Isolation And Identification Of A Bacteriophage Of Staphylococcus Aureus And Its Effect On Biofilm

Staphylococcus aureus is a common pathogen of dairy cow mastitis.Because of its strong ability to form a biofilm,commonly used antibacterial drugs have limited therapeutic effects,which greatly increases the treatment complications of Staphylococcus aureus dairy cow mastitis.Phage therapy can efficiently and quickly decompose defective bacteria,and it has become a new type of therapy to replace antibiotics and has attracted wide attention.However,related studies on the mechanism of action of Staphylococcus aureus bacteriophages on biofilms are relatively boring.Therefore,in-depth research on the biofilm formation ability of milk-derived Staphylococcus aureus and the bacteriostatic ability of bacteriophages,focusing on the analysis of the effect of bacteriophages on biofilms,is of great significance for revealing the mechanism of action of Staphylococcus aureus phage on biofilms.1.Use the crystal violet staining method to evaluate the biofilm formation ability of 55 strains of milk-derived Staphylococcus aureus,detect the biofilm formation related genes and drug resistance genes by PCR technology,and perform multilocus sequence(MLST)on drug resistant strains Subtype.The results showed that 34 strains(62%)had strong biofilm forming ability,17 strains(31%)had weak biofilm forming ability,and 4 strains(7%)had no biofilm forming ability.The detection rate of aap,Alt,Eno,Ica A and Ica D genes in strains with strong biofilm formation ability is relatively high.Among 55 strains of bacteria,17 strains(31%)detected mec A resistance genes,among which ST97(29%)and ST398(24%)were dominant strains.Bacteriophage have the ability to remove biofilms,which can destroy the structure of biofilms and remove bacteria in biofilm.2.In this study,a lytic phage of Staphylococcus aureus was isolated from cattle farm sewage and named it vB?Sau M?SDQ,and its biological characteristics and antibacterial effect were tested.The results show that the optimal multiplicity of infection of the phage is 0.01,the incubation period is 15 minutes,and the activity is stable under the conditions of lower than50? and p H 4.0?11.0,and the activity is stable in the presence of non-ionic and zwitterionic detergents.And it has obvious antibacterial effect on the bacteria in the culture medium and milk,suggesting that vB?Sau M?SDQ has good stability and antibacterial ability.3.Detect the changes in the number of bacteria in the biofilm after vB?Sau M?SDQ treatment by the plate coating method,and observe the changes in the biofilm after the phage treatment by fluorescence microscope and scanning electron microscope to verify the removal effect of the phage on the biofilm.The results show that vB?Sau M?SDQ can reduce the number of bacteria in biofilm and destroy the integrity of the biofilm.This indicates that vB?Sau M?SDQ can effectively remove the biofilm,suggesting that bacteriophages have great application potential in the prevention and control of bacterial biofilm.4.Analyze the bacterial gene expression changes in biofilm before and after vB?Sau M?SDQ treatment by RNA-Seq sequencing technology to explore the effect of bacteriophages on the biofilm.The sequencing results showed that a total of 1029 differentially expressed genes were obtained.GO enrichment analysis showed that differential genes were mainly enriched in biological processes such as ribosomal structural components,r RNA binding and protein synthesis.KEGG enrichment analysis shows that differential genes are distributed in the cell's protein synthesis,cell process and material metabolism pathways.The impact on protein synthesis is mainly manifested in the up-regulation of ribosome structure and RNA polymerase synthesis gene expression;The impact on cell processes is mainly manifested in the expression changes of enzyme synthesis genes during DNA replication,cell division and quorum sensing;The impact on material metabolism is mainly manifested in the expression changes of enzyme synthesis genes in carbohydrate metabolism,energy metabolism,and nucleic acid metabolism pathways.It shows that the bacterial protein synthesis,cell division,and metabolism in biofilm are accelerated after phage treatment,suggesting that the physiological state of the bacteria in biofilm becomes active after phage treatment,which destroys the stability of the biofilm structure.