| Staphylococcus aureus is one of the most common pathogens and the etiological agent of a variety of diseases in humans and animals.S.aureus can cause necrotizing pneumonia in rabbits,resulting in high mortality in farmed rabbits and leading to large economic losses during the course of rabbit production.Although some antibiotics have been confirmed to have a certain therapeutic effect on rabbit necrotizing pneumonia,with the extensive use of antibiotics,S.aureus has become increasingly resistant to an increasing number of antimicrobial agents.Effective methods for treating S.aureus diseases are becoming limited.Therefore,there is an urgent need to develop new therapeutic drugs and treatments.Phage therapy offers a promising alternative to combat staphylococcal infections and shows potential as an alternative to traditional antibiotic therapy.A single phage particle can be parasitized to a specific bacterial species.Phages multiply by infecting bacteria,and finally release the progeny of the phage to lyse the bacteria.An increasing number of studies have shown that phages can kill pathogens in animals,reflecting the therapeutic potential of phages.However,to our knowledge,phage therapy has not been used to treat and prevent the rabbit necrotizing pneumonia caused by S.aureus.To investigate whether phage has a therapeutic effect on rabbit necrotizing pneumonia caused by S.aureus,a serious studies were carried out in this paper.Firstly,we isolated and identified many strains of S.aureus from the lungs of the rabbits suffering from pneumonia.The drug susceptibility test results showed that most of these strains showed different degrees of drug resistance profiles.They were resistant to benzylpenicillin,erythromycin,clindamycin and sand star antibiotics.In addition,all these strains were sensitive to linezolid.Then,a novel phage named VB-Sav M-JYL01 was isolated from the sewage samples by using S.aureus S6 which isolated from rabbit lungs as a host.And the general biological characteristic of the phage was determined.The phage VB-Sav M-JYL01 forms circular translucent plaques(1-2 mm)on TSB plates that contain lawns of S.aureus S6.In addition,it exhibited an icosahedral head and aretractable tail under TEM,with the diameter of the head measuring approximately 90 ± 10 nm and the length of the tail measuring approximately 190 ± 10 nm which indicated that the phage VB-Sav M-JYL01 belonged to the Myoviridae family.The phage titers were highest when the MOI was 0.01,reaching approximately 9.8 × 109 PFU/m L.The one-step growth curve of the phage VB-Sav M-JYL01 showed the latent period of this curve was approximately 20 min,and the burst size was approximately 100 PFU/cell,which indicated that the phage had high lysis activity.Moreover,the host spectrum of the phage showed that the phage had a broader host spectrum with a lysis rate of 75.7%(50/66).Furthermore,the whole genome of the phage VB-Sav M-JYL01 was sequenced.The results showed that the full length of the phage was 141,384 bp and encodes 219 putative open reading frames(ORFs).The genome of VB-Sav M-JYL01 lacked bacterial virulence-,antibiotic resistance-and lysogenesis-related genes which laid a foundation for the use of the phage in therapeutic research.Finally,we evaluated the therapeutic effect of the phage VB-Sav M-JYL01 on the rabbit model of necrotizing pneumonia.We find that a single intranasal administration of VB-Sav M-JYL01(3 × 109 PFU)could effectively improve the survival rate at 48 h to 90%(9/10)compared with the survival rate of 10%(1/10)and 80%(8/10)observed with the PBS or linezolid treatment,respectively.The bacterial countin the lungs of rabbits treated with the phage VB-Sav M-JYL01 was 4.18 × 104 CFU/g at 24 h,which was significantly decreased compared to that of rabbits treated with PBS(7.38 × 107 CFU/g)or linezolid(3.12 × 105 CFU/g).In addition,the phage treatment significantly alleviated lung tissue damage.The levels of total proteins,Panton-Valentine leukocidin(PVL),alpha-toxin(Hla)and cytokines in the lungs of the rabbits treated with the phage were significantly lower than those of the rabbits treated with PBS and similar to those of the rabbits treated with linezolid.These data demonstrate the potential utility of phage as an alternative for preventing rabbit necrotizing pneumonia caused by S.aureus. |
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