ww.nature.com/scientificreports/ PCoA: Paired NAF V5. Skin from BC PCoA: Paired NAF vs Skin from HC PCoA: Paired NAF vS Skin from Bc Adonis, p-value=0. 2734 Adonis, p-value=0.0313 Adonis, p-value= 1.0000 AF from Bc 爸am■4△ 口 NAF from Hc △●△●△●△●△ ●△●△●△ △ sain 5 Left 0.6△skmn5Rk Pc1(20.17%) Pc121.74%) Pc1(33.97%) gure唾 le skin and Naf microbial arison.(a)PCoA plot of Bray-Curtis dissin between paired skin and NAF samples collected from both HC and BC(n=9). (b)PCoa plot of Bray-Curtis dissimilarity between paired skin and NAF samples collected from HC(n=6).(c)PCoA plot of Bray-Curtis dissimilarity between paired skin and NaF samples collected from BC(n=3). All subjects with sequences obtained from both skin and NAF are shown. The paired community wide testing was performed ing Adonis test with the strata parameter to allow permutation only within patients and not across While microbiome studies of Naf have never been reported, breast milk has been studied for its commu- nity composition 430. Both NAF and milk are secreted by the epithelial cells lining the breast ducts. Breast milk contains bacteria that are orally transmitted to the breastfed infant with a key function to populate and establish the neonate's gastrointestinal flora. The bacteria present in milk belong mostly to the phylum Firmicutes( Staphylococcus, Clostridium, Lactobacillus), Actinobacteria(Propionibacterium, Corynebacterium Proteobacteria( Ralstonia, Sphingomonas, Pseudomonas, Bradyrhizobiaccea), and Bacteroidetes(Prevotella)3. 14.30. Similarly, in NAF samples from both HC and BC, we found Clostridium and Prevotella, as well as an unclassified genus from the Sphingomonadaceae family. Thus, the NAF and milk microbiomes exhibit some overlap Though the existence of bacteria in breast tissue has previously been described, the current study establishes the presence of bacterial DNA in the breast ductal system and thereby raises the question where do breast ductal bac- teria originate?", Ductal spaces may be populated by bacteria from oral sources, resulting from sexual activity or breast-feeding. An entero-mammary pathway by which some bacteria from the gut could reach the mammary gland via an endogenous route has been postulated by others. Alternatively, it is tempting to speculate that skin bacteria from the nipple surface might reach the breast ducts through the ductal orifices via retrograde migra tion since the nipple skin and NAF microbiome had no significant OTUs by paired wilcoxon signed-rank test However, amongst the samples from HC, the nipple skin and the naf had significantly different community omposition. Additional studies with larger sample sizes are required to investigate this preliminary trend. Two individual OTUs showed differences in relative abundances between NaF from HC and BC. First the enus Alistipes(Otu00009)was present only in the NAF from BC, and was absent in the NAF collected from HC. listipes has been included among other bacteria to have an increased relative abundance in colorectal cancer2. Another orgaundant in NAF from HC than from BC. The consensus sequence from this OTU was queried against sm of interest, an unclassifed genus from the Sphingomonadaceae family(otu00007), was rela he NCBi refseq_rna using the default blaSt algorithm(Basic Local Alignment Search Tool) 3. The OtU matched two strains of Sphingobium yanoikuyae(NR_1137301 and NR_115241)with 100%identity. Notably, in our previous study, four out of the eight OTUs enriched in paired normal compared to estrogen-receptor positive (ER+) breast tumor tissue belonged to the Sphingomonadaceae family with Sphingobium yanoikuyae having the greatest statistical significance Members of the Sphingomonadaceae family are known for their ability to degrade aromatic hydrocarbons Polycyclic aromatic hydrocarbons have been associated with breast cancer. It is interesting that a microbe capa ble of degrading aromatic hydrocarbons, such as estrogen, would be comparatively higher in the healthy state and lower in the estrogen-dependent breast tumor 6. This may be especially relevant because the sex hormones have far higher concentration in the nipple aspirate fluid than in the serum. In the present study, the metabolism of aromatic hydrocarbons by Sphingomonadaceae may have a protective role and account for the greater abundance of these organisms in the NAF from healthy women, a possibility that warrants further investigation. If bacterial metabolism were related to breast cancer, then certain metabolic pathways would be discern le between the naf collected from HC and bC. PicruSt analysis infers the microbial gene contents and their functional profiles using a database of annotated sequences. None of the 18 selected KEGG pathwa were statistically significant at 5% alpha level after correcting for multiple hypothesis testing. The pati 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 path way,"Beta-Glucuronidase(k01195)"was the only KEGG bacterial ortholog predicted by the NAF microbiome The predicted Beta-Glucuronidase levels were higher in NAF from BC than from HC Beta-Glucuronidase is known to be a procarcinogenic enzyme in gastrointestinal cancer, and has been used as a marker to evaluate dietary effects on colon carcinogenesis.3. In addition, bacterial Beta-Glucuronidase activi has also been reported to be higher in patients with colon cancer than healthy controls. Notably, the NAF OTUs SCIENTIFIC REPORTS 6: 28061 DO1: 10.1038/srep28061 5www.nature.com/scientificreports/ Scientific Reports | 6:28061 | DOI: 10.1038/srep28061 5 While microbiome studies of NAF have never been reported, breast milk has been studied for its commu￾nity composition13,14,30. Both NAF and milk are secreted by the epithelial cells lining the breast ducts. Breast milk contains bacteria that are orally transmitted to the breastfed infant with a key function to populate and establish the neonate’s gastrointestinal flora14. The bacteria present in milk belong mostly to the phylum Firmicutes (Staphylococcus, Clostridium, Lactobacillus), Actinobacteria (Propionibacterium, Corynebacterium), Proteobacteria (Ralstonia, Sphingomonas, Pseudomonas, Bradyrhizobiaccea), and Bacteroidetes (Prevotella)13,14,30. Similarly, in NAF samples from both HC and BC, we found Clostridium and Prevotella, as well as an unclassified genus from the Sphingomonadaceae family. Thus, the NAF and milk microbiomes exhibit some overlap. Though the existence of bacteria in breast tissue has previously been described, the current study establishes the presence of bacterial DNA in the breast ductal system and thereby raises the question ‘where do breast ductal bac￾teria originate?’9,12. Ductal spaces may be populated by bacteria from oral sources, resulting from sexual activity or breast-feeding. An entero-mammary pathway by which some bacteria from the gut could reach the mammary gland via an endogenous route has been postulated by others31. Alternatively, it is tempting to speculate that skin bacteria from the nipple surface might reach the breast ducts through the ductal orifices via retrograde migra￾tion since the nipple skin and NAF microbiome had no significant OTUs by paired Wilcoxon signed-rank test. However, amongst the samples from HC, the nipple skin and the NAF had significantly different community composition. Additional studies with larger sample sizes are required to investigate this preliminary trend. Two individual OTUs showed differences in relative abundances between NAF from HC and BC. First, the genus Alistipes (Otu00009) was present only in the NAF from BC, and was absent in the NAF collected from HC. Alistipes has been included among other bacteria to have an increased relative abundance in colorectal cancer32. Another organism of interest, an unclassified genus from the Sphingomonadaceae family (Otu00007), was rela￾tively more abundant in NAF from HC than from BC. The consensus sequence from this OTU was queried against the NCBI “refseq_rna” using the default BLAST algorithm (Basic Local Alignment Search Tool)33. The OTU matched two strains of Sphingobium yanoikuyae (NR_113730.1 and NR_11524.1) with 100% identity. Notably, in our previous study, four out of the eight OTUs enriched in paired normal compared to estrogen-receptor positive (ER+) breast tumor tissue belonged to the Sphingomonadaceae family with Sphingobium yanoikuyae having the greatest statistical significance9 . Members of the Sphingomonadaceae family are known for their ability to degrade aromatic hydrocarbons34. Polycyclic aromatic hydrocarbons have been associated with breast cancer35. It is interesting that a microbe capa￾ble of degrading aromatic hydrocarbons, such as estrogen, would be comparatively higher in the healthy state and lower in the estrogen-dependent breast tumor36. This may be especially relevant because the sex hormones have far higher concentration in the nipple aspirate fluid than in the serum16. In the present study, the metabolism of aromatic hydrocarbons by Sphingomonadaceae may have a protective role and account for the greater abundance of these organisms in the NAF from healthy women, a possibility that warrants further investigation. If bacterial metabolism were related to breast cancer, then certain metabolic pathways would be discern￾able between the NAF collected from HC and BC. PICRUSt analysis infers the microbial gene contents and their functional profiles using a database of annotated sequences. 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” path￾way, “Beta-Glucuronidase (k01195)” was the only KEGG bacterial ortholog predicted by the NAF microbiome. The predicted Beta-Glucuronidase levels were higher in NAF from BC than from HC. Beta-Glucuronidase is known to be a procarcinogenic enzyme in gastrointestinal cancer, and has been used as a marker to evaluate dietary effects on colon carcinogenesis37,38. In addition, bacterial Beta-Glucuronidase activity has also been reported to be higher in patients with colon cancer than healthy controls39. Notably, the NAF OTUs Figure 4. Nipple skin and NAF microbial composition comparison. (a) PCoA plot of Bray-Curtis dissimilarity between paired skin and NAF samples collected from both HC and BC (n=9). (b) PCoA plot of Bray-Curtis dissimilarity between paired skin and NAF samples collected from HC (n=6). (c) PCoA plot of Bray-Curtis dissimilarity between paired skin and NAF samples collected from BC (n=3). All subjects with sequences obtained from both skin and NAF are shown. The paired community wide testing was performed using Adonis test with the strata parameter to allow permutation only within patients and not across