ww.nature.com/scientificreports/ A Nipple Skin from HC(n=B) b 亡 Nipple Skin from HO(m=8) Phylum Distribution Adonis test, p-value=0. 945 nonparametric t-test, p-value=0. 929 ge 凸春 ■ Pc1(225%) Sequencing Depth mposition.(a)PCoA plot using Bray-Curtis dissimilarity based on genus- level OTUs from nipple skin swab samples collected from either HC or BC. (b)Number of bacterial OTUs observed on the nipple skin swabs as a function of sequencing depth as assessed by diversity rarefaction curves. The difference in diversity was compared by a non-parametric t-test using the average of observed OTUs depicts the average phylum-level percentages of the nipple skin microbiome measured from HC andBC randomly sampled ten times at 918 sequencing depth. Error bars represent standard deviation. (c) Bar ch present in human breast milk and breast tissue have recently been characterized using next-generation sequenc ing and pan-pathogen array technologies Until now, the potential role of the local breast ductal microbiome with breast cancer has not been explored. this study, we used 16S rRNA gene sequencing to characterize the microorganisms present in nipple aspirate fluid (NAF). NAF is constantly secreted and absorbed by the epithelial cells lining the breast ducts and can be obtained non-invasively from at least one duct in a majority of women by applying negative pressu syringe attached to a suction cup. NAF collected from breast cancer patients has been shown to have a cantly different proteomic profile compared to NAF collected from healthy volunteers. Here, we collected NAF from healthy control ith a history of breast cancer(BC)(all were ductal carcinoma to investigate the breast ductal microbiome. Results Nipple skin microbiome from HC vS. BC. Nipple/areola skin was sampled with a sterile cotton swab as a control to compare to NAF. DNA extracted from the nipple skin samples was sequenced, the reads were clustered into unique Operation Taxonomic Units(OTUs), and then the OTUs classified to the genus taxonomic level. The relative differences amongst the nipple skin communities were calculated using the Bray-Curtis index and graphically visualized using Principal Coordinates Analysis(PCoA), whereby a shorter distance between points indicates increasing similarity in microbial composition. Though the skin varies according to body site and is expected to randomly vary across individuals, we hypothesized that the nipple skin microbiome would be inde pendent of breast cancer history. The nipple skin microbiome from HC(n=8)and BC(n=5)did not cluster parately and did not have a significant difference in bacterial composition(Adonis, p-value =0.945)(Fig. la) The rarefaction curves, which plot the number of unique species as a function of the number of reads sampled, reached a plateau for the nipple skin samples as well as for the other sample types(post-Betadine skin and NAF), indicating that our sampling depth provided sufficient coverage to capture most members of the bacterial com- munities(Figs 1b, 2b and 3b). The bacterial diversity was not significantly different between the nipple skin sam pled from HC vS BC (nonparametric t-test, p-value=0.929, Fig. 1b) In nipple skin samples from both HC and BC, bacterial composition at the phyla level was predominantly Proteobacteria(average 36.5%6), Firmicutes(average 33. 8%), and Bacteroidetes(average 19.5%)(Fig. 1c). Of all he OTUs in the nipple skin samples, Alistipes(recently reclassified Bacteroides putredinis)was the most abun dant(average 11.8%), followed by an unclassified genus from the Sphingomonadaceae family(average 11.3%), Rhizobium(average 6.7%), and an unclassified family from Acidobacteria Gp4(average 4.9%) None of the OTUs rom the skin swabs were significantly different when comparing their relative abundance between the HC and C groups(Kruskal-Wallis test, Supplemental Table S1). In summary, the nipple skin microbiome from HC and BC were not significantly distinguishable by their community composition, their diversity, or their individual OTUs, indicating that the nipple skin microbiome is independent of breast cancer history (Fig. 1) Microbiome in post-Betadine treated skin swab from HC vS BC. Betadine is an iodine-based broad trum disinfection agent used to clean skin before surgeries and other procedures. The nipple skin was treate ith Betadine to avoid contamination of NAF samples with normal nipple skin flora. We found that Betadine leaves a small residual flora that is detected by the extremely sensitive methodology used in this study. As a base line control, we collected post-Betadine samples of the nipple skin to characterize the residual microflora Reads from the post-Betadine skin swabs were clustered into OTUs and classified to the genera level. We calculated the Bray-Curtis dissimilarity index and performed PCoA to visualize community-wide differences amongst the ost-Betadine skin swabs. The post-Betadine skin microbiome from HC (n=5)and BC (n=7)did not separat SCIENTIFIC REPORTS 6: 28061 DO1: 10.1038/srep28061www.nature.com/scientificreports/ Scientific Reports | 6:28061 | DOI: 10.1038/srep28061 2 present in human breast milk and breast tissue have recently been characterized using next-generation sequenc￾ing and pan-pathogen array technologies9,12–15. Until now, the potential role of the local breast ductal microbiome with breast cancer has not been explored. In this study, we used 16S rRNA gene sequencing to characterize the microorganisms present in nipple aspirate fluid (NAF). NAF is constantly secreted and absorbed by the epithelial cells lining the breast ducts and can be obtained non-invasively from at least one duct in a majority of women by applying negative pressure with a syringe attached to a suction cup16. NAF collected from breast cancer patients has been shown to have a signifi￾cantly different proteomic profile compared to NAF collected from healthy volunteers17. Here, we collected NAF from healthy control women (HC) and women with a history of breast cancer (BC) (all were ductal carcinomas) to investigate the breast ductal microbiome. Results Nipple skin microbiome from HC vs. BC. Nipple/areola skin was sampled with a sterile cotton swab as a control to compare to NAF. DNA extracted from the nipple skin samples was sequenced, the reads were clustered into unique Operation Taxonomic Units (OTUs), and then the OTUs classified to the genus taxonomic level. The relative differences amongst the nipple skin communities were calculated using the Bray-Curtis index and graphically visualized using Principal Coordinates Analysis (PCoA), whereby a shorter distance between points indicates increasing similarity in microbial composition. Though the skin varies according to body site18 and is expected to randomly vary across individuals, we hypothesized that the nipple skin microbiome would be inde￾pendent of breast cancer history. The nipple skin microbiome from HC (n= 8) and BC (n= 5) did not cluster separately and did not have a significant difference in bacterial composition (Adonis, p-value= 0.945) (Fig. 1a). The rarefaction curves, which plot the number of unique species as a function of the number of reads sampled, reached a plateau for the nipple skin samples as well as for the other sample types (post-Betadine skin and NAF), indicating that our sampling depth provided sufficient coverage to capture most members of the bacterial com￾munities (Figs 1b, 2b and 3b). The bacterial diversity was not significantly different between the nipple skin sam￾pled from HC vs. BC (nonparametric t-test, p-value=0.929, Fig. 1b). In nipple skin samples from both HC and BC, bacterial composition at the phyla level was predominantly Proteobacteria (average 36.5%), Firmicutes (average 33.8%), and Bacteroidetes (average 19.5%) (Fig. 1c). Of all the OTUs in the nipple skin samples, Alistipes (recently reclassified Bacteroides putredinis19) was the most abun￾dant (average 11.8%), followed by an unclassified genus from the Sphingomonadaceae family (average 11.3%), Rhizobium (average 6.7%), and an unclassified family from Acidobacteria Gp4 (average 4.9%). None of the OTUs from the skin swabs were significantly different when comparing their relative abundance between the HC and BC groups (Kruskal-Wallis test, Supplemental Table S1). In summary, the nipple skin microbiome from HC and BC were not significantly distinguishable by their community composition, their diversity, or their individual OTUs, indicating that the nipple skin microbiome is independent of breast cancer history (Fig. 1). Microbiome in post-Betadine treated skin swab from HC vs. BC. Betadine is an iodine-based broad spectrum disinfection agent used to clean skin before surgeries and other procedures. The nipple skin was treated with Betadine to avoid contamination of NAF samples with normal nipple skin flora. We found that Betadine leaves a small residual flora that is detected by the extremely sensitive methodology used in this study. As a base￾line control, we collected post-Betadine samples of the nipple skin to characterize the residual microflora. Reads from the post-Betadine skin swabs were clustered into OTUs and classified to the genera level. We calculated the Bray-Curtis dissimilarity index and performed PCoA to visualize community-wide differences amongst the post-Betadine skin swabs. The post-Betadine skin microbiome from HC (n=5) and BC (n=7) did not separate Figure 1. Nipple skin microbial composition. (a) PCoA plot using Bray-Curtis dissimilarity based on genus￾level OTUs from nipple skin swab samples collected from either HC or BC. (b) Number of bacterial OTUs observed on the nipple skin swabs as a function of sequencing depth as assessed by diversity rarefaction curves. The difference in diversity was compared by a non-parametric t-test using the average of observed OTUs randomly sampled ten times at 918 sequencing depth. Error bars represent standard deviation. (c) Bar chart depicts the average phylum-level percentages of the nipple skin microbiome measured from HC and BC