ww.nature.com/scientificreports/ b Adonis test, p-value=. D02 nonparametric t-test, p-value=D,65 NAF from Hc NAF from BC PC1(18% Sequencing Depth F. Rikenellaceae: G. Alistipes (otu00009) e, F. sphingomonadaceae: G unclassified (otu00007) NAF from Hc NAF from B NAF from Hc NAF from BC Figure 3. NAF microbial composition (a) PCoA plot of Bray-Curtis dissimilarity based on genus-level OTUs of NAF collected from either HC (n=9)or BC(n=6).(b)Number of bacterial OTUs observed in the NAF 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 934 sequencing depth Error bars represent standard deviation. (c) Bar chart depicts the average phylum-level percentages of the NAF microbiome measured from HC and BC. (d) Strip-plot comparing relative abundances of the genus Alistipes(Otu00009)between HC and BC group. (e)Strip-plot comparing relative abundances of the unclassified genus from the Sphingomonadaceae family(otu00007)between HC and BC group there was no difference in bacterial diversity(paired t-test, p-value =0.52), and none of the OTUs were signifi antly different(Paired wilcoxon signed-rank test, Supplemental Table S5) Functional prediction based on NAF microbiome comparing HC vS BC. To investigate whether the microbes present in the breast ducts have a metabolic activity related to malignancy or health, we examined the gene sequences associated with 18 selected KEGG metabolic pathways that have been implicated in color cancer20-27 using the PICRUSt package 2. None of the 18 selected KEGG pathways were statistically significant at 5% alpha level after correcting for multiple hypothesis testing. The pathway"flavone and flavonol biosynthesis (ko00944)produced the lowest p-value(Kruskal-Wallis test, adjusted p-value=0.066)(Fig 5a) Of the 12 KEGG orthologs that comprise the flavone and flavonol biosynthesis pathway, Beta-Glucuronidase(k01195)"was th only KEGG bacterial ortholog predicted by the NAF microbiome. Furthermore, the gene "Beta-Glucuronidase" is inferred from a combination of eight OTUs(Table 1)and the combined relative abundances of these eight OTUs were statistically higher in NAF from BC than HC(Kruskal-Wallis test, p-value =0.0036)(Fig 5b) Di IscUSSIon This study establishes the existence of a microbiome in the breast ductal system, by detecting the presence of bacterial DNA in NAF. Analysis of the beta-diversity demonstrates that the NAF microbial cor position is different in women having had a history of breast cancer. Previous microbiome studies of breast tis- sue have described bacteria belonging mainly to the phylum Proteobacteria, Firmicutes, Actinobacteria, and Bacteroidetes. 25. Our studies on NAF show a similar microbial composition, with Firmicutes and Bacteroidetes. While previous studies on NAF have attempted to define biomarkers to help etection and prevention of breast cancer using cellular morphology, protein expression, growth factors, and hormones in the intraductal microenvironment, this is the first study to identify bacteria in the ductal syster SCIENTIFIC REPORTS 6: 28061 DO1: 10.1038/srep28061www.nature.com/scientificreports/ Scientific Reports | 6:28061 | DOI: 10.1038/srep28061 4 there was no difference in bacterial diversity (paired t-test, p-value= 0.52), and none of the OTUs were signifi￾cantly different (Paired Wilcoxon signed-rank test, Supplemental Table S5). Functional prediction based on NAF microbiome comparing HC vs. BC. To investigate whether the microbes present in the breast ducts have a metabolic activity related to malignancy or health, we examined the gene sequences associated with 18 selected KEGG metabolic pathways that have been implicated in colon cancer20–27 using the PICRUSt package28. None of the 18 selected KEGG pathways were statistically significant at 5% alpha level after correcting for multiple hypothesis testing. The pathway “flavone and flavonol biosynthesis (ko00944)” produced the lowest p-value (Kruskal-Wallis test, adjusted p-value= 0.066) (Fig. 5a). Of the 12 KEGG orthologs that comprise the “flavone and flavonol biosynthesis” pathway, “Beta-Glucuronidase (k01195)” was the only KEGG bacterial ortholog predicted by the NAF microbiome. Furthermore, the gene “Beta-Glucuronidase” is inferred from a combination of eight OTUs (Table 1) and the combined relative abundances of these eight OTUs were statistically higher in NAF from BC than HC (Kruskal-Wallis test, p-value=0.0036) (Fig. 5b). Discussion This study establishes the existence of a microbiome in the breast ductal system, by detecting the presence of bacterial DNA in NAF. Analysis of the beta-diversity demonstrates that the NAF microbial community com￾position is different in women having had a history of breast cancer. Previous microbiome studies of breast tis￾sue have described bacteria belonging mainly to the phylum Proteobacteria, Firmicutes, Actinobacteria, and Bacteroidetes9,12,15. Our studies on NAF show a similar microbial composition, with Firmicutes, Proteobacteria, and Bacteroidetes. While previous studies on NAF have attempted to define biomarkers to help early detection and prevention of breast cancer using cellular morphology, protein expression, growth factors, and hormones in the intraductal microenvironment29, this is the first study to identify bacteria in the ductal system. Figure 3. NAF microbial composition. (a) PCoA plot of Bray-Curtis dissimilarity based on genus-level OTUs of NAF collected from either HC (n=9) or BC (n=6). (b) Number of bacterial OTUs observed in the NAF 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 934 sequencing depth. Error bars represent standard deviation. (c) Bar chart depicts the average phylum-level percentages of the NAF microbiome measured from HC and BC. (d) Strip-plot comparing relative abundances of the genus Alistipes (Otu00009) between HC and BC group. (e) Strip-plot comparing relative abundances of the unclassified genus from the Sphingomonadaceae family (Otu00007) between HC and BC group