The Identification And Molecular Biology Of Bacteria From PMH And The Study Of Its Pathogenesis

Background: Progressive macular hypomelanosis (PMH) is a common skin disorderthat is often misdiagnosed and is characterized by ill-defined nummular, hypopigmented,nonscaly macules on the front and back of the trunk, with confluence of the macules inand around the middle, There is no itch, pain, or preceding inflammation.Progressivemacular hypomelanosis of the trunk is firstly described by Guillet et al in people ofmixed racial (white and black) ancestry originating from the French Caribbean. Thisentity was also described or at least recognized independently by several investigatorsfrom different parts of the world.In China,it was reported using the following variety ofdescriptive terms:pityriasis versicolor kind hypopigmentation.There is currently lackof accepted diagnostic criteria, and the disease pathogenesis is still not clear,fourhypotheses have been offered:○1“melting” of genes.○2"genodermatosis"○3"Fungusinfection"○4Bacterial infection. Now, study shows a relation between the presence of Pacnes and the hypopigmented macules, but there is little study about the bacteria and itsrelationship between progressive macular hypomelanosis.Objective: To identify and analyze the nine isolates from the patients of progressivemacular hypomelanosis；To investigate the effects on the production of cytokinesIFN-γ、 IL-6、 TNF-α from Propionibacterium acnes in human keratinocytes(HaCaT cells). Methods: The biopsy specimens from lesional skin were cultured under aerobicconditions and anaerobic conditions, respectively. The bacteria was identified by ATBRapid ID32A system and16RsDNA sequencing and to establish its phylogenetic tree.Human keratinocytes (HaCaT cells) were cultured in vitro and co-cultured with theisolates from the patients progressive macular hypomelanosis and acnes and NCTC737.Then the supernatants were harvested after24h of the HaCat cells co-culture withthe bacteria, to analyze the changes of cytokines by enzyme-linked immunosorbentassay (ELISA).Results:1. Observed under wood lampLesions of35patients in41(85.4%)were observed the red fluorescent,all normal skinnot. In295patients with acne vulgaris, the red fluorescent was found in90cases ofance(90/195)and17cases of papular acne (17/183).2. Isolation and identification of bacteriaColony forming was only observed under anaerobic blood agar plate from the lesionsskin. The bacteria is a gram-positive, non-spore-forming, anaerobic bacteria and showsred fluorescent under wood lamp and is identified as Propionibacterium acnes with acommercial identification kit (Rapid ID32A). We also obtained9isolates from acnevulgaris. The bacterias from two groups were sequenced by16SrDNA gene sequencing.The sequencing results analyzed by Blast on NCBI, similarity is99%, and one isolateswas identified as Lysinibacillus, one was identified as Enterobacter.3.16sDNA sequencing analysis and phylogenetic tree16SrDNA gene sequencing was performed on eighteen isolates (from9acne and9PMH patient).Sixteen isolates (9from PMH groups and7from acnes groups) wereidentified as Propionibacterium acnes and one isolates was identified as Lysinibacillus,one was identified as Enterobacter. The phylogenetic tree was established about those isolates and Propionibacterium acnes Standard strains(DSM16379,NR040847.1) andPropionibacterium granulosum(DSM20700,NR025276.1).4. Changes of cytokines in co-cultured supernatantThe secretions of IFN-γ、 IL-6from the supernatants of PMH groups weresignificantly increased than that NCTC groups: IFN-γ(388.73±5.39vs259.35±11.44);IL-6(171.87±2.72vs152.87±5.39), had significant changes (P＜0.05).The secretions of IFN-γ、IL-6from the supernatants of PMH groups weresignificantly increased than that blank control groups:IFN-γ(388.73±5.39vs187.87±6.68), IL-6(171.87±2.72vs88.63±3.17), had significant changes (P＜0.05). The bacteria from PMH patients inhibited the secretion of TNF-α(104.11+13.27pg/ml), a blank control (124.02+7.4pg/ml), NCTC737groups (117.96+8.3pg/ml) decreased, but the difference was not statistically significant (P>0.05).Conclusion: The bacterial species cultured from the patients of PMH may bePropionibacterium acnes; The hypopigmentation may be related with the ability ofHaCaT cells stimulated by those bacterias.