| Staphylococcus aureus,Gram-positive bacteria of Staphylococcus,causing food poisoning,endocarditis,staphylococcal scald skin syndrome and toxic shock syndrome,is a common zoonotic pathogen.At present,antibiotics are mainly used to treat the disease caused by Staphylococcus aureus,but the abuse of antibiotics has led to the development of antibiotic resistant Staphylococcus aureus,which brings difficulties in clinical treatment.In this study,five Staphylococcus aureus phages were isolated from feces of swine by the method of phage enrichment,named 4086-1,4086-2,4086-3,4086-4 and 4086-6,respectively.Five Staphylococcus aureus virulent phages belong to Podoviridae family of Caudovirales,characterized by icosahedral head and a non-contracted short tail under the electron microscopy.In addition,the result of restriction enzymes(Eco R I,Hpa I,Rsr II,Sac I,Sph I,Bgl II,etc.)on five phage genome indicated that they belonged to differernt genus.The results of the host range showed that 4086-1,4086-2,and 4086-3 could infect S.aureus CVCC 546,S.aureus 4086,S.aureus Ben,S.aureus ATCC 43300,however 4086-4 and 4086-6 could only lyse S.aureus CVCC546,S.aureus 4086.The results of one-step growth curve indicated that the latent period of phage4086-1,4086-2,4086-3,4086-4 and 4086-6 were 10 min,10 min,20 min,20 min and 10 min respectively,and the burst period were 90 min,100 min,80 min,80 min and 90 min respectively,and the burst size were 365.7 PFU/infected cell,400 PFU/infected cell,19.4 PFU/infected cell,149.4 PFU/infected cell and 52.4 PFU/infected cell respectively.The results of analyzing the effects of different physical and chemical factors on phages showed that phage 4086-1,4086-2,4086-3,4086-4 and 4086-6 were all sensitive to heat,and five phages could survive stably in the range of-20-42?,but inactivate completely above 62? for 40 min and75% ethanol for 30 min.The five phages survived stably in the environment of p H 4-10,which indicated that the phages had strong acid-alkali tolerance.After treatment with 80% acetone for 30 min,the titers of phage 4086-1,4086-2,4086-3,4086-4,and 4086-6 were decreased by 1,1,1,1.5and 2 orders of magnitude respectively,but not sensitive to DMSO and chloroform.The results of the effects of different detergents on the bacteriophage indicated that all five phages were inactivated completely after treatment with 0.1% SDS for 30 minutes,however,there was no significant difference in the titers before and after treatment with 0.1% Sarkosyl,0.1% CTAB and0.1% benzalkonium bromide for 30 minutes,respectively.The effect of osmotic pressure on theactivity of bacteriophages was determined.The results showed that the titers of five bacteriophages had no significant difference,as the concentration of sodium chloride in the solution increased.It indicated that the five bacteriophages had certain tolerance to osmotic pressure.The results of the effects of divalent ion(calcium and magnesium ions)on the bacteriophage suggested that 4086-1and 4086-3 had the highest titer in LB medium supplemented with calcium ions,4086-2 and4086-6 had the highest titer in LB medium added with calcium ion and magnesium ion at the same time,4086-4 had the highest titer in the LB medium supplemented with magnesium ions.The ability of lysing bacteria of the phages was observed by ananlyzing the dynamic changes of S.aureus after bacteriophage infection.The results showed that the number of S.aureus significantly decreased after infecting by different MOI(MOI 0.01,0.1,1.0,10)bacteriophages.The number of S.aureus increased after after infection for 8 hours,which indicated that some bacteriophage-resistant Staphylococcus aureus were developed.The effect of phages on the formation of S.aureus ATCC 43300 biofilm were analyzed.The results showed that phage 4086-1 had the strongest ability to scavenge biofilm.The mastitis in mouse model was established using S.aureus ATCC 43300(methicillin-resistant Staphylococcus aureus).After treatmentthe with bacteriophage,the results showed that the number of bacteria in breast tissue and the inflammatory infiltration in acinus of breast tissue were significantly decreased.The levels of cytokines in breast tissue were detected by ELISA.The results indicated that the levels of TNF-? and IL-6 decreased significantly after treatment with bacteriophage and antibiotic compared to control groups.Moreover,the levels of pro-inflammatory factors were significantly lower in bacteriophage treatment group than those in the antibiotic treatment group.These results indicated that the inflammatory responses could be effectively alleviated after bacteriophage treatment.In conclusion,five S.aureus phages were isolated and their biological characteristics were identified in this study,which provided important experimental data for their application.By analyzing the effect of bacteriophage on S.aureus biofilm in vitro and treating murine mastitis,it laid a foundation for further treating the infections of drug-resistant S.aureus. |
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