ww.nature.com/scientificreports/ 合F Post-Betadine skin from HC (n=5) b Post-Betadine skin from HC (n=5) Phylum Distribution nonparametric t-test, p-value=0. 15 seeN吧宁 口 □ Addobaster Pc1(21.7%) Sequencing Depth skin from Hc skin from bc Figure 2. Post-Betadine skin microbial composition (a) PCoa plot using Bray-Curtis dissimilarity based on genus-level OTUs from post-Betadine skin samples from either HC (n=5)or BC(n=7).(b)Number of bacterial OTUs observed on the post-Betadine skin 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 1164 sequencing depth. Error bars represent standard deviation. (c). Bar chart depicts the average phylum-level percentages of the post- Betadine skin swab microbiome asured from HC and bc into distinct clusters(Adonis, p-value =0.478)(Fig 2a), and the bacterial diversity between the two groups wer not significantly different(nonparametric t-test, p-value=0. 151)(Fig 2b). The post-Betadine swab microbiom is primarily composed of bacteria belonging to the phylum Proteobacteria(average 49.4%), Firmicutes(averag 23.8%6, and Bacteroidetes(average 13.0%)(Fig. 2c). None of the OTUs was significantly different between the ost-Betadine skin samples collected from HC and BC( Kruskal-Wallis test, Supplemental Table S2). As expected, he post-Betadine nipple skin microbiome from HC vS BC was not significantly different by comparing commu nity composition, by comparing species diversity, or by comparing relative OTU abundances, indicating that the NAF microbial composition from HC vS. o assess whether there is a difference in microbial compo- sition between the NAF from HC and BC, we calculated sample-to-sample variations by Bray-Curtis dissimilarity and then visualized the compositional differences by PCoA(Fig 3a). We found that the NAF microbiome from HC and bc clustered separately with a significant difference(Adonis, p-value =0.002). The Adonis test indi- cates that having had a history of breast cancer significantly affects the NAF microbial composition and explains approximately 13. 5% of the variation among the samples. The bacterial diversity of naf was not significantly different between those collected from HC vs those from BC(nonparametric t-test, p-value=0.65)(Fig. 3b) The most abundant bacteria in NAF samples were those belonging to the phylum Firmicutes(averaging 329% Bacteroidetes differences in relative abundance between NAF from HC and from BC by Kruskal-Wallis test( Supplemental Table $3). The genus Alistipes(Otu00009)was only present in the NAF from BC(p-value=0.0068)(Fig. 3d In contrast, an unclassified genus from the family Sphingomonadaceae(Otu00007)was present in NAF from both HC and BC, but was relatively more abundant in the NAF from HC compared to BC (p-value=0.02846) demonstrated significant clustering betwe nipple skin and the post-Betadine controls, the microbiome from NAF Fig 3e). Unlike the microbiome from health states Microbial composition from nipple skin vs NAF. To address whether the microbes in NAF correspond to those on the overlying nipple and areolar skin, we compared the microbiome between the nipple skin and NAF Because of the variability of microbiome across individuals, the analysis was limited to paired NAF and corresponding nipple skin samples collected from the same breast. We calculated the sample-to-sample variation by the Bray-Curtis dissimilarity and visualized it using PCoA(Fig. 4a). There was no compositional difference between the microbes present on nipple skin and in NAF when combining all HC and BC samples(Adonis, p-value=0.2734)(Fig 4a). However, a history of breast cancer is a confounding variable, so the samples from HC group and BC group were analyzed separately and the Bray-Curtis dissimilarity recalculated for each group ( Fig. 4b, c) Within the HC sample pairs(n=6), the nipple skin and NAF microbiome were significantly different by paired Adonis test using the strata parameter(p-value=0.0313). However, there was no differe the nipple skin diversity (paired t-test, p-value=0.62), and none of the OTUs were significantly different be d NAF groups( Paired Wilcoxon signed-rank test, Supplemental Table S4). Within the BC samples(n=3), the paired comparison between the nipple skin and NAF microbiome was not significantly different(Adonis, p-value=1.000). Comparing the nipple skin and NAF sample types from BC, SCIENTIFIC REPORTS 6: 28061 DO1: 10.1038/srep28061www.nature.com/scientificreports/ Scientific Reports | 6:28061 | DOI: 10.1038/srep28061 3 into distinct clusters (Adonis, p-value=0.478) (Fig. 2a), and the bacterial diversity between the two groups were not significantly different (nonparametric t-test, p-value=0.151) (Fig. 2b). The post-Betadine swab microbiome is primarily composed of bacteria belonging to the phylum Proteobacteria (average 49.4%), Firmicutes (average 23.8%), and Bacteroidetes (average 13.0%) (Fig. 2c). None of the OTUs was significantly different between the post-Betadine skin samples collected from HC and BC (Kruskal-Wallis test, Supplemental Table S2). As expected, the post-Betadine nipple skin microbiome from HC vs. BC was not significantly different by comparing commu￾nity composition, by comparing species diversity, or by comparing relative OTU abundances, indicating that the post-Betadine controls are independent of breast cancer history (Fig. 2). NAF microbial composition from HC vs. BC. To assess whether there is a difference in microbial compo￾sition between the NAF from HC and BC, we calculated sample-to-sample variations by Bray-Curtis dissimilarity and then visualized the compositional differences by PCoA (Fig. 3a). We found that the NAF microbiome from HC and BC clustered separately with a significant difference (Adonis, p-value= 0.002). The Adonis test indi￾cates that having had a history of breast cancer significantly affects the NAF microbial composition and explains approximately 13.5% of the variation among the samples. The bacterial diversity of NAF was not significantly different between those collected from HC vs. those from BC (nonparametric t-test, p-value=0.65) (Fig. 3b). The most abundant bacteria in NAF samples were those belonging to the phylum Firmicutes (averaging 42.1%), Proteobacteria (averaging 32.9%), and Bacteroidetes (averaging 14.5%) (Fig. 3c). Two OTUs showed differences in relative abundance between NAF from HC and from BC by Kruskal-Wallis test (Supplemental Table S3). The genus Alistipes (Otu00009) was only present in the NAF from BC (p-value= 0.0068) (Fig. 3d). In contrast, an unclassified genus from the family Sphingomonadaceae (Otu00007) was present in NAF from both HC and BC, but was relatively more abundant in the NAF from HC compared to BC (p-value= 0.02846) (Fig. 3e). Unlike the microbiome from the nipple skin and the post-Betadine controls, the microbiome from NAF demonstrated significant clustering between HC and BC with two differentially abundant OTUs between the two health states. Microbial composition from nipple skin vs. NAF. To address whether the microbes in NAF correspond to those on the overlying nipple and areolar skin, we compared the microbiome between the nipple skin and NAF. Because of the variability of microbiome across individuals10, the analysis was limited to paired NAF and corresponding nipple skin samples collected from the same breast. We calculated the sample-to-sample variation by the Bray-Curtis dissimilarity and visualized it using PCoA (Fig. 4a). There was no compositional difference between the microbes present on nipple skin and in NAF when combining all HC and BC samples (Adonis, p-value= 0.2734) (Fig. 4a). However, a history of breast cancer is a confounding variable, so the samples from HC group and BC group were analyzed separately and the Bray-Curtis dissimilarity recalculated for each group (Fig. 4b,c). Within the HC sample pairs (n= 6), the nipple skin and NAF microbiome were significantly different by paired Adonis test using the strata parameter (p-value= 0.0313). However, there was no difference in bacterial diversity (paired t-test, p-value= 0.62), and none of the OTUs were significantly different between the nipple skin and NAF groups (Paired Wilcoxon signed-rank test, Supplemental Table S4). Within the BC samples (n= 3), the paired comparison between the nipple skin and NAF microbiome was not significantly different (Adonis, p-value=1.000). Comparing the nipple skin and NAF sample types from BC, Figure 2. Post-Betadine skin microbial composition. (a) PCoA plot using Bray-Curtis dissimilarity based on genus-level OTUs from post-Betadine skin samples from either HC (n=5) or BC (n=7). (b) Number of bacterial OTUs observed on the post-Betadine skin 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 1164 sequencing depth. Error bars represent standard deviation. (c). Bar chart depicts the average phylum-level percentages of the post-Betadine skin swab microbiome measured from HC and BC