Research in the past few years has provided evidence that the breast tissue harbors a diverse and unique community of microbes (4). There are many theories regarding the origins of the breast microbiota, ranging from translocation of microbiota from the skin through the nipple, to the translocation of microbiota during lactation (4). Whatever the origin, the breast tissue contains a high diversity of bacteria. Using Shannon's diversity index (SDI), an index used to characterize species diversity, studies have concluded that the mammary microbiota has an average SDI of 3.6 (12). This is a relatively high diversity since the gut and oral cavity, which are considered to have a variety of bacterial communities, have an SDI of 3.9 and 6.5 respectively, while the vagina which is known to have a low diversity of bacteria has an SDI between 0.46 and 2.9 (10).

The knowledge that breast tissue has its own unique microbiome has sparked interest as to its involvement in the development of BC (4). Even though this area of research has yet to be fully explored, there have been various studies that have proved to be fruitful.

A study conducted by Urbaniak et al (10) was conducted to investigate if healthy mammary tissues from women in Canada and Ireland contained their own microbiome. In this study, breast tissue samples were collected from women undergoing breast surgery to either remove a cancerous or benign tumor or to have breast reductions. Interestingly, the results showed that the breast tissues have their own unique microbiome. By using 16S rRNA sequencing and culture, the researchers identified various bacteria and found the most abundant phyla in normal breast tissues to be Proteobacteria, Firmicutes and Actinobacteria (10) (Table I). Interestingly, while Proteobacteria were seen in abundance in breast tissue, they are normally known to be a minority in other body sites, such as the vagina, bladder and GI tract. This could be because Proteobacteria have adapted to the fatty acid environment of the breast tissue (10). More specifically, the Canadian samples showed that the most abundant taxa were from the genera Bacillus, Acinetobacter Enterobacteriaceae, Pseudomonas, Staphylococcus, Propionibacterium, Comamonadaceae, Gammaproteobacteria and Prevotella (Table I). On the other hand, the Irish samples demonstrated that the most abundant taxa were Enterobacteriaceae, Staphylococcus Listeria welshimeri, Propionibacterium and Pseudomonas (10) (Table I).

Xuan et al (13) reported that in addition to the presence of different species between tumor and normal breast tissues, tumor tissues seem to have a dramatic reduction in bacterial load compared to normal tissues. In addition, the inverse correlation between bacterial load and tumor stage implies that bacterial load could be used in conjunction with current methods to monitor the progression of BC and to facilitate staging of the disease. However, further research is warranted to evaluate and determine a possible role of the bacterial load in the diagnosis or staging of BC. Furthermore, in the study by Xuan et al (13) it was observed that one third of antibacterial genes were downregulated in tumor tissues compared to healthy tissues. These results suggest that bacteria may play a role in regulating immune responses within healthy breast tissue and these responses may be disrupted during tumorigenesis (13).

Following on from previous research, Urbaniak et al (14) used 16S rRNA amplicon sequencing and showed that the bacterial profiles differ between tissues from healthy controls (women undergoing breast reduction or breast enhancement surgeries), normal adjacent tissue from women with BC and tumor tissue from women with BC. The researchers found that women with BC had higher relative abundances of Bacillus, Enterobacteriaceae and Staphylococcus, among others. The researchers also reported that Escherichia coli (a member of the Enterobacteriaceae family) and Staphylococcus epidermidis, isolated from BC patients, were shown to induce DNA double-stranded breaks in HeLa cells using the histone-2AX (H2AX) phosphorylation assay. They also found that microbial profiles are similar between normal adjacent tissue and tissue sampled directly from the tumor in BC women. Interestingly they also found that Lactococccus, Streptococcus and Prevotella were higher in the tissues of healthy women compared to BC patients (Table I). Interestingly, all of the aforementioned bacteria have shown to confer some protective properties against BC. Lactococcus spp. is known to have anti-carcinogenic properties through potentially activating natural killer (NK) cells and enhancing cellular immunity, thus being potentially protective against BC. Streptococcus, especially S. thermophilus has also been shown to have protective properties against cancer by producing anti-oxidant metabolites that neutralize reactive oxygen species and thus protect the host from DNA damage. In addition, Prevotella is able to produce the short-chain fatty acid (SCFA) propionate, which has shown to have anti-inflammatory properties (14).

In another study, Chan et al (15) investigated the composition of nipple aspirate fluid (NAF) on BC survivors and reported a higher level of β-glucuronidase levels as well as a higher abundance of Alistipes in the NAF of BC survivors compared to healthy controls. On the other hand, they found an unclassified genus of the Sphingomonadaceae family in the NAF of healthy individuals. Additionally, they also found Sphingobium yanoikuyae to be more abundant in normal breast tissue when compared to ER⁺ breast tumor tissue (Table I). While the benefits of Sphingomonadaceae are not well known, this family has also been known to break down aromatic hydrocarbons as well as polycyclic aromatic hydrocarbons, both of which are known to be associated with BC (15,16). Due to their ability to metabolize aromatic hydrocarbons, they may confer protective properties against BC, as they were only observed in NAF of healthy breast tissue. However, more research is warranted to confirm this association (15).

Hieken et al (17) explored the different microbiota composition between benign vs. malignant breast tumor tissue. The study reported significant differences in the breast tissue microbiome between women with benign vs. women with malignant disease. Specific genus-level taxa that were significantly enriched in breast tissue from women with malignant disease included Fusobacterium, Atopobium, Gluconacetobacter, Hydrogenophaga and Lactobacillus (17) (Table I).

In a study by Thompson et al (18), the researchers aimed to compare the microbiota in BC tissues compared to non-cancerous adjacent tissues. They found Proteobacteria to be enriched in breast tumor tissue samples while Actinobacteria were found in abundance in non-cancerous adjacent tissues. In addition, Thompson et al (18) found E. coli to be prevalent in cancerous and adjacent non-cancerous tissues but with a higher abundance in non-cancerous adjacent tissue of BC women (Table I). This was inconsistent with the observations by Urbaniak et al (14) who reported higher levels of E. coli in BC compared to healthy controls. Thompson et al (18) also observed an increase in Streptococcus pyogenes, in tumor breast tissue samples among others. Other recent studies have also established a relationship between Streptococcus spp. and β-glucuronidase/β-glucosidase enzymes that are responsible for promoting the re-circulation of estrogen by breaking down the estrogen-glucoronate conjugate (18,19). Therefore, S. pyogenes may increase systemic estrogen levels which have been continuously proven to increase the risk for BC (18) (Table I).

In another study, Wang et al (20) observed the microbiome of breast tissue, urine and oral rinse from BC patients and compared them with those of healthy women (women who underwent cosmetic procedures such as bilateral reduction mammoplasty and mastopexy). Unlike previous studies, they found no significant differences either in the overall diversity or in the number of microbes between cancerous and healthy samples. Additionally, tissues from healthy participants were shown to have an increased abundance of Methylobacterium while in BC patients the same bacterium was observed to be significantly decreased (20) (Table I).

In addition, Parida and Sharma (16) reported that Enterobacteriaceae (specifically E. coli), and Bacillus cereus were more abundant in tumor tissues than in normal breast tissues. E. coli, (B2 phylotype), has cancer-promoting properties as it has the ability to produce colibacin, a known genotoxin. Colibacin is known to induce double-stranded DNA breaks and has not only been implicated in colorectal cancer but has now also been linked to BC (16). DNA damage induced by microbes may not be enough for tumorigenesis to occur, but if coupled with other molecular errors the result could be detrimental to host health (14). On the other hand, even though B. cereus does not necessarily induce DNA damage, it still possesses pro-carcinogenic properties. For example, B. cereus is able to metabolize progesterone into 5-alpha-3,20-dione (5aP), which has been seen to be much higher in BC and has been observed in vitro to induce BC cell proliferation (14,16).

Some of the limitations of the studies investigating the microbiota in normal and malignant breast tissue are the low number of participants as well as the fact that the microbiota from BC tissues was commonly compared to that of women with benign disease or women that underwent breast reduction or breast augmentation surgeries (Table I). It is questionable whether the tissues assessed by women undergoing breast reduction should be considered as ‘healthy.’ The potential absence of epithelium or stroma in these fatty tissues could render these not directly comparable to a breast cancer which is comprised predominantly of epithelium and stroma, or healthy breast tissue that has a varying abundance of these cell types.

Xuan et al (13) investigated the potential role of breast microbiota on ER⁺ BC by comparing the DNA of the microbiota found in the breast tumor tissue compared to the normal adjacent tissue from the same patients and healthy women that underwent reduction mammoplasty. Sphingomonas yanoikuyae was the most abundant bacterium found in normal adjacent breast tissue, while the bacterium Methylobacterium radiotolerans was abundant in ER⁺ breast tumor tissues (13). Interestingly, while S. yanoikuyae was detected in almost half of normal adjacent tissue samples, it was not detected at all in the paired corresponding tumor tissues, whereas M. radiotolerans was detected in both the normal adjacent and the tumor tissue samples. This observation suggests that S. yanoikuyae may have a protective function in the breast, potentially due to its ability to express certain ligands that activate important cancer immuno-surveillance mediators (13) (Table II).

Wang et al (20) observed that the microbial diversity and abundance was higher in hormone-positive BC than in hormone-negative BC. This could be due to the ability of some microbes to metabolize and regulate the bioavailability of steroid hormones in the former (16,20). Additionally, they found Methylbacterium to be significantly decreased in hormone-positive BC when compared to hormone-negative BC (20) (Table II).

A study by Banerjee et al (21) was carried out to investigate the specific microbial signatures in 4 different BC subtypes i.e., HER-2-positive (HER-2⁺), hormone receptor-positive (PR⁺/ER⁺), triple-negative (PR⁻, ER⁻, HER-2⁻) and triple-positive (PR⁺, ER⁺, HER-2⁺) BC. In addition, women who had undergone breast reduction surgery were used as a control. The researchers observed that, among others, Sphyngomonas and Mycobacterium, were present in all four BC subtypes. Additionally, they found that each subtype had a unique microbial signature; Triple-negative had the least complex microbial signature, while PR⁺/ER⁺ was associated with the most complex microbial signature. Additionally, higher levels of Brevundimonas were noted in PR⁺/ER⁺ and in triple-positive compared to PR⁻/ER⁻ and triple-negative subtypes. In hormone-negative BC (PR⁻ and ER⁻) an increased abundance of Mycobacterium was observed (21) (Table II). Identifying BC subtype signatures through microbiota composition could pave way for potential BC screening techniques. Nevertheless, it is evident that investigations regarding microbiota residing in normal breast tissue and its role in BC development is still in its infancy, but can potentially have a huge role in developing screening and therapy techniques for BC.

Overall, the limitations of the studies investigating the microbiota composition in different BC subtypes were similar to the studies comparing the microbiota in BC vs. normal tissue; i.e. the number of participants in these studies was low and they used women that underwent cosmetic surgeries as healthy controls (Table II).

The dysregulation of the gut microbiota has been associated with BC since 1990, where a study found that women with BC had an abundance of multiple different microbes in the GI tract (Table III) (23). Since then, more recent studies have continuously showed that the gut microbiome composition does indeed change in women with BC compared to those without (22).

A more recent study by Zhu et al (24) showed that the composition and functions of the gut microbial community differ between post-menopausal BC patients and post-menopausal healthy controls. In this study, the researchers performed a comprehensive shotgun metagenomic analysis of 18 pre-menopausal BC patients, 25 pre-menopausal healthy controls, 44 post-menopausal BC patients and 46 post-menopausal healthy controls. The results of the study showed that the microbial diversity was higher in BC patients than in controls. Relative species abundance in gut microbiota did not differ significantly between pre-menopausal BC patients and pre-menopausal controls. However, the relative abundance of 45 species differed significantly between post-menopausal BC patients and post-menopausal controls: 38 species were enriched in post-menopausal BC patients including Escherichia coli, Klebsiella sp_1_1_55, Prevotella amnii, Enterococcus gallinarum, Actinomyces sp. HPA0247, Shewanella putrefaciens, and Erwinia amylovora. On the other hand, 7 species were less abundant in post-menopausal BC patients compared to healthy controls including Eubacterium eligens and Lactobacillus vaginalis (24).

Interestingly Zhu et al (24) reported that Erwina amylovora and Shewanella putrefaciens were shown to have a positive correlation, albeit a weak one, with estradiol, suggesting potential involvement of both microbes in the metabolism of estrogen. Due to their potential involvement in estrogen metabolism, such gut microbiota could be used in the future as novel biomarkers for BC (24) (Table III).

In the same study, Zhu et al (24) also observed that Eubacterium eligens and Roseburia inulinivorans were more abundant in post-menopausal healthy controls than in post-menopausal women with BC. Interestingly, R. inulinivorans is a bacterium with known anticarcinogenic properties; i.e. it produces butyrate which has anti-inflammatory properties mainly by inhibiting the activation of nuclear factor (NF)-κB in intestinal epithelial cells through tumor necrosis factor (TNF)-α (24,25). Therefore, a reduction in butyrate in the colon caused by decreased levels of R. inulinivorans in post-menopausal women can increase inflammation and therefore increase their risk of BC (24). It was also observed that in both pre-menopausal women and post-menopausal women, genes of the iron transport system were increased, however, in post-menopausal women genes involved in lipopolysaccharide (LPS) biosynthesis were also seen to be increased. Both the iron transport system and LPS biosynthesis increase systemic inflammation and therefore increase the risk for BC (24) (Table III).

It has recently been proposed that the microbiota in the GI tract may act as an endocrine organ, due to the gut's ability to produce various metabolites such as SCFAs and to regulate hormone metabolism, all of which affect distal organs through the blood stream (26). Some of these microbial metabolites have been shown to influence the progression of BC (27). A well-documented microbe metabolite linked to BC is cadaverine, a biogenic amine whose biosynthesis is decreased in early stage BC (27,28). Cadaverine is considered an anticancer bacterial metabolite because of its ability to suppress the aggressiveness, metastasis and progression of tumor cells. Cadaverine has also been shown to induce the metabolism of tumor cells that have begun glycolysis by decreasing cellular oxygen consumption (27,28). Additionally, other bacterial metabolites such as lithocholic acid (LCA), a secondary bile acid, have been observed to inhibit BC progression by creating oxidative and nitrosative stress (27). More specifically, LCA inhibits epithelial-mesenchymal transition, metastasis and BC progression by activating nuclear factor erythroid 2-related factor 2 (NRF2) and has pro-apoptotic effects on BC tumor cells (27,28).

SCFAs are products of fibre being fermented in the colon by certain bacteria (27). The most predominant SCFAs are butyrate, propionate, and acetate, which are well known for their anticancer effects (27). These metabolites have shown to be protective against cancer as they are involved in mediating cell cycle arrest, and inducing apoptosis (28). Therefore, changing the composition of the gut microbiome will inevitably also change the anti-carcinogenic metabolites being produced, thereby preventing or promoting BC progression (27).

As previously discussed, the gut microbiome may be considered an endocrine organ, due partly to its role in estrogen metabolism. The microbiota in the GI tract is known to be one of the most important regulators in estrogen circulation (27). This is especially relevant in hormone-positive BC, as there is an undisputable relationship between endogenous estrogen burden and the development of BC in post-menopausal women (there is a similar relationship with pre-menopausal women but the correlation has not proven to be as strong) (28,29).

Estrogen metabolism in the GI tract is attributed to what is currently being referred to as the estrobalome. The estrobalome is defined as the aggregate of bacterial genes that result in enzymes that can metabolize estrogens (28,30). Free-circulating estrogens are subjected to hepatic first-pass metabolism where they are conjugated and then eliminated via urine or feces (30). However, a significant portion of the conjugated estrogen is reabsorbed back into circulation before it gets excreted. This suggests that bacteria in the gut possess β-glucuronidase/β-glucosidase activity allowing them to deconjugate estrogens, permitting them to become biologically active again and re-enter the circulation (30). The re-circulated estrogens then interact with breast tissue facilitating cellular growth and thus contributing to the initiation and progression of BC, as well as increasing the risk of ER-positive BC in post-menopausal women (27,28). This is especially relevant as certain bacteria that have been previously shown to be abundant in BC are coincidentally also related to an increase in activity of β-glucuronidase/β-glucosidase activity. Such bacteria include S. pyogenes, Clostridia spp., Baccilus spp. and E. coli (18,31). It was also further seen that the relationship between microbiota and estrogen metabolism can be used to predict the risk of BC in post-menopausal women due to the diversity and composition of gut microbiota being associated with patterns of estrogen metabolism (32). Therefore, a change in microbiota composition can alter normal host function, favoring BC development.

A study by Rosean et al (33) used a mouse model for hormone receptor-positive (HR⁺) mammary cancer in order to investigate whether commensal dysbiosis, more specifically, pre-existing dysbiosis in the microbiome can influence the progression and outcome in HR⁺ BC. The researchers demonstrated that a pre-established disruption of commensal homeostasis results in enhanced circulating tumor cells and subsequent dissemination to the tumor-draining lymph nodes and in distant sites such as the lungs; all of which promote the poor outcomes seen in HR⁺ BC. Due to the results being so promising, Buchta Rosean et al (33) concluded that commensal dysbiosis may have therapeutic implications and could potentially be used as a biomarker for HR⁺ BC (33). The study by Buchta Rosean et al (33) currently presents one of the strongest evidence to date that gut dysbiosis may promote breast cancer metastasis, and further studies should be conducted to investigate this association.

Diet is a well-established modifiable risk factor for BC, as changes in diet have been linked with increasing overall health and survival rates in patients that already have BC (34). A diet rich in fat has been shown to affect β-glucuronidase activity by increasing bile acid secretion, thus promoting the growth of E. coli and Enterobacter, both of which are potent β-glucuronidases and thus contribute to the estrogen burden (30,35). However, a fibre-rich diet can potentially be onco-protective; soluble fibre is fermented by SCFAs and creates a favorable environment for beneficial bacteria such as Bifidobacterium (30).

Hassan et al (37) investigated the role of Enterococcus feacalis and Staphylococcus hominis in vitro. They isolated these bacteria from the breast milk of healthy women and conducted the rest of the investigation in various antibiotic-free media. They used three different cell forms of the bacteria (live, heat-killed and cytoplasmic fractions) and found that all three forms of the bacteria caused a significant decrease in cell proliferation via induction of both cell cycle arrest and apoptosis in BC cells such as in MCF-7 cells (37).

Another study by Esfandiary et al (38) investigated the effect of the probiotic strains Lactobacillus crispatus SJ-3C-US and Lactobacillus rhamnosus GG on MDA-MB-231 cell lines. They found that both strains induced cytotoxic effects on triple-negative BC cells and that L. rhamnosus especially, downregulated genes associated with hypoxia, such as SLCA2A1 (38), which is known to code for the production of GLUT 1. By downregulating the production of GLUT 1, metastasis can be prevented through the MMP-2 and JNK pathways (9).

Lactobacillus plantarum has also shown promise in various studies, such as in the study conducted by Bharti et al (2015), where the highest cytotoxic effect on MCF-7 cancer cell lines was seen with the administration of L. plantarum (39). Kadirareddy et al (40) demonstrated that L. plantarum induced apoptosis in MDA-MB-231 cells by the production of conjugated linoleic acid which downregulated the NF-κB pathway causing proteasome degradation of IκBα, inhibition of p65 nuclear translocation, release of cytochrome c from the mitochondria and finally overexpression of Bax protein (40). These are but a few examples of in vitro studies in which probiotics strains have been used to investigate their potential role in breast cancer prevention.

The efficacy of orally administered probiotics in tumor cell inhibition was also confirmed by animal studies. For example, in an in vivo mouse model, the intake of milk fermented with Lactobacillus helveticus R389 played a role in delaying breast tumor cell development. The latter was associated with increased regulatory cytokines such as interleukin (IL)-10 and decreased IL-6 levels in serum and mammary mouse cells, as well as by induction of cellular apoptosis. IL-10 is known to induce the expression of TNF-α related apoptosis-inducing ligand (TRAIL) and death receptor 4 (DR4), as well as recruiting lymphocytes, all of which contribute to the apoptosis of mammary cells (41).

Additionally, Lakritz et al (42) observed the effect that Lactobacillus reuteri has on the development of mammary cancer in two different mouse models. In the first model, Swiss mice were fed Westernized chow (mimicking a westernized diet) thereby putting the mice at increased risk for the development of mammary tumors. The second animal model used was FVB strain erbB2 (HER2) mutant mice which are genetically susceptible to mammary tumors. Both of these mouse models were exposed to L. reuteri through their water supplies and compared to controls. Interestingly, exposure to L. reuteri in the experimental groups was found to be sufficient in delaying the onset of pre-neoplastic features in both models. This hypothesis is consistent with other research studies supporting that lactic acid-producing bacteria decrease the risk of BC in women (42–44). In addition to inhibiting early stage tumorigenesis, L. reuteri has also been reported to increase the sensitivity of BC cells to apoptosis (27,42). This study demonstrates that by modulating the tumor microenvironment through the use of probiotics, one can potentially regulate systemic immune cell responses impacting cancer progression. Such findings may have future implications in the public health sector since such administrations could be used to decrease BC risk (42).

Additionally, a study conducted by Soltan Dallal et al (45) investigated the effect that orally administrated Lactobacillus casei has on NK cytotoxicity and production of cytokines in the spleen cells of BALB/C mice that have invasive ductal carcinoma. The results showed that L. casei was able to decrease the growth rate of tumors, prolong survival, stimulate Th1 cytokine production and increase NK cell cytotoxicity in the spleen cells of mice with invasive ductal carcinoma demonstrating its potential in BC therapy (45).

Yazdi et al (46) conducted a unique study in which BALB/c mice were administered with Lactobacillus brevis that was enriched with selenium nanoparticles. The results showed that this not only improved the prognosis of mice with highly metastatic BC, but it also increased NK cytotoxicity, IFN-γ and IL-17 as well as decreased tumor metastasis to the liver (46). Subsequently, another study confirmed that the oral administration of Lactobacillus acidophilus significantly increased the survival time of BALB/c mice with mammary tumors. L. acidophilus promoted the proliferation of immune cells and increased the production of IFN-γ and decreased the production of IL-4. IFN-γ is crucial in the activation of NK cells, which are known to be the first line defence against tumor cells (36). High levels of IFN-γ not only increase NK cytotoxicity but may also increase tumor cell visibility [through its role in major histocompatability complex (MHC) expression], as well as inhibiting intra-tumoral angiogenesis (36).

In another study by Zamberi et al (47), BALB/c mice were injected with triple-negative 4T1 BC cells, and were treated with Kefir water that is naturally enriched with L. acidophilus, L. casei, and Lactobacillus lactis. The results showed a decrease in cell mitosis and an increase in apoptosis in mice that drank the water enriched with the specific probiotic strains. In addition, they found that the various strains also inhibited the inflammation in the tumor environment, modulated the immune system and regulated genes and proteins of angiogenesis (47). Moreover, Zamberi et al (47) and the Soltan Dallal et al (45) demonstrate how especially L. casei, not only decreased the growth of tumors, but also increased apoptosis and inhibited inflammation, further illustrating the emerging importance of this microbe in BC prevention and possibly treatment.

While the aforementioned studies presented promising results, it should be noted that some of the studies had limitations. For example, most of the studies were either conducted in vitro or using mouse models and therefore could not investigate the different factors that affect the diversity of the microbiome in humans, such as diet, age or genetic background (48).

In their very comprehensive perspective review Suez et al (48), reports that even though probiotics are commonly used by the general public there are still conflicting clinical results for many probiotic strains and formulations. Furthermore, the researchers stress the importance of how large scale randomized blind clinical trials should be designed to assess a number of questions such as gut colonization by probiotics, the importance of specific bacterial strains and their interactions with the indigenous microbiome and the safety and impact on the host.

Evidence from the literature discusses how the safety and adverse effects of probiotics have neither been extensively researched nor correctly documented in most of the clinical trials. In fact, it has been observed that the use of probiotics in critically ill adults in intensive care units, young infants, or neonates with a very low birth weight is associated with an increased risk of infection. In addition, patients that underwent antibiotic treatments showed increased colonization by probiotic strains, which was linked to persistent dysbiosis induced by long term probiotics (48). A systematic review by Befeta et al (49), reported that most clinical trials did not report the adverse effects accurately and support how the increasing use of probiotics both in clinical practice and over-the-counter increases the urgency to design and conduct clinical trials that adequately report and document any adverse effects caused by probiotics (49).

Therefore, it is important to note that research on the effects of probiotics on BC is still at its infancy and larger and more elaborate studies should be conducted to conclude if they have a protective long-term anticancer effects and to find the optimum dosage, strain and regimen for a treatment with probiotics to be effective.