| Mastitis is one of the most harmful and common diseases in dairy farm.It is reported that more than 25% of clinical mastitis in dairy cows is caused by Staphylococcus aureus.At present,antibiotics are still the main treatment of mastitis in dairy cows.However,its disadvantages are also obvious,such as low cure rate,drug resistance,and antibiotic residues in milk.Bacteriophage(phage)is a simple,widespread and specific biological antibacterial agent.Phage has the advantages of high specificity,high environmental abundance and long-term preservation.At present,there are many studies on the phage therapy of Staphylococcus aureus mastitis,but there are no reports on the safety mechanism of its practical application and the side effects on intestinal microecology.In this study,the whole genome sequencing of phage was combined with mouse transcriptome analysis to evaluate the safety of phage in the treatment of S.aureus mastitis.The mechanism of bacteriophage’s effective inhibition of S.aureus mastitis was discussed by combining intestinal microflora with inflammatory factor analysis.The purpose is to provide theoretical basis and practical basis for the application of phage therapy in this field.The comprehensive contents and results are as follows:(1)A highly pathogenic and multidrug-resistant S.aureus was isolated.By using it as host bacteria,two phages with good cleavage performance and broad spectrum were screened.There were 34 strains of pathogenic bacteria isolated from 55 milk samples of dairy cows with clinical mastitis.Then their physical and chemical properties were analyzed.The detection rate of Staphylococcus aureus was 17.65%among them.In these S.aureus,Sau-XJ-21 shown the most antibiotic resistance and high pathogenicity and toxicity.Two lytic phages named vBSM-A1 and vBSP-A2 were isolated by using Sau-XJ-21 as the host.The results showed that the two phages were belonged to tailed phages order,herelleviridae family.All of them have a wide host spectrum and can lyse S.aureus from different sources(bovine and human)and different regions(Xinjiang,Zhejiang and Dalian).Single bacteriophage and cocktail(volume 1:1)can effectively inhibit the growth of Sau-XJ-21 in vitro,and the effect of cocktail is better than either of the single phage group.(2)Two phages were sequenced by using Illumina genome analyzer.It was clarified that the two phages did not contain virulence gene,lysogenic gene and virulence transposon and other pathogenic genes.The whole genome analysis,functional gene annotation,tRNA morphological structure prediction and taxonomic status analysis of the two phages were carried out by using the bioinformatics software and programs such as GeneMarkS,CGView,tRNAscan-SE,Cluster X and Mega 7.0.The phage lyase Lys-vBSM-A1 was analyzed for primary structure,physical and chemical properties,hydrophobicity,transmembrane region,signal peptide prediction,secondary structure prediction and 3-D model by using ExPASy,TCDB,PortScale,Signal 5.0,Phyre2,and Swiss-Model.The results of whole genome sequencing showed that both phage vBSM-Al and vBSP-A2 were double stranded linear DNA,the total length of vBSM-Al was 140654 bp,while the vBSP-A2 was 136528 bp.The GC content of phage vBSM-Al and vBSP-A2 were 30.33%and 30.45%respectively.There was no virulence gene,lysogenic gene,virulence transposon and other pathogenic factors found in both of them,which had gene level application safety.The whole genome sequence of the both two phages has been submitted to genebank with the genebank ID of MK584893.1 and MK656892.1,respectively.Both phages contained conserved lyase named Lys-vBSM-Al,with the length of 267 aa,and two typical active domains,namely,c4olkb domain and c4olsa domain.(3)The therapeutic effect of phage mixture in the mastitis mouse model and the regulation and improvement of intestinal microecology were systematically evaluated.The effect of phage perfusion on mouse transcriptome expression and the potential mechanism of action were analyzed.The results showed that the phage cocktail can effectively alleviate the mastitis in mouse model,and its effect is similar to that of ceftiofur sodium treatment group.The intestinal microflora of mice showed that use of ceftiofur sodium will destroy the original dominant strains in the intestine and indirectly form new dominant strains that may be harmful.While the diversity of mice intestinal microflora in phage cocktail group was similar to healthy control group.In order to test the safety of phage perfusion in mammary glands of mice,the transcriptome was sequenced by using Illumina HiSeq high-throughput sequencing technology,and the differential expression gene GO classification and differential expression gene KEGG enrichment analysis were performed.The results showed that compared with the PBS control group,the phage perfusion group had significantly different expression of some genes related to inflammation(such as apoptosis,cell aggregation and antioxidant activity,etc.)At the same time,some pathways related to inflammatory factors(such as TNF signaling pathway and NF-κB signaling pathway)showed significant enrichment.It is suggested that phages may induce some immune and inflammatory responses in mice.According to the results of morphological observation,histopathological analysis of mammary gland,detection of inflammatory factor TNF-α and phage load test after 24 hours,it is revealed that the inflammatory tendency of phage perfusion is slight,and the phage titer will decrease over time,which will not affect the normal vital signs of mice.In conclusion,this paper studies the efficacy of phage cocktail in the treatment of mastitis in mice,reveals the advantages of phage cocktail compared with antibiotics from the aspect of intestinal microflora and microecology abundance in mice,and proves the safety of phage application from the perspective of whole genome,transcriptome,inflammatory factors,apparent state of mice and phage load of mammary gland.It is revealed that the phage cocktail is a kind of biosafety preparation which can effectively inhibit the growth of Staphylococcus aureus in vitro and in vivo,and has a high application prospect and feasibility. |
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