Rivalidad entre competidores actuales

The studies described above present a rationale for how distally located bacteria may contribute to disease progression or prevention. Below I discuss the possible role of bacteria within the breast in modulating this risk.

In a large scale analysis of 140,000 women in 47 studies from 30 different countries it was shown that for every 12 months of breast-feeding the risk of breast cancer decreased by 4.3% (Collaborative Group on Hormonal Factors in Breast Cancer, 2002). This study did not account for the different molecular subtypes of breast cancer, but another international study, conducted to assess the role of breast feeding in hereditary cancer, showed that women with BRCA1 mutations who breast fed for 1 year or longer had a 45% reduced risk of breast cancer, though these protective effects were not seen in patients with BRCA2 mutations (Jernström et al., 2004; Tryggvadottir et al., 2003). Interestingly, an in vivo model with Caveolin-3-/- mice, which have constitutive milk production, showed that mutants were substantially more protected against mammary tumour development than their wild-type counterparts, after tumour injection (Sotgia et al., 2009).

There are many theories as to why lactation could be protective, such as reduction of estrogen levels (Jernström et al., 2004; Lyons et al., 2009), excretion of carcinogens from the breast and differentiation of mammary epithelial cells (Lipworth et al., 2000). Hormone modulation, however, may not be a significant contributor, as breast-feeding is protective in estrogen receptor negative, progesterone receptor negative (ER-PR-) and triple negative tumours (Gaudet et al., 2011; Ursin et al., 2005).

We raise the possibility that bacteria present in the mammary glands during lactation could act independently or in concert with other factors to afford protection. In a study comparing the immunomodulatory potential of two species of Lactobacillus isolated from breast milk (L. fermentum CECT5716 and L. salivarius CECT5713) with two non-milk derived species (L. fermentum NCIMB7017511 and L. salivarius NCIMB11795) it was shown that the milk-derived strains induced higher levels of IL-10 and activated NK cells from PBMC (Pérez-Cano et al., 2010), two factors that are protective against breast cancer. Possibly, the strains of bacteria growing in breast milk have been selected to provide an evolutionary advantage to women to help control and/or prevent the progression of breast cancer. This will be a difficult hypothesis to prove but one worthy of consideration.

High-throughput sequencing to analyze the human

1.5

microbiota

The first description of microbes was reported in 1673 by the Dutch scientist Antonie van Leeuwenhoek, with his work considered to be the start of the field of Microbiology. Another pivotal landmark was the development of solid media by Robert Koch in 1881, which allowed him to isolate disease-causing organisms thereby determining the agents responsible for tuberculosis, cholera and anthrax. While culture analysis is routinely used in labs across the world and has been indispensable to microbiologists over the last 130 years, culture methods have limited our understanding of the composition and function of complex microbial communities, as most organisms cannot be grown in the lab (Amann et al., 1995; Stewart, 2012). It is for this reason, that culture-independent methods, most notably high throughput sequencing (HTS), has revolutionized the field of microbial

ecology, allowing us to study the human microbiota and its function at various body sites, in healthy and diseased patients and at different stages of human development (Coburn et al., 2015; Costello et al., 2013; Gill et al., 2006; Grice et al., 2009; Human Microbiome Project Consortium, 2012; Hummelen et al., 2010; Hunt et al., 2011; Koenig et al., 2011; Macklaim et al., 2013; Turnbaugh et al., 2009; Wolfe et al., 2012).

1.5.1

16S rRNA gene targeted sequencing

The 16S rRNA gene is part of the small ribosomal subunit of bacteria, an evolutionary conserved, ubiquitous molecule, that makes it useful for phylogenetic studies (Woese and Fox, 1977). While 16S rRNA gene amplicon sequencing is the most popular method to identify and compare bacteria within samples, some groups have opted to utilize other targets, such as cpn60 (Links et al., 2012).

HTS has allowed for the unprecedented ability to process 100-1000s of samples at a time, generating millions of reads at a reasonable cost, however, it is limited by the length of sequence it can produce and the resolution of the phylogenetic assignment. The current version of the Illumina MiSeq platform generates a maximum read length of 600bp, up from 100bp when it was first developed. Because of this, HTS studies do not sequence the full 16S rRNA gene (1.5kb) but specific regions of it, called hypervariable regions. These hypervariable regions (V1-V9), flanked by conserved regions from which amplification primers are designed, are as effective as the full length version at assigning taxonomy and estimating community richness (Andersson et al., 2008; Gloor et al., 2010; Liu et al., 2007).

Currently there is no consensus in the field as to which hypervariable region should be sequenced, because the “best one” will depend on the sequencing platform, databases used for assigning taxonomy and the type of samples used (Soergel et al., 2012). Stool microbiota studies typically use V4 or V3-V5 (which span the V4 region). V3, V4 and V4/V5 have been shown to be more accurate in determining taxonomy compared to V2, V5 alone and V6, regardless of sequencing platform (454 and Illumina MiSeq) or database used to assign taxonomy (i.e. RDP, Greengenes, NCBI) (Claesson et al., 2010; Huse et al., 2008; Liu et al., 2008; Mizrahi-Man et al., 2013). However, in terms of estimating diversity (in the stool), all regions tested performed equally well (Liu et al., 2008). Since V4 can detect a broad range of bacteria and archaea it has been chosen as the target for the Earth Microbiome Project (www.earthmicrobiome.org). V6 is commonly

used in vaginal microbiota studies as it can differentiate Lactobacillus species, which dominate the healthy vagina, as well as picking up the various organisms found in women with bacterial vaginosis (Gloor et al., 2010).

V6 was the region of choice for the milk and tissue studies described herein, because unlike primers for the V4 region, it does not cross-react with eukaryotic mitochondrial DNA (personal observations and also noted in (Huys et al., 2008)). In stool samples, where there is a higher proportion of bacterial vs eukaryotic DNA this is not a problem. But in tissue, where eukaryotic DNA predominates, amplification needs to be as specific as possible so as to not saturate the reads with eukaryotic sequences. In addition, V6 but not V4 directed primers, detect Propionibacterium (Kuczynski et al., 2012) a common skin organism that we expected to be present in our samples.

In order to more readily interpret the massive amount of data generated with HTS, 16S rRNA gene sequences that are similar to each other are clustered together into operational taxonomic units (OTUs) which can then be used to estimate microbial diversity (Chen et al., 2013; Schloss and Westcott, 2011). This is based on the assumption that sequences that are similar to each other represent organisms that are more phylogentically related (He et al., 2015). Sequences are typically clustered into OTUs with 97% similarity (considered the threshold for separating species)(Schloss and Handelsman, 2005), with the most abundant sequence in that group being the representative one used for taxonomy. A table of OTUs and counts for each sample is then used for downstream analysis such as beta diversity (diversity between samples), alpha diversity (diversity within a sample) and comparing relative abundances between groups.

Scope and objectives

1.6

The Human Microbiome Project (HMP), funded by the National Institutes of Health, was developed in order to comprehensively characterize the presence and function of bacteria colonizing different body sites, under different conditions. This initiative, and a similar one in Europe, was undertaken because of the overwhelming evidence showing how important microbes can be in maintaining health and how significant aberrations can lead to detrimental outcomes (Peterson et al., 2009). Phase 1 of the project was published in 2012 and reported the diversity of bacterial communities from 242 healthy American adults (males and females) from 15-18 body sites (Human Microbiome Project Consortium, 2012). Unfortunately and surprisingly, this large-scale study did not include analysis of human milk.

While there were milk microbiota studies prior to the HMP and others conducted after its 2012 publication, most of these utilized culture, denaturing gradient gel electrophoresis and/or PCR specific amplification. At the start of this thesis there had only been 1 human milk microbiota study utilizing HTS to characterize its composition (Hunt et al., 2011) and none to determine what factor(s) could shape this bacterial community.

My first objective was thus to characterize the human milk microbiota in Canadian women and to determine what factors shape its composition. In Chapter 2, I present a case report following a woman over 4 months for Hodgkins lymphoma to determine how drugs given distally could affect the milk microbiota- in this case we looked at the effects of the ABVD chemotherapeutic regime (adriamycin, bleomycin, vinblastine, dacarbazine). In Chapter 3, I look at the effects of common pregnancy related factors and its impact on the milk microbiota- specifically gestation (pre-term vs term birth), mode of delivery (cesarean vs vaginal) and gender of the child (male vs female vs twins).

Considering that milk is not sterile, that there is exposure of the nipple to the external environment and that the nutrient rich fatty environment of the breast could support bacterial growth, I hypothesized that breast tissue, irrespective of lactation and pregnancy, could also have its own indigenous microbiota. My second objective was thus to characterize, using HTS and culture, the bacteria in breast tissue. In Chapter 4, I present our findings from tissue samples analyzed from women from two distant demographics: Canada (n=43) and Ireland (n=39).

After showing that a breast microbiome exists, my third objective was to examine whether this microbiome could have a role in the development of breast cancer. The first

aim within this objective was to determine whether bacterial profiles differ between women with breast cancer and those who are disease free (Chapter 5). The rationale for this comes from studies showing differences in microbial profiles between healthy individuals and those with colorectal cancer, with these differences driving disease development. The second aim within this objective was to explore how the breast microbiota could promote disease progression. I did this by looking at the ability of bacteria isolated from breast cancer patients to induce DNA damage using the γH2AX

assay (Chapter 5) and by sequencing the genome of a Bacillus cereus strain isolated from a women with stage III invasive breast cancer (Chapter 6). Bacillus cereus was chosen for this analysis as Bacillus species were higher in cancer patients compared to controls and they were the most abundant organism cultured from breast cancer patients.

In a series of Appendices, I describe my work showing (i) the optimization of DNA extraction protocols for low biomass samples, (ii) histological work to try and identify where in the breast bacteria were located and lastly (iii) the properties of Lactobacillus

rhamnosus GR-1 (a probiotic) when grown in bovine milk, with an emphasis on a

pathway involving breakdown of lactosylceramide to ceramide, a pro-apoptotic signaling molecule involved in controlling aberrant cell growth.

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