Staphylococcus aureus and Staphylococcus epidermidis strain diversity underlying pediatric atopic dermatitis

Abstract

The heterogeneous course, severity, and treatment responses among patients with atopic dermatitis (AD; eczema) highlight the complexity of this multifactorial disease. Prior studies have used traditional typing methods on cultivated isolates or sequenced a bacterial marker gene to study the skin microbial communities of AD patients. Shotgun metagenomic sequence analysis provides much greater resolution, elucidating multiple levels of microbial community assembly ranging from kingdom to species and strain-level diversification. We analyzed microbial temporal dynamics from a cohort of pediatric AD patients sampled throughout the disease course. Species-level investigation of AD flares showed greater Staphylococcus aureus predominance in patients with more severe disease and Staphylococcus epidermidis predominance in patients with less severe disease. At the strain level, metagenomic sequencing analyses demonstrated clonal S. aureus strains in more severe patients and heterogeneous S. epidermidis strain communities in all patients. To investigate strain-level biological effects of S. aureus, we topically colonized mice with human strains isolated from AD patients and controls. This cutaneous colonization model demonstrated S. aureus strain-specific differences in eliciting skin inflammation and immune signatures characteristic of AD patients. Specifically, S. aureus isolates from AD patients with more severe flares induced epidermal thickening and expansion of cutaneous T helper 2 (T_H2) and T_H17 cells. Integrating high-resolution sequencing, culturing, and animal models demonstrated how functional differences of staphylococcal strains may contribute to the complexity of AD disease.

Fig. 1. Bacterial communities shift during AD disease progression

(A) Objective SCORAD for each patient at baseline, flare, and post-flare. Higher SCORAD corresponds to more severe disease. *** P<0.001 (B) Mean Shannon diversity +/− SEM in controls and AD disease states. Colors correspond to disease state. Volar forearm (Vf), antecubital crease (Ac), inguinal crease (Ic), popliteal crease (Pc), forehead (Fh), occiput (Oc), and retroauricular crease (Ra). (C) Shannon diversity versus objective SCORAD for combined antecubital (Ac) and popliteal creases (Pc) (AcPc) of AD patients. Partial correlation (adjusting for disease state). (D) Mean relative abundance of bacterial genera in AcPc for controls and AD disease states. (E) Mean relative abundance of predominant genera in AcPc for disease states, Flare (F) and Post-flare (PF). (F) Proportion of Staphylococcus versus objective SCORAD for AcPc of AD patients, partial correlation (adjusting for disease state).

Fig. 2. Staphylococcal species increase during AD disease flare

(A) Mean relative abundance of staphylococcal species within the total bacterial population in combined antecubital (Ac) and popliteal creases (Pc) (AcPc) of AD patients and controls. (B) Mean relative abundance of most abundant Staphylococcus species in AcPc for disease states, Flare (F) and Post-flare (Pf). (C) Correlation of S. aureus (left) and S. epidermidis (right) mean relative abundance and objective SCORAD for AcPc of patients, partial correlation (adjusting for disease state). (D) Comparison of S. aureus to S. epidermidis relative abundance by patient for all sites. Patient’s objective SCORAD indicated in parenthesis. Shape corresponds to physiological characteristic of the body site, color to the predominant species, and size to the magnitude of disease severity (objective SCORAD). Patients in the top row have a higher predominance of S. epidermidis, while bottom row patients are S. aureus-predominant.

Fig. 3. S. aureus-predominant individuals are often colonized with a single S. aureus strain

(A) Dendogram of 61 representative S. aureus strains based on SNVs in the core genome. Strains labeled in red were isolated from patients in (B). Colored blocks correspond to genomes of the same clade. Phylogenetically distant clade F1 is shown as an outgroup as it was recently reclassified as S. argenteus (32). (B) For S. aureus-predominant individuals, S. aureus clade relative abundances in bilateral antecubital (Ac) and popliteal creases (Pc) for AD disease states, flare and post-flare. Colors correspond to those in (A). (C) For combined samples of all sites/timepoints of individuals in (B), barcharts show the number of SNVs per individual that are mono, bi, and triallelic. (D) Venn diagram showing the number of genes shared between isolates from patients in (B), indicated in red in (A).

Fig. 4. S. epidermidis-predominant individuals are colonized by a heterogenous community of S. epidermidis strains

(A) Dendogram of S. epidermidis strains based on SNVs in the core genome. Strains isolated from patients in our study are labeled in red. Similar colors represent closely related strains that were grouped into 14 clades. Starred (*) isolates are nosocomial in origin (B) For S. epidermidis-predominant individuals, S. epidermidis strain relative abundances in combined antecubital (Ac) and popliteal creases (Pc) for AD disease states, flare and post-flare. Colors correspond to those in (A). (C) Heatmap shows mean relative abundance of each clade across all sites in S. aureus and S. epidermidis-predominant AD patients, healthy adults (HA), and healthy children (HC). (D) In principal component analysis, clades A20, A29, and A30 drive separation between S. epidermidis-predominant AD patients and healthy adults.

Fig. 5. Topical application of AD isolates induce AD-like immune responses in murine models

(A) Mice were topically associated with staphylococcal monocultures every other day 4 times before sacrifice on the 8th day. (B) Representative histological images of the ear pinnae of mice associated with tryptic soy broth TSB, S. aureus AD04.E17, HC.B1, USA300, or S. epidermidis A10.A30. Dotted line indicates separation between the epidermidis and dermis. Scale bar 50 μm. (C) Epidermal thickness of ears post topical association of patient AD isolates. Color indicates origin and species of the isolate. (D) Absolute numbers of skin eosinophil (E) Absolute numbers of skin TCRβ⁺ CD4⁺ cells. (F) Absolute numbers of skin IL-13⁺ CD4⁺ cells. (G) Absolute numbers of skin IL-17A⁺ CD4⁺ cells. (H) Frequencies of IL-13⁺ and IL-17A⁺ CD4⁺ cells from mice in (B). Results are cumulative data from 2 or 3 independent experiments, 3 mice per group. *p<0.05, **p<0.01, ***p<0.001 as calculated by ANOVA with multiple comparison correction.