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

Min Hye Kim, Mina Rho, Jun Pyo Choi, Hyun Il Choi, Han Ki Park, Woo Jung Song, Taek Ki Min, Sang Heon Cho, Young Joo Cho, Yoon Keun Kim, Sanghwa Yang, Bok Yang Pyun, Min Hye Kim, Mina Rho, Jun Pyo Choi, Hyun Il Choi, Han Ki Park, Woo Jung Song, Taek Ki Min, Sang Heon Cho, Young Joo Cho, Yoon Keun Kim, Sanghwa Yang, Bok Yang Pyun

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.

Keywords: Atopic dermatitis; extracellular vesicle; metagenomic analysis.

Conflict of interest statement

There are no financial or other issues that might lead to conflict of interest.

Copyright © 2017 The Korean Academy of Asthma, Allergy and Clinical Immunology · The Korean Academy of Pediatric Allergy and Respiratory Disease

Figures

Fig. 1. Comparison of bacterial composition in…
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…
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…
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…
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…
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.

References

    1. Sabin BR, Peters N, Peters AT. Chapter 20: atopic dermatitis. Allergy Asthma Proc. 2012;33(Suppl 1):S67–S69.
    1. Lee JH, Son SW, Cho SH. A comprehensive review of the treatment of atopic eczema. Allergy Asthma Immunol Res. 2016;8:181–190.
    1. Leung DY, Boguniewicz M, Howell MD, Nomura I, Hamid QA. New insights into atopic dermatitis. J Clin Invest. 2004;113:651–657.
    1. Pyun BY. Natural history and risk factors of atopic dermatitis in children. Allergy Asthma Immunol Res. 2015;7:101–105.
    1. Bieber T. Atopic dermatitis. N Engl J Med. 2008;358:1483–1494.
    1. Ahn K. The prevalence of atopic dermatitis in Korean children. Allergy Asthma Immunol Res. 2016;8:1–2.
    1. Lee JH, Han KD, Kim KM, Park YG, Lee JY, Park YM. Prevalence of atopic dermatitis in Korean children based on data from the 2008–2011 Korean National Health and Nutrition Examination Survey. Allergy Asthma Immunol Res. 2016;8:79–83.
    1. Niebuhr M, Langnickel J, Draing C, Renz H, Kapp A, Werfel T. Dysregulation of toll-like receptor-2 (TLR-2)-induced effects in monocytes from patients with atopic dermatitis: impact of the TLR-2 R753Q polymorphism. Allergy. 2008;63:728–734.
    1. Wollenberg A, Feichtner K. Atopic dermatitis and skin allergies - update and outlook. Allergy. 2013;68:1509–1519.
    1. Kong HH, Oh J, Deming C, Conlan S, Grice EA, Beatson MA, et al. Temporal shifts in the skin microbiome associated with disease flares and treatment in children with atopic dermatitis. Genome Res. 2012;22:850–859.
    1. Breuer K, Wittmann M, Bösche B, Kapp A, Werfel T. Severe atopic dermatitis is associated with sensitization to staphylococcal enterotoxin B (SEB) Allergy. 2000;55:551–555.
    1. Chen YE, Tsao H. The skin microbiome: current perspectives and future challenges. J Am Acad Dermatol. 2013;69:143–155.
    1. Human Microbiome Project Consortium. Structure, function and diversity of the healthy human microbiome. Nature. 2012;486:207–214.
    1. EL Andaloussi S, Mäger I, Breakefield XO, Wood MJ. Extracellular vesicles: biology and emerging therapeutic opportunities. Nat Rev Drug Discov. 2013;12:347–357.
    1. Pyun BY. Extracellular vesicle: an unknown environmental factor for causing airway disease. Allergy Asthma Immunol Res. 2016;8:179–180.
    1. Shin TS, Kim JH, Kim YS, Jeon SG, Zhu Z, Gho YS, et al. Extracellular vesicles are key intercellular mediators in the development of immune dysfunction to allergens in the airways. Allergy. 2010;65:1256–1265.
    1. Hong SW, Kim MR, Lee EY, Kim JH, Kim YS, Jeon SG, et al. Extracellular vesicles derived from Staphylococcus aureus induce atopic dermatitis-like skin inflammation. Allergy. 2011;66:351–359.
    1. Hong SW, Choi EB, Min TK, Kim JH, Kim MH, Jeon SG, et al. An important role of α-hemolysin in extracellular vesicles on the development of atopic dermatitis induced by Staphylococcus aureus. PLoS One. 2014;9:e100499.
    1. Hanifin JM, Rajka G. Diagnostic features of atopic dermatitis. Acta Derm Venereol Suppl (Stockh) 1980;92:44–47.
    1. Severity scoring of atopic dermatitis: the SCORAD index. Consensus Report of the European Task Force on Atopic Dermatitis. Dermatology. 1993;186:23–31.
    1. Edgar RC. Search and clustering orders of magnitude faster than BLAST. Bioinformatics. 2010;26:2460–2461.
    1. Caporaso JG, Kuczynski J, Stombaugh J, Bittinger K, Bushman FD, Costello EK, et al. QIIME allows analysis of high-throughput community sequencing data. Nat Methods. 2010;7:335–336.
    1. Drago L, De Grandi R, Altomare G, Pigatto P, Rossi O, Toscano M. Skin microbiota of first cousins affected by psoriasis and atopic dermatitis. Clin Mol Allergy. 2016;14:2.
    1. Seite S, Flores GE, Henley JB, Martin R, Zelenkova H, Aguilar L, et al. Microbiome of affected and unaffected skin of patients with atopic dermatitis before and after emollient treatment. J Drugs Dermatol. 2014;13:1365–1372.
    1. Choi Y, Park H, Park HS, Kim YK. Extracellular vesicles, a key mediator to link environmental microbiota to airway immunity. Allergy Asthma Immunol Res. 2017;9:101–106.
    1. Kim JH, Lee J, Park J, Gho YS. Gram-negative and gram-positive bacterial extracellular vesicles. Semin Cell Dev Biol. 2015;40:97–104.
    1. Campos JH, Soares RP, Ribeiro K, Andrade AC, Batista WL, Torrecilhas AC. Extracellular vesicles: role in inflammatory responses and potential uses in vaccination in cancer and infectious diseases. J Immunol Res. 2015;2015:832057.

Source: PubMed

Sponsorer og samarbejdspartnere

Medicinske tilstande

Narkotikainterventioner

3
Abonner