| BackgroundProbiotics may plays an important role in the connection between Chinese traditional medicine and western modern medicine.Probiotics could improve health by inhibiting the harmful bacteria.Similarly,Chinese medicine advocate that right qi restrains evil qi.Skin is a diversified ecosystem colonized by a diverse microbiota.The colonized microbiota can fortify the microbial barrier,chemical barrier,physical barrier,innate and adaptive immune barrier.When the balance between commensals and pathogens is disturbed,skin disease or even systemic infectious disease then result.Atopic dermatitis（AD）,a chronic inflammatory dermatosis often occurs in children and adolescents,is characterized by skin itchy,skin dry,and skin recurrent flare.The disease etiology of AD is very complex and mainly involved the pathological changes of immunological dysregulation and skin barrier damage.In microbiology,the skin microbiota of AD patients is characterized by serious dysbacteriosis and increased Staphylococcus aureus colonization.The skin microbiota may serve as a potential indicator for the diagnosis,treatment and therapy monitoring of AD disease.Therefore,it is of great significance to characterize the microbial diversity and normal variation between healthy individuals and AD patients.It is also of great significance to the modernization of traditional Chinese medicine theories of dispelling evil and supporting right and securing the root and radically reform.ObjectiveThis study intends study on skin microbial diversity,microbiota cutotypes and probiotic microbe.i.Based on culture and non-culture technology,the human skin microbial diversity and component differences of antecubital fossa skin microbiota between AD patients and healthy individuals were investigated to establish a quantitative counting culture method for skin microbiota typing,to obtain the baseline and normal variation in the microbiota of healthy individuals in southern China,so as to provide a basis for the diagnosis,treatment and therapy monitoring for AD disease.ii.The probiotic microbial resources that inhibit the growth of Staphylococcus aureus and restore the skin barrier are explored,based on the component differences of skin microbiota between AD patients and healthy individuals.iii.The functional potential of probiotic microbe is explored,so as to provide theoretical mechanism for traditional Chinese medicine lotion through skin microbiota in treating the skin flare of AD patients,and provide novel explanation for the traditional Chinese medicine theory of right qi restrains evil qi.Methods1.The AD patients that fulfilled the Williams diagnostic standard and healthy individuals,aged from 12 to 60 years old,were incorporated in the study for microbial diversity of human antecubital fossa.Skin swabs were collected from antecubital fossa.For microbiota profiling,the V4 hypervariable region of the 16S rRNA gene was amplified by polymerase chain reaction.Library preparation and sequencing using Illumina MiSeq platform according to the standard protocols by GENEWIZ,Inc.（Suzhou China）.The taxonomic frequency profiles were created to reflect the community’s operational taxonomic unit composition at phylum and genera levels.Alpha diversity,beta diversity,and microbial differences between AD patients and healthy individuals were analyzed according to the data of 16S rRNA amplicon sequencing.Ⅱ.In total 62 AD patients that fulfilled the Williams diagnostic standard,and 59 healthy individuals,aged from 12 to 60 years old,were incorporated in the study to analyze the microbial diversity and cutotypes.The skin samples were then quantitative cultured in the normal atmosphere environment of 30℃ for 5 days.Monoclonal colonies were selected by "experience picking method" and "all picking method" according to former literature.The microbial identification were performed by MALDI-TOF mass spectrometry and 16SrRNA sequencing.The proportion and density of each microbial taxa were calculated according to the identification results and selection area.For the HV group,the skin samples of the elbow fossa,where AD skin inflammation is pre valent,and the adjacent areas of the medial forearm and medial bind arm were collected in this study to compare the differences of microbial composition.For the AD group,the skin samples of lesioned and non-lesioned skin were collected in this study to compare the differences of microbial composition.The relevance were analyzed between the key microbial taxa and the skin damage indicator of TISS scores.For all skin samples,the cluster analysis of each microbial percentage was performed using IBM SPSS Statistics 25.0 software by selecting the systematic cluster analysis-intergroup linkage method.The cutotypes were then performed based on the cluster analysis results.Ⅲ.The S.aureus strains were collected for the molecular typing,virulence genes distribution and drug resistance analysis,isolated from the skin of outpatients and inpatients meeting Williams’ diagnostic criteria in the Department of Dermatology of Guangdong Provincial Hospital of Traditional Chinese Medicine between October 2019 and October 2022.After eliminating the duplicate strains,genomic DNA was extracted for genome sequencing.The obtained non-duplicate strains were perform the multi-locus sequence typing,Staphylococcal protein A（spa）typing,and SCCmec typing.The encoded virulence factor genes were analyzed by the online VFanalyzer tools by matching with the virulence factor database.The drug sensitivity test was performed by both instrumental and manual methods.Statistical analysis of the experimental results was performed using SPSS v.22.0.Ⅳ.Coagulase negative staphylococci（CoNS）isolates that inhibited the growth of Staphylococcus aureus were screened from healthy human skin.The genomic DNA of the strains was extracted and then sequenced using an Illumina novaseq platform.Functional annotations were assigned using eggnog-mapper and the eggNOG database.The biosynthetic gene clusters of the strains were determined using the online tool antiSMASH bacterial version 6.0 with default parameter.Statistical analysis was performed using SPSS v.22.0.Ⅴ.One Sphingosinicellaceae-like strain designated as SZY PN-1T,isolated from the skin of cubital fossa of a healthy volunteer,was performed the polyphasic taxonomic analysis.Traditional biochemical tests and chemotaxonomic features were carried out with related type strain.The genetic analyses were performed by the 16S rRNA gene squence alignments,the average nucleotide identity（ANI）,G+C content and the phylogenetic tree based on the gene sequencing.The biosynthetic gene clusters of strain SZY PN-1T,were determined using the online tool antiSMASH bacterial version 6.0 with default parameter.To investigate whether SZY PN-1T could promote wound healing,the effect of its fermentation solution on the migration ability of HaCaT cells was analyzed using a cell scratch assay.Ⅵ.The beneficial Staphylococcus hominis,which can inhibit Staphylococcus aureus,was screened from the healthy volunteers of AD patients’ families.The screened probiotics was prepared into bacterial suspension with sterile saline,and then used to treat the skin flare of AD disease by microbiota transplantation.The therapeutic effect was evaluated by the TISS scores of skin damage.Agar diffusion method was used to analyze the antimicrobial effect against Staphylococcus aureus for compound arnebia oil,Xianglian lotion,antipruritic and diuretic granules,antipruritic granules lotion and Chinese medicine dehuuidifying paste.ResultsⅠ.In total 20 AD patients and 33 healthy individuals have been completed the 16S rRNA gene sequencing for microbial diversity of human antecubital fossa.At the phylum level,the dominant phyla were Pseudomonadota,Bacillota,Actinomycetota,Bacteroidota and Deinococcota,both in AD group and healthy control group.In comparison,phylum Bacillota was significantly enriched,while phyla and Bacteroidetes were significantly deprived in the AD group compared with the healthy volunteer group.The alpha diversity showed that HV group has higher species diversity than AD group.The beta diversity showed that the aggregation degree of HV group is higher than AD group,while the dispersion degree of AD group is higher than HV group.The microbial differences analysis showed that the colonization rate of genus Staphylococcus in AD group was significantly higher than that in HV group,while the colonization rate of genera Moraxella,Corynebacteriumy,Acinetobacter,Paracoccus,Cutibacterium,Chryseobacterium,Brevundimonas,Stenotrophomonas and Roseomonas were significantly lower than that in the HV group.Ⅱ.In total 62 AD patients and 59 healthy individuals aged from 12 to 60 years old have been incorporated in the study to analyze the microbial diversity and cutotypes.For the HV groups,26 healthy volunteers have been simultaneously collected the skin samples of elbow fossa,medial forearm and medial rear arm.At the species level,only the detection rate of Staphylococcus epidermidis in the medial forearm and the medial hind arm had found statistically significant differences in the top 10 detection rate species in the three sites.This results suggests that the species compositions were consistent in the elbow fossa,medial forearm and medial hind arm for healthy individuals.The AD group was further divided into lesion group and non-lesion group.In comparison,the counting number of whole microbial colonies,the counting number of Staphylococcus aureus,and the detection rate of Staphylococcus aureus in the AD lesion group were significantly higher than those of the AD non-lesion group.According to Spearman’s correlation analysis,the proportion and the density of S.aureus detected in AD patients were significantly and positively correlated with the severity of dermatitis.By comparing the species composition of antecubital fossa,it has been found the significantly increase of Staphylococcus aureus,while the significantly decrease of Staphylococcus hominis,Staphylococcus epidermidis,Staphylococcus capitis,Micrococcus luteus,Roseomonas mucosa,and Moraxella osloensis in the specimens of AD group.The cluster analysis based on the percentage of cultured colonies per species showed 9 clear groups for 10 and 15 relative inter-group distance.Combining the cluster analysis and the microbial characteristics,the skin microbiota in antecubital fossa can be divided into the following cutotypes,such as sterile cutotype,Sho cutotype,CoNS cutotype,M cutotype,C cutotype,and Sau cutotype.The sterile cutotype was characterized by almost sterile and little microbe.The Sho cutotype was dominated by Staphylococcus hominis.The CoNS cutotype was dominated by other CoNS other than Staphylococcus hominis.The M cutotype was dominated by Moraxella osloensis.The C cutotype was dominated by Cutibacterium.The Sau cutotype was dominated by Staphylococcus aureus.In comparison,Sho cutotype,C cutotype and M cutotype are typical healthy skin,while Sau cutotype is typical inflamed skin of AD patients.Ⅲ.A total of 90 non-repetitive Staphylococcus aureus isolates were collected from Guangdong Provincial Hospital of Traditional Chinese Medicine from October 2019 to October 2022.In total 25 ST types were identified,and 7 new sequence types were found.The major types of Staphylococcus aureus isolated from AD patients were ST7（15.56%）,ST 188（11.11%）and ST 15（11.11%）,accounting for more than one-third of all S.aureus isolates.The ninety strains of S.aureus could be identified as 48 spa types,of which five isolates could not be identified as new spa types.The major spa types were t91（16.67%）and t189（9.00%）.For the 90 non-repetitive Staphylococcus aureus isolates,77 strains（85.6%）were MSSA and 13 strains（14.4%）were MRSA.The SCCmec typing showed that only one type（type Ⅳ）was found among the 13 MRS A isolates,containing 5 isolates,and no SCCmec typing was detected in the remaining isolates.The Staphylococcus aureus strains isolated from AD inpatients were generally susceptible to antibiotic drugs,except that 74.36%isolates were resistance to penicillin,28.21%isolates were resistance to erythromycin,20.51%isolates were resistance to clindamycin.The resistance rate to other commonly antibiotics was less than 10%.Ⅳ.This study identified 14 CoNS strains with antibacterial activity against S.aureus,including 10 strains of S.epidermidis and 4 strains of S.hominis.The genome size of ten Staphylococcus epidermidis strains ranged from 2.48 to 3.02 Mb,the G+C content ranged from 31.8 to 32.1%,the number of CDS genes ranged from 2258 to 2842,the number of rRNAs ranged from 4 to 9,the number of tRNAs ranged from 37 to 56,and tmRNAs were all 1.The genome size of three Staphylococcus hominis strains ranged from 2.25 to 2.36 Mb,the G+C content ranged from 31.2 to 31.4%,the number of CDS genes ranged from 21821 to 2341,the number of rRNAs ranged from 4 to 9,the number of tRNAs ranged from 49 to 61,and tmRNAs were 1.In total 123 gene clusters from 10 categories have been predicted using antiSMASH software.The major biosynthetic gene clusters types include cyclic lactone autoinducer,Type 3 polyketide synthase,staphyloferrin A,nonribosomal peptides,staphylopine,ribosomally synthesized and posttranslationally modified peptides,cyclodipeptide synthetase,transAT-PKS/NRPS,lanthipeptide（Ⅱ）,thiopeptide.These new secondary metabolites are important to facilitate the excavation and development of novel antibiotics and antineoplastic drugs.Ⅴ.Strain SZY PN-1T was characterized as a Gram-negative,aerobic,non-motile,and non-spore-forming bacillus.After 3 days incubation at 35℃ and 5%CO2 environment,the colonies of strain SZY PN-1 T were yellow,round,and convex.Phylogenetic trees based on 16S rRNA gene and whole gene showed that strain SZY PN-1 T was clustered into an independent clade with’Sandaracinobacter sibiricus’ RB16-17 and Sandaracinobacter neustonicus JCM 30734T.The ANIb and ANIm values between SZY PN-1 T and the closest related S.neustonicus JCM 30734T were 76.5%and 84.8%,respectively.The phenotypic,phylogenetic and genome-based analyses suggested that strain SZY PN-1T represents a new species of a new genus,for which the name Sandaracinobacteroides hominis gen.nov.,sp.nov.,within the family Sphingosinicellaceae.Like other Sphingosinicellaceae species,Sandaracinobacteroides hominis SZY PN-1 T was aslo charaterized by sphingolipids in the cell wall.The sphingolipids compounds have been proved for potent antibacterial effects against S.aureus.The fermentation solution of SZY PN-1T can promote HaCaT cell migration,promote wound healing,restore skin barrier function,and alleviate AD-related immune/inflammatory responses.This suggests that Sandaracinobacteroides hominis SZY PN-1T may play an important role in the bacterial defense mechanism of healthy normal skin.Sandaracinobacteroides hominis SZY PN-1 T is expected to be developed as probiotic and used for microbial treatment of skin diseases.Ⅵ.The beneficial Staphylococcus hominis F01,which can effectively inhibit the growth of Staphylococcus aureus,was screened from one healthy volunteers of AD patients’families.When the microbiota transplantation of Staphylococcus hominis F01 was transplanted to the dermatitis site of AD patients,the skin injury symptoms of 6 AD patients were significantly improved with no side effects.Agar diffusion method was used to analyze the antimicrobial activity,and the result show that Xianglian external lotion showed obvious inhibition against Staphylococcus aureus.ConclusionThe human skin is charaterized harsh and nutrient-poor environment.The colonizing commensal microbiota are often with low requirements for nutrition.Based on 16S rRNA gene amplification sequencing,we established a rapid culture method for the study of microbial diversity and cutotypes for human skin.We found that Staphylococcus aureus was significantly increased in AD group,and the proportion and the density of S.aureus detected in AD patients were significantly and positively correlated with the severity of dermatitis.The skin microbiota in antecubital fossa can be divided into several different cutotypes.In comparison,Sho cutotype,C cutotype and M cutotype are typical healthy skin,while Sau cutotype is typical inflamed skin of AD patients.The major types of Staphylococcus aureus isolated from AD patients were ST7（15.56%）,ST188（11.11%）and ST 15（11.11%）,which are similar to the ST distribution of Staphylococcus aureus in human nasal cavity.This study obtain the baseline and normal variation in the microbiota of healthy individuals in southern China,and also provide a basis for the diagnosis,treatment and therapy monitoring for AD disease.The probiotic microbial resources that inhibit Staphylococcus aureus have been explored based on the microbial differences between AD patients and healthy individuals.Staphyococcus hominis F01 has been screened with strong antimicrobial ability against Staphylococcus aureus.Sandaracinobacteroides hominis SZY PN-1T,a novel species within the family Sphingosinicellaceae,has also been found potential probiotic for the treatment of skin diseases.This study provide theoretical basis for skin microbiota transplantation,and the micro-ecological mechanism of traditional Chinese medicine lotion in the treatment of AD dermatitis.The result of probiotics from healthy skin can inhibit pathogenic Staphylococcus aureus,also provide novel explanation for the traditional Chinese medicine theory of ’dispelling evil and supporting right’ and ’securing the root and radically reform’. |
PDF Full Text Request