Table 2.

Studies investigating changes to the breast microbiome in breast cancer. Abbreviations: ER, estrogen receptor; CNB, Core needle biopsies; HER2, herceptin receptor/erbB receptor; NAF, Nipple aspirate fluid; PR, progesterone receptor; SEB, Surgical excision biopsies; TNBC, Triple negative breast cancer.

Sample Type and Sample Size	Method	Observations	Changes to the Microbiome	Ref.
Breast tumor tissue and paired normal adjacent tissue from the same 20 patient (ER positive)	Pyrosequencing 16S V4 rDNA	The amount of bacteria, measured by the copy number of 16S rDNA, is not significantly different in paired normal tissue from breast cancer patients and healthy breast tissue from healthy individuals. The amount of bacteria, measured by the copy number of 16S rDNA, is significantly reduced in breast cancer tissue.	The most abundant phyla in breast tissue were Proteobacteria, Firmicutes, Actinobacteria and Bacteroidetes. Methylobacterium radiotolerans is relatively enriched in tumor tissue and Sphingomonas yanoikuyae is relatively enriched in paired normal tissue.	[105]
Breast tissue from 81 women with and without breast cancer from Canada and Ireland. Canadian patients: benign (n = 11), cancerous tumors (n = 27) and healthy individuals (n = 5) Irish patients: breast cancer (n = 33) and healthy individuals (n = 5)	Ion Torrent V6 16S rRNA sequencing and culture	Breast tissue contains a diverse population of bacteria. Geographical difference exist between breast tissue microbiome of Canadian and Irish subjects.	Proteobacteria and Firmicutes (specifically the class Bacilli) were the most abundant phyla in breast tissue. The most abundant taxa in the Canadian samples were: Bacillus (11.4%), Acinetobacter (10.0%), Enterobacteriaceae (8.3%), Pseudomonas (6.5%), Staphylococcus (6.5%), Propionibacterium (5.8%), Comamonadaceae (5.7%), Gammaproteobacteria (5.0%), and Prevotella (5.0%). The most abundant taxa in the Irish samples were: Enterobacteriaceae (30.8%), Staphylococcus (12.7%), Listeria welshimeri (12.1%), Propionibacterium (10.1%), and Pseudomonas (5.3%). Higher abundance of Escherichia coli was detected in women with cancer than in healthy controls.	[110]
Triple negative breast cancer (TNBC) samples (n = 100)	PathoChip array	There are unique microbial signatures in triple negative breast cancer.	Multiple viruses and other microorganisms were detected in triple negative breast cancer samples. Bacterial signatures: Brevundimonas diminuta, Arcanobacterium haemolyticum, Peptoniphilus indolicus, Prevotella nigrescens, Propiniobacterium jensenii and Capnocytophaga canimorsus (see in [107])	[107]
Nipple aspirate fluid (NAF) from healthy women (n = 23) and from women with breast cancer (n = 25)	16S V4 rRNA gene sequencing	Microbiome composition of NAF from healthy control and breast cancer are significantly different. Beta-glucuronidase levels are higher in NAF from breast cancer than from healthy control.	The most abundant phyla in NAF were Firmicutes, Proteobacteria, and Bacteroidetes. In NAF from breast cancer genus Alistipes was more abundant and an unclassified genus from the Sphingomonadaceae family in NAF from healthy women.	[108]
Breast tissues from patients with benign (n = 13) and invasive breast cancer (n = 15). The invasive cancers were stage I in 10 patients (67%) and stage II in 5 (33%). Tumors were histologic grade I in 43% and grade II in 57%. The invasive cancers were all ER and PR positive, and a minority (29%) were HER2 positive.	16S V3-V5 rDNA hypervariable taq sequencing	Breast tissue microbiome is different in women with malignant disease and in women with benign disease.	The most abundant phyla in breast tissue were Firmicutes, Actinobacteria, Bacteroidetes and Proteobacteria. Breast cancer malignancy correlated with enrichment in taxa of lower abundance including the genus Fusobacterium, Atopobium, Gluconacetobacter, Hydrogenophaga, and Lactobacillus.	[109]
Breast tissue from 58 women: benign (n = 13), cancerous tumors (n = 45), and healthy individuals (n = 23)	16S V6 rRNA sequencing	Different microbiome profile exist between breast tissue from healthy women and women with breast cancer. Normal tissues from women with benign tumors are more similar to normal adjacent tissues from cancer patients than to normal tissues from healthy women.	Breast cancer patients had higher relative abundances of Bacillus, Enterobacteriaceae and Staphylococcus. Lactococccus and Streptococcus were higher in healthy women than in breast cancer patients.	[106]
Breast tissue from 39 breast cancer patients (n = 17 tumor, n = 22 normal) and breast tissue from 24 healthy patients	16S V3-V4 rRNA sequencing	Microbiome of tumor and paired normal tissues from the same breast cancer patient are similar. Breast tissue from cancer and non-cancer patients have significantly different microbiome.	Decreased relative abundance in the genus Methylobacterium (phylum Proteobacteria) was found in breast cancer patients.	[111]
Breast tissue from tumor (n = 668) and normal adjacent tissue (n = 72) from The Cancer Genome Atlas (TCGA)	16S V3-V5 RNA sequencing data	The microbial composition is associated with alterations in the host expression profiles.	The most abundant phyla in breast tissues are Proteobacteria, Actinobacteria, and Firmicutes. Proteobacteria was increased in the tumor tissues and Actinobacteria abundance increased in non-cancerous adjacent tissues. Mycobacterium fortuitum and Mycobacterium phlei are species differentially abundant in the tumor samples. Geneset enrichment suggested that Listeria spp was associated with the expression profiles of genes involved with epithelial to mesenchymal transitions. H. influenza was associated with the proliferative pathways: G2M checkpoint, E2F transcription factors, and mitotic spindle assembly.	[112]
Breast cancer tissues [ER or PR positive (n = 50), HER2 positive (n = 34), triple positive (n = 24), triple negative (n = 40)] and breast tissue from healthy individuals (n = 20)	PathoChip array	There are unique viral, bacterial, fungal and parasitic signatures in each breast cancer type. Triple negative and triple positive samples showed distinct microbial signature, while the ER positive and HER2 positive samples shared similar microbial pattern.	Unique and common microbial signatures in the major breast cancer types are summarized in Table 1 in [113] All four breast cancer types had dominant signatures for Proteobacteria followed by Firmicutes. Actinomyces signatures was also detected in each breast cancer types.	[113]
Fresh tissue samples of both cancer and paired healthy tissues from core needle biopsies (CNB; n = 12) and surgical excision biopsies (SEB; n = 7). 3 patients underwent both procedures	hypervariable region of the 16S-rRNA gene (V3)	More similarities than differences exist between tumors and adjacent normal tissues from CNB and SEB specimens. There are more differences between subjects than between healthy and cancerous tissues collected from the same patient.	In breast tissue Proteobacteria are the most abundant phylum followed by Firmicutes, Actinobacteria and Bacteroidetes. Presence of genus Ralstonia is associated with breast tissue. The relative abundance of Methylobacterium was different in certain patients.	[114]
Breast tissue from benign (n = 22) and malignant (n = 72) breast cancer patients (Chinese cohorts)	16S V1-V2 rRNA sequencing	Microbiome profile is different in benign and malignant diseases. Microbiome composition is different in histological grades of malignant breast tissue. There is a specific correlation of microbial biomarkers and microbial pathways with advanced disease. Glycerophospholipid metabolism and ribosome biogenesis pathways were upregulated in grade III tumor compared to grade I and II. Flavonoid biosynthesis was significantly lower in grade III compared to grade I and II.	The enriched microbial biomarkers in malignant tissue included genus Propionicimonas and families Micrococcaceae, Caulobacteraceae, Rhodobacteraceae, Nocardioidaceae, and Methylobacteriaceae. The relative abundance of family Bacteroidaceae decreased and the relative abundance of genus Agrococcus (family Microbacteriaceae) increased with the development of malignancy. Genus Propionicimonas and five families Micrococcaceae, Caulobacteraceae, Rhodobacteraceae, Nocardioidaceae and Methylobacteriaceae were abundant in malignant disease compared to benign disease.	[115]