To investigate the distribution of M. furfur in different body sites, we considered the normalized hits FPM as an estimate of M. furfur relative abundance. We statistically analyzed the relative abundance of M. furfur in skin samples with different physiological conditions (Figure 1). We also explored the effect of sample occlusion, subject age and subject gender on the relative abundance of M. furfur in the samples.
Optimization of metrics:
We started by testing each of the nine biomarkers to assess their diagnostic ability, reproducibility, and thus reliability in detecting M. furfur sequences within metagenomes.
The results obtained from aligning each of the nine markers, and their combination, as BLAST query sequences were quite reproducible (Figure 3). Pearson correlation analysis of number of BLAST hits, represented as normalized hits FPM, indicated a high correlation near 1 in most cases (Pearson correlation coefficients > 0.9, reaching 10.0 at instances). Two sequences with accession number KC141972.1, KC141971.1 gave exactly the same results (Figure 3).
Since the results obtained from normalized hits (FPM) for all genes were quite correlated with results from those of single genes (Figure 2 and Figure 3), as well as with best hits for all genes, we estimated the abundance as all gene FPM, as these values were comparable for small or large data sets, since they were normalized for the number of sequence reads.
Effect of skin site on the distribution of M. furfur in representative skin metagenomes (Figure 4A)
The type/location of the skin site significantly affected the relative abundance of M. furfur among the samples (Kruskal-Wallis rank, p-value = 1.261x10-06).
Among the 11 skin sites (Figure 1 and Table 1) included in our analysis, the retro auricular crease, which is a sebaceous area behind the ear, had the highest relative abundance of M. furfur, followed by the antecubital fossa, a moist joint area, then the forehead, a sebaceous and exposed area. Next came the palm, which is an intermittently moist and exposed area (Figure 4A).
The back and the subclavius area, which are both sebaceous areas that are covered most of the time had intermediate abundance. Finally, the axilla, plantar heel, toe web space, umbilicus and volar forearm areas had the lowest M. furfur abundance. Interestingly, all these sites are neither sebaceous nor in a joint area and they are covered by clothes most of the time.
Expectedly and reassuringly, the control samples had the lowest number of hits, confirming the specificity of the methodology. These were included in the published study to normalize the reading errors in the results.
Effect of skin nature on M. furfur abundance:
The relative abundance of M. furfur was significantly higher in the sebaceous skin sites, followed by the rarely intermittently moist sites, then the moist sites, and finally the dry sites (Kruskal-Wallis test, p-value = 1.523x10-08, Figure 4B).
From another perspective, whether the skin is occluded or exposed significantly affected the relative abundance of M. furfur (Kruskal-Wallis rank, p-value = 0.0004654). The highest abundance was observed in the exposed sites, followed by occluded then intermittently occluded (Figure 4C).
Sex and age had no significant effect on the relative abundance of M. furfur among different skin sites (Kruskal-Wallis rank, p-value = 0.6458, 0.1498, respectively) (Figure 5).