Effect Of The Fermentaion Broth Of Eurotium Cristatum On Staphylococcus Aureus And Its Biofilm

Staphylococcus aureus(S.aureus)biofilm related infection has been one of the clinical problems.It is extremely important to find a safe and non-toxic substance which can effectively inhibit the growth of S.aureus and its biofilm to prevent the formation of S.aureus biofilm and treat biofilm related infection.In this study,S.aureus isolated from clinic was used as experimental strain,strong biofilm producing strain was screened out,and the resting model of S.aureus biofilm in vitro was established.The fermentation broth of Eurotium cristatum(E.cristatum)was used as the object of study to explore its inhibitory effect on S.aureus and its intervention effect on biofilm.The purpose of this study is to provide clues and evidence for finding new drugs to treat S.aureus biofilm associated infections.PART ONE: IDENTIFICATION OF BIOFILM FORMATION ABILITY OF S.AUREUS AND ESTABLISHMENT OF ITS MODEL IN VITROIn this part,58 clinical isolates of S.aureus were used as the experimental objects,and the biofilm forming ability of the strains wasqualitatively and quantitatively analyzed by Congo red plate method and crystal violet semi-quantitative method.Then,the biofilm model was established by using the strong biofilm producing strain.Crystal violet staining,scanning electron microscopy(SEM)and the formation curve of biofilm were used to observe the formation and morphology of biofilm.The results showed that the positive rate of biofilm of 58 clinical isolates of S.aureus was 51.72%.Among them,5 were strong biofilm-producing strains,25 were weak biofilm-forming strains and 28 were no biofilm-forming strains.The biofilm model of S.aureus could be successfully cultured in vitro in 6-well plate with cover glass as carrier,and the strong biofilm-producing strain 2280 could form a mature biofilm after72 hours of culture.Crystal violet staining showed that the mature biofilm was connected in lamellar shape with a certain thickness and a small gap between cells.Under SEM,the mature biofilm adhered to the surface of the carrier in large numbers,and the extracellular matrix formed a membrane to wrap the bacteria.Based on the results of crystal violet staining,SEM and the growth curve of biofilm,we found that the formation of biofilm could be divided into five stages: reversible attachment stage(0-12 h),irreversible adhesion stage(12-24 h),mutual adhesion stage(24-48 h),biofilm maturation stage(48-72 h)and biofilm dispersal stage(> 72 h).PART TWO: THE EFFECT OF THE FERMENTATION BROTH OF E.CRISTATUM ON S.AUREUSIn this part,the sensitivity of the test strains to commonly used antimicrobial agents was determined by automatic drug susceptibility analysis system,and the inhibition effect of the fermentation broth on S.aureus was determined by disk diffusion method and tube butterfly method.The minimal inhibitory concentration(MIC)and minimal bactericidal concentration(MBC)of broth and antibiotics against S.aureus 2280 were determined by broth dilution and plate method.The results showed that the positive bacterial biofilm producing strains showed higher resistance rate to antibiotics,and the strength of the biofilm producing strains was related to the type of drug resistance.The stronger the biofilm producing strains were,the more kinds of drug resistance they had,and the difference was significant(P=0.008).Of the 5 strains of S.aureus with strong biofilm production,4 strains were resistant to more than 6kinds of drug.The results of disc diffusion test showed that except penicillin,the diameter of inhibition zone of biofilm positive bacteria to clindamycin,azithromycin,oxacillin and fermentation broth was smaller than that of biofilm negative bacteria.The diameter of inhibition zone of biofilm positive bacteria to fermentation broth of E.cristatum was 8.77mm,and the diameter of inhibition zone of biofilm negative bacteria to fermentation broth was 11.2mm.The results of tube-butterfly test showed that the diameter of inhibition zone of fermentation broth was 12.87 mm and 15.72 mm for biofilm positive and biofilm negative strains respectively.The disc diffusion method and the tube butterfly method showed similar experimental results:the fermentation broth of E.cristatum could form the inhibition zone around the S.aureus strain.The MIC and MBC of the fermentation broth against S.aureus 2280 were 0.625mg/ml and 2.5mg/ml,respectively.This part of the study shows that the fermentation broth has a certain inhibitory effect on S.aureus.PART THREE: EFFECTS OF FERMENTATION BROTH OF E.CRISTATUM ON BIOFILM OF S.AUREUSIn the early and mature stages of biofilm formation of S.aureus 2280,different concentrations of fermentation broth of E.cristatum were added to intervene,and crystal violet adhesion test and fluorescence microscopy were used to observe the biofilm formation after intervention.The results showed that the fermentation broth inhibited the formation of biofilm of S.aureus at early stage,when the concentration of fermentation broth was higher than 1 MIC with the adhesive capacity of biofilm in 96-well plate decreased sharply.The inhibitory effect of fermentation broth on biofilm of S.aureus at early stage increased with the increase of the concentration of fermentation broth,when the concentration of fermentation broth was higher than 4 MIC,the inhibition effect of fermentation broth on biofilm of S.aureus at early stage increased with the increase of the concentration of fermentation broth.A bacterial biofilm was formed on the surface of the cover slide carrier.For mature S.aureus biofilm,the elimination of fermentation broth was not obvious.The fluorescence images showed that the number of dead bacteria in biofilm increased with the increasing concentration of fermentation broth(1/4MIC~ 16MIC).When the drug concentration reached 16 MIC,although a large number of bacteria died,most of the biofilm structure was still intact.The results suggested that the fermentation broth of E.cristatum had the best inhibitory effect on biofilm formation in the early stage of biofilm formation and the metabolites of fermentation broth of E.cristatum had the potential to be developed as a drug for the treatment of S.aureus infection.In this study,the effects of the fermentation broth of E.cristatum on S.aureus and its biofilm were preliminarily studied,which provided a theoreticalbasis for the treatment of S.aureus infection by the fermentation broth,and provided a new idea for the application and development of the fermentation broth in the field of medicine and pharmacy.