Microbiota of Human Breast Tissue

Clinical samples and study design (Canadian samples).Ethical approval for this study was obtained from Western Research Ethics Board and Lawson Health Research Institute, London, Ontario, Canada. Patients provided written consent for sample collection and subsequent analyses.

(i) Tissue collection and processing.Breast tissue was collected from 43 women (aged 18 to 90) undergoing breast surgery at St. Joseph's Hospital in London, Ontario, Canada. Thirty-eight of those women underwent lumpectomies or mastectomies for either benign (n = 11) or cancerous (n = 27) tumors, and 5 women underwent breast reductions and had no history of breast cancer. For those women with tumors, the tissue obtained for analysis was collected outside the marginal zone, approximately 5 cm away from the tumor. After excision, the fresh tissue was immediately placed in a sterile vial on ice and homogenized within 30 min of collection. As an environmental control, a tube filled with sterile phosphate-buffered saline (PBS) was left open for the duration of the surgical procedure and then processed in parallel with the tissue samples.

Tissue samples were homogenized in sterile PBS using a PolyTron 2100 homogenizer at 28,000 rpm. The amount of PBS added was based on the weight of the tissue in order to obtain a final concentration of 0.4 g/ml. Fresh homogenate and the environmental controls were then plated on different agar plates for culture analysis and the remaining amount aliquoted and stored at −80°C until DNA extraction.

(ii) DNA isolation.After tissue homogenates were thawed on ice, 400 μl (equivalent to 160 mg of tissue) was added to tubes containing 1.2 ml of ASL buffer (QIAamp DNA stool kit; Qiagen) and 400 mg of 0.1-mm-diameter zirconium-glass beads (BioSpec Products). Mechanical and chemical lyses were performed by bead beating at 4,800 rpm for 60 s at room temperature and then 60 s on ice (repeated twice) (Mini-Beadbeater-1; BioSpec Products), after which the suspension was incubated at 95°C for 5 min. Subsequent procedures were performed using the Qiagen QIAamp DNA stool kit according to the manufacturer's protocol, with the exception of the last step, in which the column was eluted with 120 μl of elution buffer. DNA was stored at −20°C until further use.

Clinical samples and study design (Irish samples).Ethical approval for this study was obtained from the University College Cork Clinical Research Committee. Patients provided written consent for sample collection and subsequent analyses.

(i) Tissue collection.Breast tissue was collected from 38 women (aged 20 to 85) undergoing breast surgery at Cork University Hospital or South Infirmary Victoria Hospital, Cork, Ireland. Thirty-three women underwent lumpectomies or mastectomies for cancerous tumors (taken at least 5 cm away from the primary tumor site), while 5 women underwent breast reductions and had no history of breast cancer. Once collected, the specimens were placed in sterile cryotubes, flash frozen in liquid nitrogen within 45 min of collection, and then stored at −80°C until DNA extraction.

(ii) DNA isolation.Tissue samples were thawed on ice and the genomic DNA extracted using the Gene-Elute mammalian genomic DNA miniprep kit (Sigma-Aldrich) as per the manufacturer's protocol with the exception of the elution step, where the column was eluted with 70 μl of elution buffer.

Ion Torrent V6 16S rRNA sequencing. (i) PCR amplification.At the London, Canada, lab, the genomic DNA isolated from Canadian and Irish clinical samples was amplified using the barcoded primers V6-LT and V6-RT (see Table S1 in the supplemental material), which amplify the V6 hypervariable region of the 16S rRNA gene. The PCR was carried out in a 40-μl reaction mixture containing 5 μl of DNA template (or nuclease-free water as a negative control), 1.5 mM MgCl₂, 0.8 μM each primer, 4 μl of 10× PCR buffer (Invitrogen), 0.2 mM deoxynucleoside triphosphates (dNTPs), 0.05U Taq polymerase (Invitrogen), and 0.15 μg/μl of bovine serum albumin. Thermal cycling was carried out in an Eppendorf Mastercyler under the following conditions: initial denaturation at 95°C for 2 min followed by 25 cycles of 95°C for 1 min, 55°C for 1 min, and 72°C for 1 min. After amplification, the DNA concentration was measured with the Qubit 2.0 fluorometer (Invitrogen) using the broad-range assay. Equimolar amounts of each PCR product were then pooled and purified using the QIAquick PCR purification kit (Qiagen). The PCR-purified sample was then sent to the London Regional Genomics Center, London, Ontario, Canada, for V6 16S rRNA sequencing using the Ion Torrent platform as per the Center's standard operating procedure.

(ii) Sequence processing and taxonomic assignment.Custom Perl and Bash scripts were used to demultiplex the reads and assign barcoded reads to individual samples. Reads were kept if the sequence included a perfect match to the barcode and the V6 16S rRNA gene primers and were within the length expected for the V6 variable region. Samples with more than 600 reads were kept, while those with fewer than 600 were discarded. Reads were then clustered by 97% identity into operational taxonomic units (OTUs) using UClust v. 3.0.617 (13). OTUs that represented ≥2% of the reads in at least one sample were kept, while those that did not meet the cutoff were discarded to account for the high error rate intrinsic to Ion Torrent sequencing. Taxonomic assignments for each OTU were made by the Ribosomal Database Project (RDP) SeqMatch tool (14). From the top 20 matches to the RDP named-isolate database, the full taxonomy was retained for matches with the highest S_ab score. For multiple top matches with equal scores, the highest common taxonomy was retained (e.g., genus level if multiple species matched equally well). Since the maximum number of matches displayed per sequence is 20, the RDP taxonomic assignments were verified by BLAST against the Greengenes named-isolate database with an output of 100 hits (15). Taxonomy was assigned based on hits with the highest percent identities and coverage. If multiple hits fulfilled this criterion, classification was reassigned to a higher common taxonomy. In instances where the highest percent identity/coverage yielded a single match, if this was less than 90% and the S_ab score from RDP was less than 0.7, taxonomy was assigned at the family level instead of at the genus level. The sequences for each OTU and the RDP/Greengenes classification are shown in Data Set S1 in the supplemental material. The Ion Torrent V6 16S rRNA sequences obtained in this study have been deposited in the Short Read Archive (SRA) at NCBI.

(iii) Data analysis.The QIIME pipeline (16) was used to (i) calculate weighted UniFrac distances and Shannon's diversity index (logarithms with base 2), (ii) summarize OTUs by different taxonomic levels, and (iii) generate unweighted-pair group method with arithmetic mean (UPGMA) trees representing hierarchical clustering of samples. The UniFrac distances were calculated by using a phylogenetic tree of OTU sequences built with FastTree (17) and based on an OTU sequence alignment with MUSCLE (18). Weighted UniFrac compares microbial profiles (presence/absence and abundance) between samples (i.e., beta diversity) (19), while Shannon's diversity index evaluates the microbial diversity within a sample (i.e., alpha diversity). The higher the Shannon's diversity index, the more diverse a sample is, and a value of zero indicates the presence of only one species (20). UPGMA trees allow one to visualize the distance matrix, and the robustness of what was observed was tested with jackknifing and bootstrapping. For beta-diversity analyses, the data set was rarified to the lowest read count/sample. Bar plots, box plots, and strip charts were all generated in R (21).

Culture analysis.In an effort to prove that bacteria in the breast were viable, 100 μl of Canadian tissue homogenate from each of the 43 samples and 100 μl of each environmental control were plated on Columbia blood agar (CBA) plates and incubated aerobically or anaerobically at 37°C. Of note, the facilities to perform culture were not available at the Irish site. DNA from single colonies was extracted using the InstaGene Matrix (Bio-Rad) and then amplified using the eubacterial primers pA/pH, which amplify the 16S rRNA gene (see Table S1 in the supplemental material). The PCR was carried out in a 50-μl reaction mixture containing 10 μl of DNA template (or nuclease-free water as a negative control), 1.5 mM MgCl₂, 1.0 μM each primer, 0.2 mM dNTP, 5 μl 10× PCR buffer (Invitrogen), and 0.05 Taq polymerase (Invitrogen). Thermal cycling was carried out in an Eppendorf Mastercycler under the following conditions: initial denaturation step at 95°C for 2 min, followed by 30 cycles of 94°C for 30 s, 55°C for 30 s, and 72°C for 1 min. A final elongation step was performed at 72°C for 10 min. After running 10 μl of the PCR mixture on a 1% agarose gel to verify the presence of amplicons, 40 μl of the PCR mixture was then purified using the QIAquick PCR purification kit (Qiagen). The purified PCR products were then sent for Sanger sequencing to the London Regional Genomics Centre, London, Ontario, Canada. Sequences were analyzed using the GenBank 16S rRNA sequence database with the BLAST algorithm (22). Taxonomy was assigned based on the highest Max score.

Statistical analysis.Statistical analysis was performed in R using the Kruskal-Wallis one-way analysis of variance followed by the Mann-Whitney U test with Bonferroni correction.