| Staphylococcus is a kind of gram-positive bacteria which exists widely in nature.Low host specificity,easy to colonize in the body of edible animals,easy to pollute the food,brings great loss to our animal husbandry industry.At present,Staphylococcus infection is mainly treated by antibiotics,but the non-standard use of antibiotics has led to the emergence of a large number of drug-resistant strains,such as Methicillin Resistant Staphylococcus aureus(MRSA)strains and Vancomycin Resistant Staphylococcus aureus(VRSA)strains,drug-resistant strains are highly adaptable and have a high pollution rate,which is a great threat to the future breeding industry.And with the introduction of the policy of reducing resistance and limiting resistance,it is necessary to find the ideal antibiotic substitutes.Bacteriophage has the characteristics of strict host specificity,high efficiency,high safety and no pollution.It can be used for the prevention and control of bacterial diseases,feed additives or food preservatives,and is one of the ideal products to replace antibiotics.The purpose of this study is to isolate and screen Staphylococcus and bacteriophages that can lysate staphylococcus,explore the biological characteristics of bacteriophages,and verify the therapeutic effect of bacteriophages on Staphylococcus infection.To provide theoretical basis and technical support for the research and application of Staphylococcus phage preparation.The specific results are as follows:1.Isolation,purification,culture and drug sensitivity test of StaphylococcusSuspected Staphylococcus infection samples were collected from aquaculture farms in Hubei,Sichuan,Guangdong,Jiangsu and Shandong.298 strains of Staphylococcus were isolated successfully by PCR,of which 122 were Staphylococcus aureus.The drug sensitivity test results showed that 298 strains of Staphylococcus were the most sensitive to doxycycline and ofloxacin,with resistance rates of 0.6% and 6%.They were also sensitive to kanamycin,flufenicol,gentamicin,cefradine and ceftriaxone,and the least sensitive to ampicillin,with resistance rates of 63%.2.Isolation,purification,biological characterization and genome analysis of phageFour strains of Staphylococcus were isolated and screened,and purified after proliferation.The morphology of four strains of phage was observed by electron microscope.The optimal number of infection and range of cleavage of the four phages were determined.Genome sequencing and analysis were conducted on the two phage strains with the widest cleavage spectrum.The results of thermal stability test showed that the titer remained relatively stable after incubating at 4℃ to 40℃ for 1 h,and the activity of phages decreased sharply after incubating at 60℃ for 1 h.After incubating at 80℃ for 1 h,the four phages basically lost their activity.The p H stability test results show that: The four strains were incubated at p H 4-12 for 1 h.The results showed that the phage activity remained stable at p H 6-8 for 1 h,and began to decline beyond this range.At p H 2,the phage activity would lose,and at p H 8-12,the decline would be relatively slow.The results of one-step growth curve showed that the four phages had strong ability of lysis and proliferation,and had potential for clinical application in the treatment of Staphylococcus infection.The results of the drop cracking experiment showed that the cracking spectra of three strains were broad,and the cracking ratios of TJP1,TJP2 and TJP4 to 298 strains of Staphylococcus were 81%,36% and 72%,respectively,while that of one strain was narrow,and the cracking ratios of TJP3 to 298 strains of Staphylococcus were 4%.Pnagus TJP1 belongs to Dubowvirus genus of Azeredovirinae,and pnagus TJP4 belongs to Kayvirus genus of Twortvirinae.No virulence gene,drug resistance gene and other related genes were found in the genome,indicating that the two strains were virulent and safe phages.3.Animal experimentsThe back fur of 40 BALB/C mice aged 6-8 weeks was removed,and the wound model was established by using a hole punch to trim the 8 mm diameter wound on the back.The mice were randomly divided into 5 groups(blank control group,phage control group,challenge group,phage treatment group and antibiotic treatment group),and the results were as follows:In the whole experiment period,a total of 4 mice died in the challenge group,but no mice in the other groups died,indicating that phage preparation can effectively reduce the mortality of Staphylococcus infection.The wound of mice in all groups showed a trend of improvement,but the rate of improvement was different.In terms of the change of wound area,from the 1st day to the12 th day,the infected wound area of mice in the phage treatment group and the antibiotic treatment group was reduced by 97% and 98%,respectively.At the 3rd day,compared with the challenge group,the wound area in the phage treatment group and the antibiotic treatment group was significantly reduced(P < 0.01).There was a significant difference in the wound area between the phage treatment group and the challenge group(P < 0.05),and a very significant difference in the wound area between the antibiotic treatment group and the challenge group(P < 0.01).There was no significant difference in the wound area between the phage treatment group and the antibiotic treatment group(P >0.05).The results showed that phage therapy had a good effect on the clinical symptoms of Staphylococcus infection,which was equivalent to antibiotic therapy.In terms of the bacterial load of infected wounds,the bacterial load(CFU/mg)in the wounds of mice in the phage treatment group and the antibiotic treatment group decreased significantly from day 1 to day 9 compared with that in the challenge group(P < 0.01),and the bacterial load in the wounds of mice in the phage treatment group and the antibiotic treatment group was 0 after day 9.No significant difference was observed in Staphylococcal load between the pnag-treated group and the antibiotic treatment group(P > 0.05),indicating that pnag-treated preparations could effectively reduce the bacterial load after Staphylococcal infection,which was comparable to the effect of antibiotic treatment.In summary,298 strains of Staphylococcus were isolated and identified in this study,and drug resistance of these Staphylococcus was analyzed.Four strains of virulent phage were isolated and observed by electron microscope.The biological characteristics,optimal infection complex number and cleavage spectrum were determined.Two strains of phage TJP1 and TJP4 with good characteristics and wide cleavage spectrum were selected for genome and phylogenetic analysis.Through animal experiments,it is verified that phage therapy can reduce the mortality rate of Staphylococcus infection,accelerate the healing speed of infected wounds,reduce the amount of bacteria in the wound,and has good therapeutic effect.It is one of the ideal alternatives to antibiotics to solve the drug-resistant strains of Staphylococcus in the future. |
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