The Study On The Effect Of Cell-Free Supernatant From Lactobacillus Plantarum On Sporotrichosis In Vitro

Background and purposeSporotrichosis is a chronic granulomatous skin disease.For Chinese patients,the pathogen of this disease is Sporotrichum globosum.There are four clinical types of the disease,including localized skin type（fixed pattern）,skin lymphatic type,skin disseminated type and skin appearance type.Most patients diagnosed with sporotrichosis require 4 to 6 months of antifungal therapy.Due to the long treatment cycle of the disease,the large toxic and side effects of antifungal drugs and the increase of drug-resistant cases in recent years,the discovery of a new drug with broad spectrum,high efficiency and low side effects has become the focus of our current research.In recent years,more and more studies have shown that Lactobacillus plantarum has obvious inhibitory effects on Aspergillus,Candida,filamentous fungi and other fungi^[1][2][3].This paper studies whether the acellular supernatant of Lactobacillus plantarum can inhibit sporotrichosis,which lays a foundation for the future study of the mechanism of anti sporotrichosis of Lactobacillus plantarum and the development of new drugs for the treatment of sporotrichosis.Methods1.Three-point inoculation method:Firstly,sporohypha（mycelium phase and yeast phase）and Lactobacillus plantarum were activated.The activated Lactobacillus plantarum was scraped and diluted with normal saline and counted to obtain different concentrations of Lactobacillus plantarum suspension.All concentrations of Lactobacillus plantarum suspension were taken from 100ul and added to MRS liquid medium respectively.Them were cultured in an oscillator and centrifuged and filtered24 hours later,the acellular supernatant corresponding to each concentration was obtained.Lactobacillus plantarum CFS and PDA Agar medium were put into plate in the experimental group,while PDAAgar medium was used in the control group.Then the spore mycelium suspension was inoculated on the prepared plate,each plate was inoculated in three places,so that the distance between each inoculation point was2cm.The mycelial phase group and yeast phase group were cultured in 28℃constant temperature incubator and 37℃constant temperature incubator respectively.The colony growth was observed and recorded 7 days later.2.Observation under scanning electron microscope:in the three-point inoculation experiment,the colonies of the experimental group and the control group which were obviously inhibited compared with the control group were fixed in glutaraldehyde and observed under scanning electron microscope after conventional treatment.3.Observation under transmission electron microscope:in the three-point inoculation experiment,the colonies of the experimental group and the control group which were obviously inhibited compared with the control group were fixed in glutaraldehyde and observed under transmission electron microscopy after conventional treatment.Results1.Three-point inoculation method:no matter the mycelium phase or yeast phase,compared with the control group,the growth of sporomycetes was inhibited.The results of mycelial phase analysis showed that when the concentration of Lactobacillus plantarum CFS was in the range of 10⁹cfu/L～10¹¹cfu/L,the colony growth diameter decreased with the increase of Lactobacillus plantarum concentration,and there was no obvious colony formation when the concentration reached 10¹²cfu/L.When observing the growth of yeast phase,when the concentration of Lactobacillus plantarum CFS was in the range of 10⁶cfu/L-10⁹cfu/L,the higher the concentration of Lactobacillus plantarum,the stronger the inhibitory effect on the growth of sporomycetes.When the concentration reached 10⁹cfu/L,there was no obvious colony growth.2.Observation under scanning electron microscope:compared with the control group,the number and morphology of fungal spores and hyphae in the experimental group changed obviously and were destroyed in different degrees.3.Observation under transmission electron microscope:Compared with the control group,the cell wall and cell membrane of the experimental group were damaged,the cytoplasm was sparse and uneven,there was no complete vacuole structure,and some cytoplasmic contents leaked out.In the sporotrichosis yeast phase experimental group,the cell wall and cell membrane structure were destroyed,the cytoplasm shrank,the density increased,and there was no vacuole structure.Conclusions1.In the three-point inoculation experiment,the cell-free supernatant of Lactobacillus plantarum had a certain inhibitory effect on both hyphal phase and yeast phase,and the inhibitory effect was enhanced with the increase of concentration.2.The experiment of scanning electron microscope showed that the acellular supernatant of Lactobacillus plantarum could destroy the integrity of mycelium or yeast mycelium and reduce the germination of conidia.3.Under transmission electron microscope,it was found that the acellular supernatant of Lactobacillus plantarum could destroy the integrity of cell wall and cell membrane of spore mycelium or yeast phase,changing its cytoplasmic matrix..