| Atopic Dermatitis(AD)is a recurrent inflammatory skin disease characterized by pruritus,which is highly associated with colonization of Staphylococcus aureus and decreased skin microbial diversity.Staphylococcus aureus has an aggravating effect on AD and is therefore considered a reasonable therapeutic target.In view of the poor efficacy of the current broad-spectrum antibiotic therapy and the problems of bacterial resistance,the use of microbial interspecific competition against Staphylococcus aureus will be an effective microbial therapy.In this study,human skin microorganisms were isolated,cultured and identified.The antagonistic effect of different skin microorganisms on Staphylococcus aureus was studied and the representative strains with strong antagonistic effect were screened out.The whole genome of representative strains was sequenced.Skin inflammation in AD mice was ameliorated by applying representative strains to mouse skin.The results are as follows:1.1.Culture group of skin microorganisms.Samples are taken from different skin areas of the human body using disposable swabs.After simple culture,single colony isolation,lineation purification,morphology and 16 S r RNA molecular biological identification,a total of tens of thousands of single colonies were obtained,and 86 representative strains were selected.After DNA extraction,strains were amplified and sequenced directly and BLAST.After comparison and identification,they belong to 6bacterial genera and 20 species.The six genera are staphylococcus,Pseudomonas,Bacillus,Klebsiella,Brebacter and micrococcus.2.The inhibitory effect of skin microorganisms on cell growth and biofilm formation of Staphylococcus aureus was studied by co-culturing cell-free supernatant of skin microorganisms with Staphylococcus aureus USA300 at 21% and 1% oxygen levels The results showed that 11 strains,mainly Bacillus subtilis and Staphylococcus epidermidis,had a growth inhibition rate greater than 30% at 21% oxygen level.Bacillus subtilis TOS-89 had the highest inhibition rate on staphylococcus aureus cell growth,reaching 75%.There were 9 strains with growth inhibition rate greater than 30%at 1% oxygen level,mainly Bacillus subtilis,Staphylococcus epidermidis and Staphylococcus aureus.Bacillus subtilis TOS-89 had the highest inhibition rate on staphylococcus aureus cell growth,reaching 39%.Among the inhibitory effects of cellfree supernatant of skin microorganisms on the formation of Staphylococcus aureus USA300 biofilm,bacillus and Staphylococcus strains showed relatively good inhibitory effects,with the highest inhibitory rates of 64% and 53%,respectively.The direct interaction between the screened bacteria and Staphylococcus aureus was investigated by bacteriostatic zone method.The results showed that a total of 12 strains had significant inhibitory effect on Staphylococcus aureus USA300,and Bacillus subtilis TOS-90 had the largest bacteriostatic zone of 1.22±0.08 cm.Brevibacterium sp.37TOS-91 had the largest R value(inhibition zone diameter/colony diameter)of3.22±0.69.According to the above experimental results and related research literature,three representative antagonistic strains were selected for the subsequent antagonistic study of Staphylococcus aureus USA300.Staphylococcus warneri TOS-23,Staphylococcus epidermidis GS-68 and Bacillus subtilis TOS-90,respectively.3.Improvement effect of skin microorganisms on atopic dermatitis in mice.The mouse model of AD was constructed by DNFB solution and Staphylococcus aureus USA300,and the antagonistic bacterial suspension was applied on the skin of the mice for intervention treatment.The treatment group consisted of four groups,They were normal group(CT),model group(AD),Bacillus subtilis TOS-90 treatment group(BS),Staphylococcus epidermidis GS-68 treatment group(SE)and Staphylococcus warneri,respectively TOS-23 treatment group(SW)and mixed bacteria treatment group(M).The results showed that the treatment effect of BS group was relatively better,the dermatitis score SCORAD,ear thickness and colonization of Staphylococcus aureus USA300 decreased significantly(P<0.001).Histological HE staining showed that BS group had less epidermal damage than AD group.Sequencing of skin microbiome with16 S r RNA showed that the diversity and richness of skin microbiome in BS group were increased compared with that in AD group(P<0.05).Lefse analysis of different species between AD and BS groups showed that the dominant bacteria in AD group was Staphylococcus,while the dominant bacteria in BS group were Corynebacterium,Prevotella and Bacteroides.The results showed that the treatment group improved the skin inflammation in mice with atopic dermatitis by inhibiting Staphylococcus aureus USA300 and increasing the diversity of skin microorganisms.4.Complete genome sequencing of Bacillus subtilis TOS-90.The total genome length of Bacillus subtilis TOS-90 was 4.69 Mb,GC content was 45.94%,and 5870 coding genes were predicted.According to COG functional annotation results,3502 genes had clear protein functional classification.According to KEGG functional annotation,Bacillus subtilis TOS-90 possessed biosynthetic genes of streptomycin,siderophotic group non-ribosomal peptide,neomycin,vancomycin,cephalosporin,neomycin,kanamycin and gentamicin.According to CAZy results,57 carbohydrate active enzymes were annotated.422 signal peptide proteins were predicted by Signal P,and the proportion of signal protein was 7.19%.Anti SMASH database was used to predict gene clusters of secondary metabolites.A total of 18 secondary metabolite gene clusters were found,10 of which were related to antibiotic synthesis.In conclusion,in this study,human skin symbiotic bacteria were applied to atopic dermatitis in mice,effectively improving the skin symptoms of atopic dermatitis in mice,and providing a theoretical basis for microbial therapy for atopic dermatitis. |
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