Molecular analysis of human forearm superficial skin bacterial biota

Abstract

The microbial ecology of human skin is complex, but little is known about its species composition. We examined the diversity of the skin biota from the superficial volar left and right forearms in six healthy subjects using broad-range small subunit rRNA genes (16S rDNA) PCR-based sequencing of randomly selected clones. For the initial 1,221 clones analyzed, 182 species-level operational taxonomic units (SLOTUs) belonging to eight phyla were identified, estimated as 74.0% [95% confidence interval (C.I.), approximately 64.8-77.9%] of the SLOTUs in this ecosystem; 48.0 +/- 12.2 SLOTUs were found in each subject. Three phyla (Actinobacteria, Firmicutes, and Proteobacteria) accounted for 94.6% of the clones. Most (85.3%) of the bacterial sequences corresponded to known and cultivated species, but 98 (8.0%) clones, comprising 30 phylotypes, had <97% similarity to prior database sequences. Only 6 (6.6%) of the 91 genera and 4 (2.2%) of the 182 SLOTUs, respectively, were found in all six subjects. Analysis of 817 clones obtained 8-10 months later from four subjects showed additional phyla (numbering 2), genera (numbering 28), and SLOTUs (numbering 65). Only four (3.4%) of the 119 genera (Propionibacteria, Corynebacteria, Staphylococcus, and Streptococcus) were observed in each subject tested twice, but these genera represented 54.4% of all clones. These results show that the bacterial biota in normal superficial skin is highly diverse, with few well conserved and well represented genera, but otherwise low-level interpersonal consensus.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Phylogenetic analysis of bacterial 16S rDNA detected in normal skin from six subjects. (A) From 1,221 clones, sequences representing eight bacterial phyla and 182 SLOTUs were observed. Alignments were done with Greengenes, and misalignments were manually curated in ARB (48), evolutionary distances were calculated with the Jukes–Cantor algorithm, and phylogenetic trees were determined by the Neighbor-Joining method; with 1,000 trees generated, bootstrap confidence levels are shown at tree nodes for values ≥70%. (B) Phylogenetic tree of the 628 clones within the phylum Actinobacteria. SLOTU designations are located at the termination of each branch. The 16S rDNA clones and cultivated non-type strains represent potential bacterial species; these clones are represented by the nearest species, followed by the GenBank accession number of the best-matched sequence. Unknowns are represented by the serial number of the clone used in this study, followed by the closest match, and percent sequence identity. (C) Phylogenetic tree of the 345 clones representing the phyla Firmicutes, Deinococcus-Thermus, Thermomicrobia, and unclassified organisms. Designations are as described for B. The clones representing Deinococcus-Thermus, Thermomicrobia, and unclassified were from the same subject. (D) Phylogenetic tree of the 248 clones representing the phyla Proteobacteria, Bacteroidetes, and Cyanobacteria. Designations are as described for B.

Fig. 2.

Collector's curves of observed and estimated SLOTU richness of pooled forearm skin samples from six healthy subjects. (A) Each curve reflects the series of observed (Sobs) or estimated (Chao1) richness values obtained as the 1,221 16S rDNA clones are added to the data set in an arbitrary order. As reported for the human colon (23), as an increasing proportion of SLOTUs have been encountered, the Chao1 curve rises less steeply; however, additional SLOTUs continue to be identified to the end of the sampling. Although 182 (95% C.I., range 169–195) SLOTUs were observed, the Chao1 score of total species richness, estimates that the skin bacterial biota in the six subjects contains ≈246 SLOTUs (95% C.I., range 217–301). Based on this prediction, the present study identified 74.0% (95% C.I., 64.8–77.9%) of the SLOTUs in this bacterial ecosystem. (B) When results from resampling of four of the subjects with 817 new clones were included, the number of SLOTUs was 247 (95% C.I., range 232–262), and the Chao1 was 328 (95% C.I., range 295–385), indicating coverage of 75.3% (95% C.I., range 68.1–78.6).

Fig. 3.

Distribution of 2,038 16S rDNA clones from left and right forearm, by phylum. At the first sampling, 1,221 clones were obtained from the six subjects, and 817 clones were later obtained from four of these six. Thus, in total, 2,038 clones were studied, with percents amongst the 10 phyla indicated by the color designations. For the four individuals sampled twice (subjects T1 and T2), there was little difference in the overall phylum distribution between the two time points.

Fig. 4.

DPCoA of SLOTU relatedness in 20 forearm skin samples obtained from six subjects. Subjects were designated A–F, and at each sampling both left (L) and right (R) forearm skin was examined. In four subjects, new specimens were obtained 8–10 months later (e.g., designated FL2). In a representation of the first two orthogonal principal axes, based on a sample dissimilarity matrix, samples from the same subject at the same time point are plotted by using the same color, with circle size proportional to the sample's Rao diversity index. The scale (top right) indicates the relative diversity of circles, with the test sample of smallest diversity (FR) indexed as 1.0.