Metagenomic Analysis of Skin Microbiome and Circulating Bacterial Extracellular Vesicles in Atopic Dermatitis

학위논문 (박사)-- 서울대학교 대학원 : 협동과정 임상약리학전공, 2015. 8. 조상헌.Introduction: Staphylococcus aureus is the most common bacterium colonizing the skin of atopic dermatitis (AD) patients. Up to 90% of those with AD are colonized by S. aureus, which is much higher than the rate in healthy subjects. In addition to the contribution of local bacteria, systemic bacterial structure affects pathogenesis of AD. No previous study has considered the systemic bacterial contribution. Until recently, bacteria could only be identified by culture-based assays, while identify only 1% of the total bacteria and which takes days. Here, we performed metagenomic analysis to determine the proportion of colonizing or pathogenic bacteria in the local or systemic environment of AD patients compared to healthy controls. Methods: Twenty-seven patients with AD and 6 healthy control subjects were enrolled. Skin washing fluids were obtained from moistened gauze positioned on skin lesions of AD patients and the cubital fossa of normal controls. Blood and urine samples were also obtained. Genomic DNA was extracted from all samples. The 16s ribosomal RNA gene was amplified using the universal primer and sequenced using a next generation sequencer. Sequenced data were analyzed. Results: S. aureus was dominant in the skin from AD patients and hardly detected in skin from control subjects. Staphylococcus comprised 66.2% and 0.2% of the total bacterial DNA of AD patients and normal controls, respectively. Alcaligenaceae family and Sediminibacterium were the most frequent bacteria in the skin of the controls. After treatment, the proportion of Staphylococcus decreased from 38.8% to 5.7%. The proportion of Alicyclobacillus, Propionibacterium, and Streptococcus was increased from 2.2% to 6.0%, from 2.2% to 4.6%, and from 0.5% to 6.5%, respectively, in the post-treatment group. The bacterial composition of urine and serum were almost identical, and both differed appreciably from skin. The proportion of Lactococcus, Leuconostoc, and Lactobacillus were significantly increased in the control group, and Alicyclobacillus, Propionibacterium, and Streptophyta(o) were increased in AD patients. Conclusions: Metagenomics of bacterial DNA in the skin washing fluid of AD patients confirmed that S. aureus is a main skin colonizer of AD compared to normal controls and that the bacterial composition changes dramatically after treatment. S. aureus may be closely related to the pathogenesis and exacerbation of AD. Lactic acid bacteria, which are beneficial constituents of the human microbiome, were dominant in the urine of healthy controls compared to AD patients. This indicates that lactic acid bacteria might have protective roles against AD.Contents I. Introduction 1 II. Materials and Methods 4 2.1 Study subjects 4 2.2 Collection of skin washing fluid 4 2.3 Laboratory tests and measurement of AD severity 4 2.4 DNA isolation from skin washing fluid, urine, and serum 5 2.5 EV preparation from urine and serum 5 2.6 Emulsion-based PCR (emPCR) sequence analysis 5 2.7 Next generation sequence analysis 6 2.8 Phylogenetic classification 7 2.9 Evaluation of S. aureus exotoxin B specific- and S.aureus EV specific- IgG and IgE levels 7 2.10 Statistical analyses 8 III. Results 9 3.1 Clinical characteristics of AD patients and normal controls 9 3.2 Bacterial composition in skin washing fluid 9 3.3 Changes in bacterial profile proportion in skin washing fluid in AD patients before and after the treatment 10 3.4 Comparison of bacterial EV composition in urine and serum of AD patients 11 3.5 Differences of bacterial EV composition in urine of AD patients and normal controls 12 3.6 Treatment-related changes in the bacterial EV profile in urine in AD patients 13 3.7 IgG and IgE to S. aureus exotoxin B and S. aureus EV 14 IV. Discussion 15 V. References 24 VI. Figure legends 30 VII. Figures and Tables 36 VIII. Korean abstract 70Docto