisms (VO, live). The TO was measured using adenosine triphosphate (ATP) and quantitative polymerase chain reaction (qPCR) assays. The VO was measured using internal ATP, propidium monoazide (PMA)-qPCR, and flow cytometry (after staining for viable microorganisms) assays. Based on the results, it was possible to establish a ratio between spore counts and VO for each viability assay. The ATP-based spore to VO ratio ranged from 149-746, and the bacterial PMA-qPCR assay-based ratio ranged from 314-1,491 VO, per spore. The most conservative estimate came from fluorescent-assisted cell sorting (FACS), which estimated the ratio to be 12,091 VO per 1 NSA spore. Since archaeal (less than 1%) and fungal (approximately 2%) populations were negligible, the spore to VO ratios were based on bacterial population estimates. The most conservative ratio from this study can be used as a replacement for the SSB estimate on nutrient-deprived (oligotrophic) desiccated spacecraft surfaces, to estimate the VO from NSA measurements without utilizing state-of-the art molecular methods that are costly and require more biomass than is typically found on spacecraft surfaces

Effect of Chemotherapy on the Microbiota and Metabolome of Human Milk: A Case Report

BACKGROUND: Human milk is an important source of bacteria for the developing infant and has been shown to influence the bacterial composition of the neonatal gut, which in turn can affect disease risk later in life. Human milk is also an important source of nutrients, influencing bacterial composition but also directly affecting the host. While recent studies have emphasized the adverse effects of antibiotic therapy on the infant microbiota, the effects of maternal chemotherapy have not been previously studied. Here we report the effects of drug administration on the microbiota and metabolome of human milk. METHODS: Mature milk was collected every two weeks over a four month period from a lactating woman undergoing chemotherapy for Hodgkin\u27s lymphoma. Mature milk was also collected from healthy lactating women for comparison. Microbial profiles were analyzed by 16S sequencing and the metabolome by gas chromatography-mass spectrometry. FINDINGS: Chemotherapy caused a significant deviation from a healthy microbial and metabolomic profile, with depletion of genera Bifidobacterium, Eubacterium, Staphylococcus and Cloacibacterium in favor of Acinetobacter, Xanthomonadaceae and Stenotrophomonas. The metabolites docosahexaenoic acid and inositol known for their beneficial effects were also decreased. CONCLUSION: With milk contents being critical for shaping infant immunity and development, consideration needs to be given to the impact of drugs administered to the mother and the long-term potential consequences for the health of the infant

Local Bacteria Affect the Efficacy of Chemotherapeutic Drugs

In this study, the potential effects of bacteria on the efficacy of frequently used chemotherapies was examined. Bacteria and cancer cell lines were examined in vitro and in vivo for changes in the efficacy of cancer cell killing mediated by chemotherapeutic agents. Of 30 drugs examined in vitro, the efficacy of 10 was found to be significantly inhibited by certain bacteria, while the same bacteria improved the efficacy of six others. HPLC and mass spectrometry analyses of sample drugs (gemcitabine, fludarabine, cladribine, CB1954) demonstrated modification of drug chemical structure. The chemoresistance or increased cytotoxicity observed in vitro with sample drugs (gemcitabine and CB1954) was replicated in in vivo murine subcutaneous tumour models. These findings suggest that bacterial presence in the body due to systemic or local infection may influence tumour responses or off-target toxicity during chemotherapy

The microbiota of breast tissue and its association with breast cancer

In the United States, 1 in 8 women will be diagnosed with breast cancer in her lifetime. Along with genetics, the environment contributes to disease development, but what these exact environmental factors are remains unknown. We have previously shown that breast tissue is not sterile but contains a diverse population of bacteria. We thus believe that the host's local microbiome could be modulating the risk of breast cancer development. Using 16S rRNA amplicon sequencing, we show that bacterial profiles differ between normal adjacent tissue from women with breast cancer and tissue from healthy controls. Women with breast cancer had higher relative abundances of Bacillus, Enterobacteriaceae and Staphylococcus. Escherichia coli (a member of the Enterobacteriaceae family) and Staphylococcus epidermidis, isolated from breast cancer patients, were shown to induce DNA double-stranded breaks in HeLa cells using the histone-2AX (H2AX) phosphorylation (γ-H2AX) assay. We also found that microbial profiles are similar between normal adjacent tissue and tissue sampled directly from the tumor. This study raises important questions as to what role the breast microbiome plays in disease development or progression and how we can manipulate this for possible therapeutics or prevention. IMPORTANCE This study shows that different bacterial profiles in breast tissue exist between healthy women and those with breast cancer. Higher relative abundances of bacteria that had the ability to cause DNA damage in vitro were detected in breast cancer patients, as was a decrease in some lactic acid bacteria, known for their beneficial health effects, including anticarcinogenic properties. This study raises important questions as to the role of the mammary microbiome in modulating the risk of breast cancer development

UV Shielding of Bacillus pumilus SAFR-032 Endospores by Martian Regolith Simulants

As exploration of the solar system advances with life detection missions on the horizon, the concern for planetary protection has grown considerably. When attempting to detect extraterrestrial life, the likelihood of false positives from terrestrial contamination must be minimized. The Exposing Microorganisms in the Stratosphere (E-MIST) balloon project aims to evaluate whether resilient terrestrial bacteria can survive stressors in a Mars-like environment. This is accomplished by sending Bacillus pumilus SAFR-032, an endospore-forming bacterial isolate from a spacecraft assembly facility, to the Earth's middle stratosphere (30-38 kilometers), where low temperature and pressure and high radiation and dryness conditions are similar to the surface of Mars. Previous ground and flight tests showed that the vast majority of SAFR-032 spores (99.99 percent) were inactivated by direct sunlight due to ultraviolet (UV) radiation. This observation led us to explore the role of dust shielding in changing microbial survivorship outcomes. To determine the dust particle distributions and density for potentially shielding microbes from UV radiation, samples of a Martian dust simulant were mixed with SAFR-032 spores. The dry heat sterilized simulant used was JSC MARS-1, weathered volcanic ash from Hawaii that displays many chemical and physical properties similar to the Martian soil as characterized by the Viking Lander 1, including reflectance spectrum, chemical composition, mineralogy, grain size, specific gravity, and magnetic properties. First, scanning electron microscopy was undertaken to visualize the aggregation of the spores with dust particles (i.e., shading effects), and samples of varying dust concentrations were subsequently irradiated with UVC light to test survivorship outcomes. After a relationship between dust concentration and spore survivorship was determined, a solar simulator capable of irradiating samples with a fuller UV spectrum (less than 280-400 nanometers) was used to perform a more robust middle stratosphere simulation. Taken together, we will use results from the ground-based irradiation studies to feed into experimental designs for the next E-MIST ultra-long duration polar balloon flight launched by NASA