Substantial alterations of the cutaneous bacterial biota in psoriatic lesions

Abstract

For psoriasis, an idiopathic inflammatory disorder of the skin, the microbial biota has not been defined using cultivation-independent methods. We used broad-range 16S rDNA PCR for archaea and bacteria to examine the microbiota of normal and psoriatic skin. From 6 patients, 19 cutaneous samples (13 from diseased skin and 6 from normal skin) were obtained. Extracted DNA was subjected to the broad range PCR, and 1,925 cloned products were compared with 2,038 products previously reported from healthy persons. Using 98% sequence identity as a species boundary, 1,841 (95.6%) clones were similar to known bacterial 16S rDNA, representing 6 phyla, 86 genera, or 189 species-level operational taxonomic unit (SLOTU); 84 (4.4%) clones with <98% identity probably represented novel species. The most abundant and diverse phylum populating the psoriatic lesions was Firmicutes (46.2%), significantly (P<0.001) overrepresented, compared to the samples from uninvolved skin of the patients (39.0%) and healthy persons (24.4%). In contrast, Actinobacteria, the most prevalent and diverse phylum in normal skin samples from both healthy persons (47.6%) and the patients (47.8%), was significantly (P<0.01) underrepresented in the psoriatic lesion samples (37.3%). Representation of Propionibacterium species were lower in the psoriatic lesions (2.9+/-5.5%) than from normal persons (21.1+/-18.2%; P<0.001), whereas normal skin from the psoriatic patients showed intermediate levels (12.3+/-21.6%). We conclude that psoriasis is associated with substantial alteration in the composition and representation of the cutaneous bacterial biota.

Figure 1. Diversity of bacterial species in 39 skin specimens from 12 subjects. Panel A. Collector's curves of SLOTU richness of pooled skin samples from six healthy subjects and six patients with psoriasis.

Each curve reflects the observed (Sobs) or estimated (Chao1) richness values obtained in 3 different groups of 16S rDNA clones. PP+PN indicates the 1,925 (1,314 from psoriatic lesion and 611 from uninvolved skin, respectively), clones from six patients with psoriasis, added to the data set in an arbitrary order. NN includes 2,038 clones from healthy persons reported in a prior study . Overall, the sequences detected from the (Total) 39 human skin specimens from these studies represent 366 different SLOTUs, whereas the Chao1 score estimates that the skin bacterial biota contains 476 SLOTUs. Based on this prediction, Good's estimator of coverage is 96.9%. Panel B. Diversity at the species level. The 20 samples from 6 healthy subjects (pink) and 19 samples from 6 patients with psoriasis (blue) are indicated by the color designations. NNT1 (n = 12) represents left and right forearm skin for six subjects, NNT2 (n = 8) represents the resampling of four subjects 8–10 months later . PN (n = 6) represents uninvolved skin from six patients with psoriasis. The samples from healthy subjects and from uninvolved skin from psoriatic patients are indicated with solid symbols, and the samples from psoriatic lesions are indicated with open symbols. All samples from the same subject are indicated by the same symbol. For each group, the horizontal line indicates the median of the number of SLOTU.

Figure 2. Phylogenetic analysis of bacterial 16S rDNA detected in 39 samples from human skin.

From 3,963 clones, sequences representing 11 bacterial phyla and 366 SLOTUs were observed. The numbers in parentheses indicate the number of SLOTUs in each phylum. Alignments were done with Greengenes, and misalignments were manually in MEGA 4.0 , 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 ≥50%. Panel A: Phylogenetic analysis of 20 samples from 6 healthy subjects. Panel B: Phylogenetic analysis of 13 samples from the lesions of 6 patients with psoriasis.

Figure 3. The most common bacterial genera (Panels A, B) and bacterial (Panels C, D) species found in human skin, based on 16S rDNA clones.

Panel A. Samples from healthy persons (n = 20) and normal skin (n = 6) of patients with psoriasis (n = 2,649 clones). The ratio of Streptococcus to Propionibacterium is 0.4. For the four most common genera, the percentage of clones is shown. Panel B. Samples from the lesions (n = 13) of patients with psoriasis (n = 1,314 clones). The ratio of Streptococcus to Propionibacterium is 5.0 Panel C. Samples from healthy persons (n = 20) and normal skin (n = 6) of patients with psoriasis (n = 2,649 clones). The ratio of Streptococcus mitis to Propionibacterium acnes is 0.2. Panel D. Samples from the lesions (n = 13) of patients with psoriasis (n = 1,314 clones). The ratio of Streptococcus mitis to Propionibacterium acnes is 2.5. Code: Propionibacterium acnes (A1, B7); Corynebacterium tuberculostearicum (A2, B1); Staphylococcus hominis (A5, B2); Streptococcus mitis (A4, B3); Staphylococcus epidermidis (A8, B9); Enhydrobacter aerosaccus (A3); Staphylococcus capitis (A6); Staphylococcus caprae (A7); Dermacoccus AF409025 (A9); Corynebacterium mucifaciens (A10); Corynebacterium simulans (B4); Rothia mucilaginosa (B5); Staphylococcus aureus (B6); Streptococcus salivarius (B8); Flavobacteriaceae DQ337018 (B10).

Figure 4. DPCoA of SLOTU relatedness in 39 human skin samples obtained from healthy subjects and psoriatic patients.

The healthy 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 AL2). The psoriatic patients were designated 1, 2, 3, 4, 6, and 8, PN indicates that sample is from uninvolved skin and PP is from psoriatic lesions. In a representation of the first two orthogonal principal axes, based on a sample dissimilarity matrix, samples from the same subjects at the same time point are plotted by using the same color, and the normal skin and psoriatic lesion samples are indicated by thin and thick circle walls, respectively. The size of each circle is proportional to the sample's Rao diversity index. The scale (bottom right) indicates the relative diversity of the populations within the circles, with the test sample of smallest diversity (2PN) indexed as 1.0.