Metagenomic Sequencing Study Of The Effect Of ALA-PDT On Microbiome Of Pilosebaceous Units In Severe Acne

Objectives:ALA-PDT is commonly used to treat severe acne.However,there is no optimal treatment parameter and remains a paucity of data of the antimicrobial effects that result from ALA-PDT in vivo.We further explored the efficacy and safety of low intensity red light ALA-PDT combined with minimally invasive removal of the contents of skin lesions in the treatment of severe acne,and used metagenomic sequencing to compare the makeup,diversity,and function of the microbiota in pilosebaceous units of patients with severe acne before and after ALA-PDT.This study provides insight into the pilosebaceous unit microbiota in severe acne and how it is modulated by ALA-PDT,which provides the basis for revealing the pathogenesis of severe acne and the mechanism of ALA-PDT.Methods:A longitudinal cohort study was performed on 11 participants with severe facial acne.All patients were given 5%ALA-PDT combined with minimally invasive removal of the contents of skin lesions,short encapsulation time(90 min)and low intensity red light irradiation(power density 50mw/cm2 irradiation,total energy 60J/cm2)every two weeks for three sessions in total.The number of skin lesions at baseline and 2,4,6,12 weeks after treatment and side effects during the whole treatment procedure were recorded.TEWL and sebum values were measured at baseline and 12 weeks after treatment.At baseline and 2 weeks after the first treatment,the contents of pilosebaceous units of lesions on forehead and cheek were collected and DNA was extracted by magnetic bead method.Metagenomic sequencing was performed by Illumina platform to analyze and compare the microbiota makeup,diversity and functional gene characteristics before and after treatment.Results:1.Efficacy and safety analysisThe best efficacy was achieved at 12 weeks after the first treatment with an effective rate of 81.8%.The side effects were transient and the VAS pain score was 3-4 points.The TEWL of 7 patients showed a decreasing trend at 12 weeks after the first treatment,but there was no statistical difference.The sebum values decreased with statistical difference at 12 weeks after the first treatment.2.Metagenomic sequencing resultsThere was a diversity of microbiota in pilosebaceous units.Actinobacteria was the most abundant phylum,followed by Proteobacteria,Finnicutes and Bacteroidetes.The majority of bacterial abundance(94%)was from two phyla:Actinobacteria and Proteobacteria.At the genus level,the predominant generas were as follows:Cutibacterium,Pseudomonas and Staphylococcus.At the species level,the most common bacteria were C.acnes,followed by S.epidermidis and P.fluorescens.At the genus level,Cutibacterium was negatively correlated with other genera,such as Pseudomonas,Klebsiella,Brevundimonas,Ralstonia,Delftia and Cupriavidus.3.Characteristic analysis of functional genesThere was a diversity of functional genes in pilosebaceous units.Cutibacterium performed the highest contribution of functional genes,followed by Staphylococcus.Functional genes related to metabolism and genetic information played dominant roles.Functional genes related to the tricarboxylic acid cycle(TCA cycle)of aerobic metabolism accounted for a large proportion.Functional genes related to sialidase,lipase,poiphyrin metabolism and ABC transporter were commonly found in the samples.4.Changes of microbiota before and after ALA-PDTTreatment with ALA-PDT led to clinical improvements in acne severity concurrent with a significant reduction in the relative abundance of C.acnes,while P.fluorescein increased significantly after ALA-PDT.There were no significant changes identified in other species.ALA-PDT administration was associated with an increase in microbiota diversity(alpha diversity)and reductions in the abundance of the functional genes involved in energy metabolism and DNA replication,especially the sialidase,lipoidase,succinate dehydrogenase genes abundance dropped significantly.Conclusions1.Minimally invasive removal of the contents of skin lesions can promote the accumulation of ALA in pilosebaceous units,and pain is mild when exposed to low intensity red light.The overall efficacy and safety of this treatment method are good.2.There is significant diversity of microbiota and functional gene in pilosebaceous units of severe acne.C.acnes is dominant species in number and function,which suggesting that it maybe related to the pathogenesis of severe acne.The abundance of gram-negative bacteria may be related to the severity of acne.The finding of colonization of P.fluoresces in pilosebaceous units for the first time is worth further study.3.ALA-PDT may play a therapeutic role by killing C.acnes,increasing P.fluorescens and the microbiota diversity,and inhibiting the function and virulence factor of microbiota,while reconstructing the balance of microbiome in pilosebaceous units of severe acne.