| Objective:(1)The purpose of this study was to investigate the epidemiological characteristics of patients with acne vulgaris.(2)Using 16 s rRNA sequence to analyze the characteristics of dominant pathogenic microorganisms in acne vulgaris lesions,providing a rapid and accurate method for identification of dominant pathogenic bacteria.(3)This study aimed to analyze the difference in bacterial abundance and structure of the microflora in skin lesions of patients with acne vulgaris and sebum contents of healthy individuals by 16 s rRNA high-throughput sequencing technology.Methods:(1)Enroll 142 cases of acne vulgaris in the dermatology clinic and fill in the clinical information registration form for epidemiological investigation.The severity of each patient was recorded according to pillsbury classification.The age,first onset age,family history,occupation were also recorded.(2)The skin lesions of 80 patients with acne vulgaris were collected and inoculated in aerobic and anaerobic media respectively.The isolated strains were identified by biochemical identification,16 S rRNA amplification and sequencing techniques.(3)The skin lesions of 30 patients with acne vulgaris and sebum contents of 30 healthy individuals were collected according to high-throughput sequencing standards.Genomic DNA extraction,PCR amplification and library construction were performed to obtain optimized sequences and perform OUT classification,Alpha diversity analysis,Beta diversity analysis,differential species statistics and community structure analysis.Results:(1)Among 142 patients with acne vulgaris,31 cases(21.83%)were mild,83 cases(58.45%)were moderate,and 28 cases(19.72%)were severe according to the severity of acne vulgaris.There was no significant difference in the first onset age of acne patients with different genders and severity.(2)After Gram staining and biochemical reaction,68 strains of acne vulgaris were isolated and matched with P.acnes.After 16 S rRNA sequencing,62 strains of P.acnes were isolated from 80 patients with acne vulgaris.Detected in the lesions of patients with acne vulgaris is given priority to Cutibacterium acnes,accounted for 55%,followed by staphylococcus epidermidis and accounted for 21%,staphylococcus aureus accounted for 8%.Stenotrophomonas maltophilia 4%,yellow lipobacteria 4%,Bacillus subtilis 3%,Bacillus cereus 2%,Goat staphylococcus 1%,O.sphaeroides 1%,Bacillus licheniformis 1%.Biochemical identification and 16 S rRNA sequencing identification showed no significant difference in the positive rate of P.acnes(P>0.05).(3)The results of this experiment showed that there were 3811687 effective sequences from the skin lesions of 30 patients with acne vulgaris and sebum contents of 30 healthy individuals with an average length of 414.37 bp.At the phylum level,the highest proportion of microorganisms in sebum contents of healthy individuals was Proteobacteria,with relative abundance of 96.95%,followed by Firmicutes,Actinobacteria and Fusobacteria,with relative abundance of 2.21%,0.79%and 0.04% respectively.At the phylum level,in acne vulgaris patients,the highest proportion of microorganisms was Actinobacteria,with a relative abundance of 39.86%,followed by Proteobacteria,Firmicutes and Fusobacteria,with a relative abundance of27.29%,25.43% and 6.26% respectively.At the genus level,the highest proportion of microorganisms in sebum contents of healthy individuals was Stenotrophomonas,accounting for 96.83%,followed by Streptococcus,Staphylococcus and Cutibacterium,accounting for 1.2%,0.99% and 0.69% respectively.The highest proportion of microorganisms in acne vulgaris was Cutibacterium,accounting for 34.38%,followed by Stenotrophomonas,Staphylococcus and Cetobacterium,accounting for 18.52%,22.86%and 6.26% respectively.At the species level,the relative abundance percentage of Cutibacterium acnes,Cutibacterium granulosum and Serratia marcescens was 0.38%,0.27% and 0.06% respectively.The relative abundance percentages of Cutibacterium acnes in skin lesions of patients with acne vulgaris were 24.37%,and the relative abundance percentages of Peptoniphilus,Cutibacterium granulosumand and Serratia marcescens were4.54%,3.85% and 2.76% respectively.By T-test analysis among groups,the difference of between microorganisms in sebum contents of healthy individuals and acne vulgaris patients was statistically significant at the phylum level,including Proteobacteria,Actinobacteria,Firmicutes,Fusobacteria,Bacteroidetes(P<0.05).The difference of between microorganisms in two groups was statistically significant at the genus level,including Stenotrophomonas,Cutibacterium,Staphylococcus,Cetobacterium(P<0.05).The difference of between microorganisms in two groups was statistically significant at the species level,including Cutibacterium acnes,Cutibacterium granulosum,Sphingomonas leidyi Corynebacterium tuberculosis,Acinetobacter nosocomialis,Paenibacillus amylolyticus(P<0.05).Through Alpha diversity analysis,Observed species index,Chao index and ACE index reflecting the abundance had statistically significant differences in the groups between the patients with acne vulgaris and the healthy individuals(P<0.05).There was no significant difference in the Shannon index,Simpson index,and PD_whole_tree between the patients with acne vulgaris and the healthy individuals(P<0.05).Through Beta diversity analysis,Weighted Unifrac and Unweighted Unifrac were used as indicators to analyze the microbial dissimilarity index in sebum contents of healthy individuals and acne vulgaris patients.The results show the difference was not statistically significant(P>0.05).In the acne vulgaris group and the healthy individuals,two distinct clusters were formed in the Clustering diagram,and the abundance and species of the microorganisms were significantly different.Anosim analysis of community structure differences found that there was significant difference in community structure between acne vulgaris group and the healthy individuals.Conclusion:(1)According to the acne Pillsbury classification,moderate acne vulgaris is the most common acne vulgaris;there is no significant difference in the initial onset age of acne patients with different gender and severity.(2)16S rRNA sequencing technology and biochemical identification have high consistency in the identification of P.acnes,and 16 S rRNA sequencing technology can efficiently and accurately identify the dominant bacteria of acne lesions-Propionibacterium acnes.(3)High-throughput sequencing revealed that the difference of microorganisms in the acne vulgaris group and the healthy individuals was statistically significant at the species level including Cutibacterium acnes,Cutibacterium granulosum,Sphingomonas leidyi,Corynebacterium tuberculosis,Acinetobacter nosocomialis,Paenibacillus amylolyticus.These species may be key microorganisms that cause the onset of acne vulgaris.There were differences in bacteria abundance in the acne vulgaris group and the healthy individuals.There were no significant differences in equilibrium degree and diversity in acne vulgaris group and the healthy individuals.There were significant differences in community structure between acne vulgaris group and the healthy individuals... |
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