Preliminary Study Of Staphylococcus Aureus Phage On Mouse Mastitis

Staphylococcus aureus(S.aureus)is a common pathogen,which can cause the cow clinical and subclinical mastitis.However,due to the increasing resistance of S.aureus to antibiotics,especially the emergence of multi-drug resistant bacteria such as methicillin-resistant S.aureus(MRSA),has made the treatment of cow mastitis become increasingly difficult.Therefore,it is imperative to find a new antibacterial therapy with high efficiency and no drug-resistance.Bacteriophage(phage),as a bacterial virus that can infect and lyse bacteria specifically,is able to eliminate specific pathogens without affecting the microbial flora balance in human or animal bodies,showing their unique antibacterial potential.Thus,in this work,S.aureus was isolated and identified from the milk of cows with mastitis,as well as the S.aureus phage was isolated and characterized using S.aureus as the indicator bacteria.Finally phage cocktail was used to the treat S.aureus infection at the cellular and mouse level.In order to isolate and identify S.aureus from milk,13 strains of S.aureus were successfully identified based on the growth characteristics of the S.aureus on blood agar plate,BP plate and the PCR amplification of specific Staphylococcus gene 16S r RNA.Through the detection of drug resistance genes and virulence genes of S.aureus,it was found that most of the S.aureus isolated from milk showed multi-drug resistance and carried different amounts of virulence genes.Further,four strains with wide drug resistance and strong virulence were selected to verify their toxic effect on cells.Through the cytotoxicity experiment of S.aureus on bovine mammary epithelial cells,it was found that these four strains of S.aureus caused different degree of cell damage.To isolate S.aureus phages,the strategy to prepare phages from the environment in which cows live were applied.Three strains of S.aureus phages were isolated from the environment sample using the double-layer agar culture method,which were named as v B?Sau M?YNP1(YNP1),v B?Sau M?FLP8(FLP8),and v B?Sau P?PNP1(PNP1),respectively.All three phages can form round,transparent and smooth edge plaque with indicator bacteria.The titers of phages YNP1,FLP8 and PNP1 were more than 3.5×10⁹ PFU/m L,8.4×10⁸PFU/m L and 2.0×10¹¹PFU/m L,respectively.Through 120 KV transmission electron microscopy,the morphology of phage YNP1 and FLP8 was similar,with icosahedral head and contractitive tail,belonging to Myoviridae.The phage YNP1 has a head with diameter of about 100 nm and a tail with length of about 125 nm.The head diameter of phage FLP8 is about 82 nm and the tail length is about 143 nm.Phage PNP1 has an icosahedral head with a diameter of about 82 nm and a non-contractitive tail with a shorter tail of about 25 nm,belonging to the Podoviridae family.To explore the biological characteristics of these phages,the physicochemical characteristics of YNP1,FLP8 and PNP1 were analyzed through double-layer agar plate method and spot test method.The results showed that phage PNP1 had a relatively wide split spectrum and could lyse 12 strains of S.aureus.YNP1 phage showed strong cleavage activity against 7 strains of S.aureus,while YNP1 phage could also lyse 1 strain of multi-drug resistant Staphylococcus gallinarum isolated from milk.Phage FLP8 has the narrowest cleavage spectrum and can only lyse its host bacteria SH-5.Phages YNP1,FLP8 and PNP1 all have strong thermal stability and wide p H tolerance range.In addition,the phages YNP1,FLP8and PNP1 were not sensitive to chloroform.After chloroform treatment at different concentrations,the three phages still maintained high titers.The effect of UV on the activity of YNP1 and FLP8 was not significant.After 1hour of UV irradiation,the change of titer was not significant,while PNP1was very sensitive to UV.After 1 hour of UV irradiation,the titer decreased by 6 orders of magnitude.The optimal Multiplicity of infection(MOI)of phage YNP1,FLP8,and PNP1 were 1,1,and 0.01,respectively.One-step growth kinetics of the phage showed that the latent period of YNP1,FLP8 and PNP1 was 10 min;and the rise period of YNP1,FLP8and PNP1 was 50 min,40 min and 110 min,respectively.The burst size of YNP1,FLP8 and PNP1 were 71,153 and 200,respectively.To elucidate the potential of phage cocktail,the bacteriolytic experiments in vitro and phage cocktail blocking experiments on bacterial infected bovine mammary epithelial cells(MAC-T)were performed.The results showed that phage cocktail had a strong cleavage effect on multi-drug resistant S.aureus,and the mutant strains resistant to phage produced the least.In the blocking experiment of bovine mammary epithelial cells infected by bacteria,phage cocktail can effectively resist the invasion of MAC-T by S.aureus and inhibit the inflammatory response of cells.In order to explore the bactericidal experiment,the mouse mastitis model was established by multi-drug resistant S.aureus JY-2.After phage treatment,the bacterial count results showed that the number of bacteria in the mammary glands of mice significantly decreased.The pathological results showed that phage cocktail can effectively improve the damage of bacteria to breast tissue after treatment.The results of ELISA on the cytokine IL-6 showed that after phage cocktail treatment,the expression level of IL-6 decreased significantly and the inflammatory response was effectively alleviated.Here,we prepared phage cocktail using isolated phages,and verified its effective bactericidal ability at the cell level and in mouse models.The result provided a reference for the treatment of drug-resistant bacterial caused bovine mastitis.