| Mastitis in dairy cattle is a common and multiple disease in dairy farming,which not only brings serious economic losses to the dairy industry but also poses a potential threat to human health.Mastitis in dairy cattle is usually caused by one or more non-specific pathogenic microorganisms,of which Staphylococcus aureus is one of the most predominant pathogens.Staphylococcus aureus is highly susceptible to drug resistance,and its infected cows often show subclinical signs of mastitis,which is difficult to prevent and control.Antibiotics are the primary choice for the treatment of mastitis in dairy cows,but with increasing bacterial resistance,the treatment is not as effective as it could be.Also antibiotic residues in milk further aggravate the economic losses of dairy farming.Phages are a class of viruses that can specifically lysing bacteria and have the advantages of high efficiency and safety as biobiostatic agents.In this study,the infection and drug resistance of S.aureus in mastitis in dairy cows were analyzed by isolating pathogens from mastitis milk samples.The phage was also isolated from clinical isolates of S.aureus as host bacteria and classified by a combination of both structural and genetic aspects through morphological observation and genome sequencing.The biological characteristics and stress resistance of phages were also analyzed to further provide basic data for the research.Exploring the effect of bacteriophage on biofilms can provide a theoretical basis for the development of bacterial environmental inhibitory drugs.Finally,the mouse model of S.aureus mastitis was established,and treated with bacteriophages,exploring the therapeutic effect of bacteriophage on bacterial infection in vivo,which can provide new ideas for the treatment of S.aureus infection.In 2020,a total of 215 pathogenic strains were isolated from 408 clinical mastitis milk samples sent from 37 large-scale dairy farms in different regions of the country,including 19 strains of S.aureus（8.84%）.A total of 37 strains of S.aureus isolated from this experiment and previously conserved in the laboratory were tested for drug resistance by paper diffusion method.The results showed that S.aureus was more resistant to ampicillin,erythromycin and norfloxacin,and showed sensitivity to tetracycline and gentamicin,and 19 strains of S.aureus showed multi-drug resistance.Three phages were isolated from the sewage,named SP107,SP688 and SP160.As observed by transmission electron microscopy,all three phage strains had long and non-contractible tails and belonged to the Caudovirales,Siphoviridae.The biological properties（host spectrum,optimal infection complex,lysis kinetics）and resistance（temperature,p H and UV stability）were investigated.SP107 and SP688 could lyse 9 strains of Staphylococcus aureus,while SP160 could only lyse its host bacteria.The optimal infection complexes were 0.1,1 and 1,000,respectively.lysis kinetics showed that all three strains could inhibit bacterial growth.all three strains were stable in activity from-20°C to54°C,stable in activity between p H 3-11,and sensitive to UV light.Whole genome sequencing of three phages was performed using Illumina Genome Analyzer sequencing and coding gene prediction genome using Gene Mark S.The results showed that phages SP107,SP688 and SP160 were all circular DNA with whole genome length of 44,644 kb,45,499 kb,42,790 kb,and GC content of 33.92%,34.44%and 34.1%,respectively.ORFs with known functions in the SP107 and SP688 genomes were annotated equally,both of which contained no virulence genes and showed applied safety.The SP160genome has two different sequences encoding lyase and a stretch of hydrolase,containing virulence genes.Considering phage biology and application safety,SP107 phage was selected for application exploration.The ability of phages to clear S.aureus biofilms in vitro was explored by crystal violet staining,and the results showed that SP107 can clear the biofilm of 4 S.aureus strains.A murine mastitis model was successfully constructed using S.aureus 107 to identify10~7CFU/50μL as the optimal infection concentration.Through clinical and anatomical observation,histopathological section observation and detection of tissue bacterial quantity,to explore the therapeutic effect of phage SP107 in mouse mastitis model,the results showed that by removing bacteria from the breast tissue,the number of bacteria in the breast tissue was effectively reduced,reducing the pathological damage caused by the inflammatory response and facilitating the self-healing process of the breast tissue.In conclusion,this experiment obtained resistant S.aureus from production and isolated three phages;Its biological characteristics and whole genome sequence were analyzed,and data support was provided for its application;By analyzing the scavenging effect of bacteriophage on S.aureus biofilm in vitro,and applying it to the treatment of mouse mastitis model,it has laid a foundation for the treatment of S.aureus resistant strain infection. |
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