| Background and Objectives:Acne vulgaris is a common skin disease.It usually causes acne rash,local skin inflammatory,resulting in redness,swelling,itching and pain.After healing,acne vulgaris scars are left which are difficult to repair,leading to changes in the appearance of the skin and affecting the quality of life.One of the main causes of acne vulgaris is the infection of P.acne,which is a common bacterial community that lives on the surface of human skin.Acne vulgaris from Multidrug-resistant P.acne can be more severe because they are more resistant to antibiotics,which can make treatment more difficult,lead to longer treatment times and incur more health care costs.In addition,it may pose a more serious public health and psychological threat,as acne vulgaris is disfiguring and prone to relapse,which may affect the patient’s social interaction,ability to learn and work,and affect the patient’s mental health.Therefore,treating acne vulgaris caused by multidrug-resistant P.acne is essential to improve patients’ quality of life.With the increasingly serious problem of antibiotic resistance worldwide,bacteriophage(phage)therapy has been paid more attention again.Phages are organisms that target lytic bacteria without affecting mammalian cells,and their lytic bacteria are not affected by drug-resistance genes.In this paper,a new phage was isolated and purified by sewage co-culture method.By studying its biological characteristics and genome information,and analyzing its curative effect in acne vulgaris rat model,we aim to further study the application of phage therapy for acne vulgaris.Methods:1.Biological characteristics analysis of phage.(1)Specific phages were screened by sewage co-culture methodPropionibacterium acnes were collected from the faces of multiple acne patients for bacterial culture identification,drug resistance gene analysis and drug sensitivity test,and the host bacterium Pacne11-13 was obtained.Pacne11-13 was co-cultured with host bacteria from untreated sewage,and specific phages were screened by double-layer AGAR plate method.(2)Morphology of phage under electron microscopeThe coarse particle morphology of phage φPa P11-13 was observed and photographed by transmission electron microscopy.The size of phage particles was measured using image-Pro Plus6.0 image analysis software.(3)General biological characteristics of phage φPa P11-13Optimal MOI value: the number of phages/number of bacteria =10.000,1.0000,0.1000,0.0100,0.0010,0.0001 were added into the test tube,and the titer of phages was measured by drip method after 24 hours of co-culture.The optimal complex number of infection was the MOI value at the highest titer of phage.Optimal adsorption time: host bacteria and bacteriophage solution were mixed and placed in a low oxygen incubator,and incubated for 5minutes,10 minutes and 15 minutes,respectively.The optimum adsorption time was when the phage titer was the lowest.One-step growth curve measurement: the titer was measured by drip method after the host bacteria and phage solution were co-cultured for 0 h,1 h,2 h,3h,4 h,5 h,6 h,7 h,8 h,9 h,10 h,11 h and 12 h.Analyze and plot one-step growth curves using Graph Pad 9.0.0.(4)Stability determination of phage φPa P11-13Uv stability: Take a sterile 16-well plate and add 300 u L pnagus supernatant to 3 Wells,which can just cover the bottom of the hole.The transparent cover of 16-well plate was removed and placed under ultraviolet lamp.The titer of each group of phages was determined by spotting method.Temperature stability: Host bacteria were mixed with phage solution and incubated in bath at 37℃,50℃,60℃,70℃,80℃ and 90℃ for 1 hour.Then,the titer of phage φPa P11-13 was measured by drop method,and the temperature stability curve of phageφPa P11-13 was drawn.Determination of p H stability: placed host bacteria and phages liquid after blending in p H is 1,2,3,4,5,6,7,8,9,10,11,12,13 EP tube,and then use drop method was developed for the determination of phage phi Pa P11-13 degrees.Chloroform stability: host bacteria and phage solution were mixed and placed in 10 u L,20 u L,50 u L chloroform.(5)Host spectrum determination of phage φPa P11-13(6)Sixteen strains of Propionibacterium acnes were selected as host bacteria,and the formation of plaque on the plate was observed by double-layer AGAR plate method.If no plaque appeared,it was considered that phage φPa P11-13 could not lysate the strain.If transparent or turbidized plaque appeared,it was considered that φPa P11-13 could lyse the strain.(6)Identification of phage φPa P11-13 proteinIsolation of phage proteins using SDS-PAGE and analysis of phage proteins using LCMS/MS system(liquid chromatography-tandem mass spectrometry).2.Whole genome sequencing of phage φPa P11-13(1)Genome sequencing of phage φPa P11-13Illumina Nova Seq platform was used to sequence the whole genome of phage φPa P11-13,including sample quality inspection,library construction,gene sequencing and base reading,original data analysis and processing,genome sequence splice,etc.(2)Gene information analysis of phage φPa P11-13Genome-wide results of phage φPa P11-13 were analyzed,and Gene Mark S,RAST and PHASTER were used to predict the open reading frame.ARDB and VFDB databases are used to predict virulence factors and pathogenic genes.Finally,collinearity analysis and phylogenetic tree construction were carried out.3.Establish an acne model to verify its antibacterial effect in vivo(1)Establishment of acne rat modelThe rat model was established by injecting host bacteria Pacne11-13 into the ear of rats.The rats were randomly divided into acne model group,antibiotic administration group,and phage administration group.There were 2 control groups(phage control group)and blank control group,with 5 SD rats in each group.(2)Therapeutic effect experimentEar bacteria load measurement: The rat ear tissue was cut into pieces with surgical scissors and put into test tubes.After homogenization,plate counting method was used to count the number of bacteria.HE staining was used to evaluate the degree of inflammation:After staining mouse ear tissues,optical microscopy was used for observation,tissue thickness was measured using a scale,and inflammatory cells were counted using Image J software.Results:1.Isolation of a new lytic phage,φPa P11-13,infecting P.acne,was isolated from the sewage management center of Xinqiao Hospital.φPa P11-13 can form transparent plaque with diameters of 1.0～5.0 mm on the double-layer agar plate,indicating a robust lytic ability against its host.Transmission electron microscopy(TEM)revealed that phage φPa P11-13 belongs to the family Siphoviridae(head diameter 60 ± 4.5 nm,tail length 170 ± 6.4 nm,tail width 14 ± 2.4 nm).The one-step growth curve showed the incubation period of phageφPa P11-13 was 5 hours,and the burst size was 26 PFU(plaque-forming unit)/cell.Moreover,it exhibited tolerance over a broad range of p H and temperature ranges but was utterly inactivated by ultraviolet(UV)irradiation for one hour.2.Whole-genome of phage φPa P11-13 and analyzed the genome information.The whole-genome sequencing results revealed φPa P11-13 had a linear ds DNA with 29,648 bp length.The G/C content was 54.08%.Non-coding RNA genes and virulence factors were not found.45 open reading frames(ORFs)were identified after online annotation.The annotation information of phage φPa P11-13 was uploaded to Gen Bank and the accession number:ON557706.The phylogenetic tree was constructed by amino acid sequence of terminal large subunit protein.The results further indicated that phage φPa P11-13 belonged to the Sipoviridae family.The characterization and genomic analysis of φPa P11-13 will develop our understanding of phage biology and diversity and provide a potential arsenal for controlling antibiotics-resistant P.acne-induced severe acne vulgaris.3.Effectiveness evaluation of φPa P11-13 against multi-drug resistant P.acne-induced acne vulgaris in rat model.Multidrug-resistant P.acne was used to establish an animal model of acne vulgaris.After treatment with specific phage φPa P11-13,the inflammatory cells,bacterial load and ear thickness of rats were significantly reduced and grateful therapeutic effect was obtained.In the course of Multidrug-resistant P.acne infection,the application of phage has a good effect on reducing inflammation.Conclusion:1.According to the isolation,identification and biological characteristics analysis of phage φPa P11-13,it was confirmed that phage φPa P11-13 was a new strain of Propionibacterium acnes,belonging to the Sipoviridae family.It has good temperature and acid-base stability,strong cracking ability,wide host spectrum,and can effectively crack multi-drug resistant Propionibacterium acnes.2.Through gene sequencing and gene annotation of phage φPa P11-13,we have a deeper understanding of its genetic information.The phage has good safety,its genome does not contain virulence factors and resistance genes,and its discovery enriches the phage library against Propionibacterium acnes.Through collinear analysis and phylogenetic tree construction,phage φPa P11-13 was further confirmed as a member of the Sipoviridae family,which provided a theoretical basis for the subsequent experiments of our research group.3.Animal experiments have proved that phage φPa P11-13 can effectively inhibit the reproduction of pathogenic bacteria and has a clear therapeutic effect on acne rats.It provides some ideas for clinical exploration of acne caused by multidrug resistant Propionibacterium acnes.In addition,it could help researchers better understand the pathogenesis of acne,so that patients can develop more effective treatments. |
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