Screening,identification And Evaluation Of Biocontrol Yeasts Against Methicillin-resistant Staphylococcus Aureus

Staphylococcus aureus is both a food-borne pathogen and an opportunistic pathogen that can colonize multiple parts of the human body and cause host infection.The methicillin-resistant Staphylococcus aureus(MRSA)has drug resistance making it more difficult to cure,and has become an important pathogen in hospitals and communities.The application of probiotics and their metabolites to prevent and control pathogenic bacteria has the advantages of low cost and reduced use of antibiotics,which has broad development and application prospects.At present,the main types of probiotics on the market are lactic acid bacteria,bacillus and yeast,but there are relatively few probiotic yeasts available compared to the other two.In order to exploit the resources of yeast and effectively prevent and control MRSA,this paper intends to screen the yeasts with anti-MRSA ability from fruits and fermented foods.The following are the main research contents and results:(1)Screening of anti-MRSA yeast and in vitro evaluation of probiotic properties of yeast.Yeasts were isolated and purified from fermented foods and fruits by selective medium,and then the yeasts with inhibitory ability to MRSA were screened from those yeasts.Finally,15 strains of yeast with anti-MRSA ability were screened from haw,grape and yellow physalis,among these strains,8 of these strains belonged to the genus Starmerella and 7 belonged to the genus of Hanseniaspora.S.bacillaris CC-PT4isolated from grape peel was found to have promising after comprehensive evaluation.The strain was tolerant to 37℃,acid,0.3%bile salts,simulated gastric fluid and simulated intestinal fluid,and had good self-aggregation ability and hydrophobicity.It was sensitive to amphotericin B,showingα-hemolysis but notβ-hemolysis.(2)Inhibitory effect of S.bacillaris CC-PT4 on MRSA.Plate streaking method and 96-well microplate method were used to test the inhibitory effect of S.bacillaris CC-PT4 on MRSA growth.The ability of S.bacillaris CC-PT4 to inhibit the formation of MRSA biofilm and to remove the formed biofilm was observed by crystal violet staining and fluorescence microscopy.The mechanism of S.bacillaris CC-PT4 on MRSA biofilm was explored by analyzing biofilm extracellular polymeric substances(EPS)and biofilm-related genes.The results showed that the cell-free supernatant(CFS)of S.bacillaris CC-PT4 had an inhibitory effect on the growth of MRSA,and the inhibitory effect enhanced with increasing fermentation time.CFS can also inhibit the formation of MRSA biofilm and has remove effect on the formed biofilm of MRSA.The RT-q PCR analysis shows that CFS could down-regulate the expression of biofilm-related genes srt A,ica A and sar A,thereby achieving the effect of inhibiting MRSA biofilm.(3)The CFS of S.bacillaris CC-PT4 was analyzed by untargeted metabolomics.Metabolites in S.bacillaris CC-PT4 CFS were detected by UHPLC-Q-TOF MS.The results showed that 30.872%of the metabolites identified in CFS were organic acids and derivatives.The significantly accumulated metabolites p-coumaric acid,hydroxyphenyllactic acid,DL-Indole-3-lactic acid and L-carvone have antibacterial or anti-biofilm abilities.The inhibitory effect of S.bacillaris CC-PT4 on MRSA is the result of the synergistic effect of various antibacterial substances in CFS.In addition,the inhibitory effect of 2-amino-1-phenylethanol on MRSA was verified by molecular docking and antibacterial and anti-biofilm experiments,which has not been reported before.(4)Whole-genome sequencing and analysis of the S.bacillaris CC-PT4 genome.The whole genome of S.bacillaris CC-PT4 was sequenced based on Illumina Nov Seq and Pac Bio Sequel sequencing platforms and bioinformatic analysis was performed.The results showed that the genome size of S.bacillaris CC-PT4 was 9.45 Mb,the GC content was 39.50%,and it contained 4510 genes and 2 secondary metabolite biosynthetic gene clusters.S.bacillaris CC-PT4 genome contains the genes associated with acid tolerance,such as F1F0-ATPase,cation/H(+)antiporter,Na(+)/H(+)antiporter,and proton ATPases and related subunits.This strain also has the genes encoding ATP-dependent bile acid permease and MFS antiporter,which are related to bile salt tolerance.In addition,the strain contains genes encoding heat shock proteins and genes related to oxidative stress,cell wall damage,DNA repair,and general stress response proteins,as well as flocculation and adhesion-related protein-encoding genes,which are help strain adapt to complex surroundings.In addition,the genome of S.bacillaris CC-PT4 may have genes encoding lysostaphin and killer toxin.There are 20genes associated with human diseases annotated by the pathogen host interactions(PHI)database and the database of fungal virulence factors(DFVF),but the sequences identity are less than 41%.S.bacillaris CC-PT4 also has genes associated with drug resistance,especially resistance to azoles.The results of previous antifungal susceptibility experiments also showed that S.bacillaris CC-PT4 was resistant to itraconazole and fluconazole,but sensitive to amphotericin B.The combined analysis of the genome and metabolites found many common metabolic pathways,involving163 metabolites and 1100 genes.Some functional metabolites in CFS,geneticin,irinotecan,citalopram,and tetrahydrofolate,were successfully associated with genes through common metabolic pathways,which further confirmed the possibility of these metabolites being synthesized.(5)Evaluation of the oral safety of S.bacillaris CC-PT4.The BALB/c mice were given a daily oral dose of 200μL 5×10~9 CFU/m L of S.bacillaris CC-PT4 for 28consecutive days to explore the effect of S.bacillaris CC-PT4 on mice according to the indicators of organ index,blood routine examination,blood biochemistry and intestinal flora.The results showed that S.bacillaris CC-PT4 had no significant effect on the organ index,blood routine and blood biochemistry of the mice.Through the analysis of intestinal flora,it was found that gavage of S.bacillaris CC-PT4 decreased the number of OTU of intestinal bacteria and intestinal fungi in mice.LEf Se analysis found that S.bacillaris CC-PT4 could significantly increase the relative abundance of Arthrobacter,Bifidobacteriales,Alistipes,Lactobacilllus,Pediococcus,Weissella,Desulfovibrio,Acinetobacter and Alkanindiges in bacterial flora.The LEf Se analysis of the intestinal fungal flora found that class Saccharomycetes and order Saccharomycetals were significantly enriched.