Fig 1.
Methods Summary (a) Schematic of swabbing locations. (b) Graphical representation of methods used.
Fig 2.
Top phyla and genera grouped by anatomic location.
Phyla (a) and species (b) representing, on average, greater than 0.1% or 1% of all sequences, respectively. Percentages represent abundance of phyla averaged across all samples. Lines between keys link taxonomy of each OTU shown. Two samples (from AL2 and AL3) exhibit abnormally high Actinobacteria (green bars in (a)), specifically Kocuria rhizophila and an unidentified Micrococcus species. Grouped by anatomic location, biological replicates (n = 4/AL) are shown with each bar representing one animal.
Fig 3.
Species overlap, composition, and alpha diversity by anatomic location (a) Venn diagram showing the overlap of the 518 identified species between anatomic locations (n = 4/AL). (b) Weighted PCoA Plots wherein each point (n = 16) represents a single swab, reveals the swabs containing abnormally high Actinobacteria, indicated by the arrows. PERMANOVA analysis revealed no significant grouping between anatomic locations (p>0.05). (c) Rarefaction curves for each swab from zero to 40,000 reads per sample showing the number of observed species at each read depth. (d) Chao1 (richness, p = 0.0054), PD_whole tree (diversity, n.s.), Shannon (diversity, n.s.), Simpson (Evenness, n.s.) indices of alpha diversity. AL1 (blue), AL2 (yellow), AL3 (green), AL4 (red). **p<0.01 (2-way ANOVA with Tukey’s Post-testing n = 4/AL).
Table 1.
Species level differences by anatomic region.
Fig 4.
(a) Number of colony forming units (CFU) for each anatomic location (n = 7/AL). One-way ANOVA revealed no significant differences between anatomic locations. (b) Representative 1-10-fold dilution of CFU plates is displayed showing a diverse polymicrobial community. Protein content is similar between anatomic locations for MEM-BTM (c) and KM-BTM (d). KM-BTM had a significant reduction in protein content compared to KM alone (p = 0.004) however MEM-BTM did not.
Fig 5.
The effect of MEM-BTM on Fibroblast and Keratinocyte migration.
(a) Fibroblasts seeded on cell migration plates were allowed to migrate overnight. Fibroblasts incubated with BTM from all locations migrated more than the negative control (p<0.04) however migration was impeded in BTM from ventral swabs (AL3, p = 0.04 & AL4, p = 0.04) compared to a-MEM control (two-tailed unpaired t-test, n = 7/AL). #, p<0.05 v. Media control; +, p<0.05 v. “Insert in” Control. (b) Respective 4x images of each location are shown and scale bars represent 1.0 mm. (c) Keratinocytes seeded on cell migration plates were allowed to migrate overnight. NHEKs incubated with BTM from all locations exhibited retraction compared to media control (p<0.05), however AL1 (p = 0.004), AL2 (p = 0.0003), and AL3 (p = 0.02) also exhibited significant retraction compared to negative control (two tailed unpaired t-test, n = 7/AL). #, p<0.05 v. Media control; +, p<0.05 v. Insert In Control. (d) Respective 2x images of each location are shown and scale bars represent 2.0 mm. Images are a representation of two independent experiments.
Fig 6.
The effect of KM-BTM on Keratinocyte viability, differentiation, and VEGF release.
(a and b) NHEKs were treated with KM-BTM, fixed, and stained with caspase-3. The percentage of cells expressing caspase-3 is higher in NHEKs treated with BTM from AL4 compared to other locations (p<0.02) and lower in AL1 (p = 0.0003) compared to incubated KM. (a, right column) Cytokeratin-14 expression was maintained when incubated in BTM from AL2-4 but not in AL1 or KM alone. For caspase-3 stain, 4x images are shown and scale bars represent 500 μm. For K-14 stain, 10x images are shown and scale bar represents 50 μm. (c) VEGF protein levels in cell supernatant were decreased following incubation in BTM from all locations (p<0.01) except for AL1 compared to incubated keratinocyte media (two tailed unpaired t-test, n = 7/AL).