Allergy Asthma Immunol Res.  2017 Sep;9(5):453-461. 10.4168/aair.2017.9.5.453.

A Metagenomic Analysis Provides a Culture-Independent Pathogen Detection for Atopic Dermatitis

• Kim MH
• Rho M
• Choi JP
• Choi HI
• Park HK
• Song WJ
• Min TK
• Cho SH
• Cho YJ
• Kim YK
• Yang S
• Pyun BY

• KMID: 2383994
• DOI: http://doi.org/10.4168/aair.2017.9.5.453

Abstract

PURPOSE
Atopic dermatitis (AD) is an inflammatory skin disease, significantly affecting the quality of life. Using AD as a model system, we tested a successive identification of AD-associated microbes, followed by a culture-independent serum detection of the identified microbe.
METHODS
A total of 43 genomic DNA preparations from washing fluid of the cubital fossa of 6 healthy controls, skin lesions of 27 AD patients, 10 of which later received treatment (post-treatment), were subjected to high-throughput pyrosequencing on a Roche 454 GS-FLX platform.
RESULTS
Microbial diversity was decreased in AD, and was restored following treatment. AD was characterized by the domination of Staphylococcus, Pseudomonas, and Streptococcus, whereas Alcaligenaceae (f), Sediminibacterium, and Lactococcus were characteristic of healthy skin. An enzyme-linked immunosorbent assay (ELISA) showed that serum could be used as a source for the detection of Staphylococcus aureus extracellular vesicles (EVs). S. aureus EV-specific immunoglobulin G (IgG) and immunoglobulin E (IgE) were quantified in the serum.
CONCLUSIONS
A metagenomic analysis together with a serum detection of pathogen-specific EVs provides a model for successive identification and diagnosis of pathogens of AD.

Keyword

Atopic dermatitis; extracellular vesicle; metagenomic analysis

MeSH Terms

Alcaligenaceae
Dermatitis, Atopic*
Diagnosis
DNA
Enzyme-Linked Immunosorbent Assay
Extracellular Vesicles
Humans
Immunoglobulin E
Immunoglobulin G
Immunoglobulins
Lactococcus
Metagenomics*
Pseudomonas
Quality of Life
Skin
Skin Diseases
Staphylococcus
Staphylococcus aureus
Streptococcus
DNA
Immunoglobulin E
Immunoglobulin G
Immunoglobulins

Figure

• 

  Fig. 1 Comparison of bacterial composition in the skin washing fluid of AD patients (case) and normal controls. (A, B) In the skin washing fluid of patients, Staphylococcus accounted for the majority of bacteria, followed by Pseudomonas and Streptococcus in the heatmap. In normal controls, Staphylococcus, Pseudomonas, and Streptococcus were hardly detected, whereas Sediminibacterium, Alcaligenaceae (f) were predominant. AD, atopic dermatitis; f, family.

• 

  Fig. 2 Changes in the bacterial proportion of skin washing fluid in patients with AD before and after treatment. (A, B) The proportion of Staphylococcus decreased after treatment. Increasing proportions following treatment were evident for Alicyclobacillus, Propionibacterium, and Streptococcus. AD, atopic dermatitis.

• 

  Fig. 3 Bacterial community analysis of skin washing fluid of AD patients with before and after treatment according to the phylogenetic hierarchy of phylum, class, order, and family. (A) In AD patients, Firmicutes was dominant before treatment and decreased following treatment, with Actinobacteria being increased after treatment. (B) Bacilli dominated before treatment and decreased after treatment, with Actinobacteria being increased after treatment at the class level. (C) Bacillales were dominant before treatment and decreased after treatment, with Propionibacteriales and Lactobacillales being increased after treatment at the order level. (D) Staphylococcaceae was dominant before treatment and was decreased after treatment, with Alicyclobacillaceae, Propionibacteriaceae and Streptococcaceae increased after treatment at the family level. AD, atopic dermatitis.

• 

  Fig. 4 Bacterial community analysis of skin washing fluid of AD patients with before and after treatment in the genus level. (A, B) Staphylococcus was reduced, while Alicyclobacillus, Propionibacterium, and Streptococcus were increased and bacterial diversity was restored after treatment compared to that before treatment. AD, atopic dermatitis.

• 

  Fig. 5 SEB-specific IgG and IgE and S. aureus EV-specific IgG and IgE levels. (A, B) SEB-specific IgG and IgE levels were significantly higher in the serum of AD patients compared with that of normal controls. (C, D) S. aureus EV-specific IgG and IgE levels were significantly higher in the serum of AD patients compared with those of normal controls. SEB, S. aureus exotoxin B; IgG, immunoglobulin G; IgE, immunoglobulin E; EV, extracellular vesicle; AD, atopic dermatitis.

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