The Strain-level Signatures And Quorum-sensing Regulation Of Skin Staphylococcus Epidermidis In Pediatric Atopic Dermatitis

Background:The skin dysbiosis is one of the most prominent characteristics in atopic dermatitis(AD),which plays an important role in the development of AD.The overcolonization of pathogenic Staphylococcus aureus(S.aureus)and decreased diversity of skin commensals were observed in patients with AD.Staphylococcus epidermidis(S.epidermidis),a conspicuous resident on the skin surface,can affect the virulence factor expression and epidermal colonization of S.aureus via accessory gene regulator(agr)quorum-sensing system.As such cross-species interactions are highly strain-dependent,the exact role and mechanism of S.epidermidis remain to be elucidated.Objective:To explore the strain-level signatures of skin S.epidermidis and its quorumsensing regulation on S.aureus colonization in AD.Methods:Skin microbiota of patients with AD were collected from the lesional(AD-L group)and the adjacent non-lesional skin(AD-NL group),while those of normal control(NC)subjects were collected from the same sites.Shotgun metagenomic sequencing was conducted to delineate the compositive signatures of AD microbiome at the community level and clarify the correlation with disease severity.Combined with culture methods,we isolated numerous S.epidermidis strains and performed whole-genome sequencing(WGS),and explored the phylogenetic diversity,function-gene difference,and agr typing of the isolates at the strain level.The metagenomic dataset was also mapped against the genomes of S.epidermidis isolates with different agr types to obtain their abundance within individuals and the correlation with the abundance of S.aureus and disease severity.Conditioned culture supernatants from clinical isolates of S.epidermidis with different agr types were cocultured with S.aureus in vitro,where the growth,agr activity and metabolism of S.aureus were then investigated.In addition,an epicutaneous S.aureus mouse model was constructed to explore the effects of different S.epidermidis agr types on the epidermal S.aureus colonization and skin inflammation in vivo.Results:Metagenomic analysis showed that the skin microbiota of AD patients presented with reduced microbial richness and increased abundances of S.aureus and S.epidermidis.The relative abundance of Staphylococcus,particular S.aureus,was positively correlated with the SCORAD index,whereas that of S.epidermidis presented with no significant correlation with AD severity.The relative abundance of S.epidermidis was in parallel with that of S.aureus in the NC group and AD-NL groups,but not in the AD-L group.The ratio of S.epidermidis to S.aureus was negatively correlated with the SCORAD index.Further WGS analysis revealed that the phylogenetic diversity of S.epidermidis isolated from AD and NC was comparable,yet with significant strain heterogeneity in functional genes.Based on the sequence variation of autoinducing peptide(AIP),the signal molecule of the agr system,S.epidermidis isolates in this study could be clustered into four agr types and two subtypes,Ⅰb and Ⅲb,which expressed the same AIP as type Ⅰ and type Ⅲ,respectively(but with a unique AIP leader peptide).S.epidermidis agr type Ⅳ was hardly detected in AD isolates,except one isolate cultured from non-lesional skin of AD.The number of S.epidermidis agr type Ⅰ was also slightly decreased among isolates of the AD-L group in comparison with isolates from NC.The comparison results between metagenomic and WGS dataset also confirmed the presence of these isolates on the skin of the study subjects.Functional experiments revealed that S.epidermidis agr type Ⅰ and type Ⅳ inhibited the S.aureus agr virulence system in vitro,and the isolates of S.epidermidis with different agr types heterogeneously affected the global gene expression of S.aureus.In the epicutaneous S.aureus mouse model,topical application of S.epidermidis agr type Ⅰ and type Ⅳ reduced the epidermal colonization of S.aureus and alleviated the skin inflammation,while agr type Ⅱ and type Ⅲ had little effect.Conclusion:This study first delineated the functional heterogeneity of S.epidermidis in AD at the strain level,which revealed that the locus variation in agr operon was one of the important reasons for the heterogeneous effects of different S.epiermidis strains on S.aureus.Thereinto,S.epidermidis agr type Ⅰ and type Ⅳ inhibited the virulence factor expression and epidermal colonization of S.aureus,and alleviated the downstream skin inflammation.The study provides new theoretical and experimental evidences for the skin dysbiosis in AD,and application of defined strains of S.epidermidis with functional agr type may become a new option for the microbiome-modulation therapy of AD.