| Mastitis in dairy cows is one of the common and frequent diseases in dairy farming,causing serious economic losses to the dairy industry every year.Streptococcus is one of the common pathogenic microorganisms of mastitis in dairy cows,with Streptococcus uberis,Streptococcus dysgalactiae,and Streptococcus agalactiae being the most common isolates from cows suffering from mastitis.The primary choice of treatment for mastitis in dairy cows is antibiotic therapy,but as bacterial resistance increases,the treatment effect becomes less than ideal,and the residual of antibiotics in milk not only aggravates the economic loss of dairy farming,but also endangers the public health safety of human beings.In the current context of "antibacterial reduction","antibacterial restriction" and "antibacterial ban",there is an urgent need to find a new antibacterial agent.Lysin,as a protein with bactericidal activity,is considered to be one of the most promising alternatives to antibiotics.Compared with traditional antibiotics,lysin has many unique advantages,such as stable protein properties,high bactericidal efficiency,less tolerance,and easy to be designed and modified artificially.In this study,we analyzed the drug resistance of isolated Streptococcus mastitis from cows and mined the genome for a new antibacterial substance,prophage lysin,with the following main findings as well as conclusions:(1)Streptococcus isolation and drug resistance analysis.In this study,streptococci were isolated,identified and analyzed for drug resistance in milk samples with mastitis from five regions(Dezhou,Shandong,Datong,Shanxi,Wuwei,Xingtai,Hebei and Shijiazhuang,Hebei).12 strains of Streptococcus spp.were isolated,including 7 strains of Streptococcus uberis,3 strains of Streptococcus dysgalactiae,1 strain of Streptococcus agalactiae,and 1 strain of Streptococcus infantarius.The drug resistance analysis revealed 10 drug-resistant strains among the 12 strains of Streptococcus,including 7 multi-drug resistant(resistant to three or more antibiotics)strains,while only 1 strain was completely sensitive.This indicates that the drug resistance problem of streptococci associated with mastitis in dairy cows has become very serious,and the drugs available for the treatment of drug-resistant streptococcal infections are very limited,and there is an urgent need to find new antibiotic alternative bactericides for the prevention and control of drugresistant streptococci.(2)Whole-genome sequencing and lysin mining.Whole-genome sequencing of 12 strains of Streptococcus sp.resulted in the mining of 20 prophage lysin genes.Phylogenetic tree construction and structural domain prediction of the mined lysins showed that there was a rich and diverse lysin library in the genome of Streptococcus mastitis-associated dairy cows,and structural domain prediction showed that several lysins had Amidase 2,Amidase 3,Amidase 5,CHAP,Glucosaminidase,Phage lysozyme 2,Glucosaminidase,and Glucosaminidase,Phage lysozyme 2and Peptidase M23,with seven different cleavage domains and four different cell wall binding domains,SH3-3,SH3-5,CW 7 and Lys M.(3)Prokaryotic expression of lysin.Based on the phylogenetic tree and structural domain prediction analysis,11 lysin species with large differences were selected for in situ expression,and11 Escherichia coli expression systems were successfully constructed to achieve the soluble expression and purification of lys-224,lys-1459,lys-1519,lys-1644 and lys-733.This laid the foundation for exploring the activity of lysin.(4)Biological characterization of lysin.The cleavage spectra of the five lysins were determined.The results showed that the five lysins had different cleavage spectra: lysin lys-224 and lys-1459 showed excellent cleavage with a broad cleavage spectrum and could span multiple species;Cleavage by lysin lys-1644 exhibited strong specificity for Streptococcus dysgalactiae.In contrast,lysin lys-733 and lys-1519 were relatively poorly cleaved and had a narrow cleavage spectrum.The effect of temperature and p H on lysin activity was further determined.The results showed that lysin lys-224 and lys-1459 showed the best cleavage effect at 4℃,and lysin lys-1644 showed the best lysis effect at 37℃,and lysin lys-733 and lys-1519 showed the best cleavage at45°C;lysin lys-1459,lys-733,lys-1519 and lys-1644 showed the best activity at p H 6;lysin lys-224,on the other hand,was more suitable for the acidic environment and showed the best activity at p H 4.This section shows that the five lysins have different biological properties,confirming that the multiple prophage lysins mined through the genome have some diversity and some of them are more tolerant to temperature and p H,which can be used for antibacterial applications in complex environments.In summary,this study isolated Streptococcus mastitis-associated streptococci from dairy cows that were highly resistant to a variety of antibiotics,and multiple multidrug-resistant strains emerged(7/12).In response to the serious problem of drug resistance in streptococci,this study successfully mined a rich library of prophage lysin by whole genome sequencing of bacteria,and successfully expressed five different lysins,and determined their cleavage profiles and biological properties,and the biological properties of the five lysins showed large differences.Among them,lys-1459 and lys-224 showed a wide lysis spectrum and strong lysis ability,and were highly tolerant to low temperature or acidic environments,which have great potential for application.This study provides an experimental basis for the clinical application of streptococcal prophage lysin,which is important for the prevention and control of mastitis streptococcal infection in dairy cattle. |
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