| Pseudomonas aeruginosa(P.aeruginosa)is a common opportunistic zoonotic pathogen.In animals,P.aeruginosa causes otitis media and urinary tract infections in dogs,mastitis in cows,hemorrhagic pneumonia in some fur-bearing animals such as minks and foxes,and endometritis in horses.At the same time,the pathogen can also cause human lung,blood,urinary tract,and burn infections,especially infection of cystic fibrosis patient’s airway one of the main pathogens.Conventional antibiotics are the first choice for treating P.aeruginosa infection.However,P.aeruginosa is inherently resistant to many antibiotic drugs and can develop acquired resistance to carbapenems,which greatly reduces the therapeutic effect of antibiotics.In recent years,due to the emergence of multi-drug resistant bacteria,phage therapy has attracted wide attention because of its completely different antibacterial mechanism from antibiotic therapy.Phages are viruses that specifically infect bacteria and are one of the most common and diverse organisms on Earth.Phages can invade bacteria and kill their host bacteria with high specificity,so they can be found anywhere the host is present.Phages can also be used as novel therapeutic agents to treat bacterial infections,especially multidrug-resistant bacteria.In contrast to antibiotics,lytic phages can specifically kill host bacteria without affecting other probiotics.P.aeruginosa lung infections are usually treated with traditional antibiotics.The use of phages and their cocktails to control P.aeruginosa infection is rarely reported.In this study,multiple P.aeruginosa obtained in the laboratory were used as host bacteria,and 20 phages of P.aeruginosa were isolated.After multigeneration purification,the titer was higher than 10~8 PFU/m L.Moreover,the lysis spectrum of all phages was measured,and three phages with wide and complementary lysis spectrum were used for subsequent studies,including P-B8,P-B31 and P-B55.The relevant studies on them are as follows:1.After observation by transmission electron microscope,the three phages were all in the Caudovirales,P-B8 belongs to the family of Siphoviridae,P-B31 and P-B55 belong to the family of Myoviridae.2.The biological characteristics of phage determine its ability to adapt to living environment and invade host bacteria.The high temperature resistance and acid-base stability of the three phages were tested.It was found that the three phages were very stable and had the potential to be used as antibacterial agents.Under the condition of water bath at 25℃to 60℃for 80 min,the three phages still maintained the ability to infect P.aeruginosa.In the p H 4 to p H 11 range for 1 h,the three strains remained stable and had bactericidal activity.From the one-step growth curve,the incubation period of the three phages was shorter,within 5 min,and the outbreak amount was higher during the outbreak period,the average of the three phages reached 350 PFU/cell.3.Phage viruses are not only different in shape and size,but also have more diverse genomes.A fuller understanding of the phage genome is one of the keys to its becoming a therapeutic agent.The three phages were amplified and concentrated,and their whole genomes were extracted.The results showed that the length of three phages were 92,835 bp in P-B8,92,420 bp in P-B31,and91,901 bp in P-B55,respectively.All of them were linear double-stranded DNA with about 49%G+C content.The three phages were highly similar to the published P.aeruginosa phages.REST software predicted that three phages had about 170 open reading frames respectively with average gene length between 470 and 478bp base pairs.And about half of the genes encode proteins match published gene that products in the NCBI database,with similarities ranging from 25 percent to100 percent.However,no phage lysogen modules,antibiotic resistance genes or putative virulence factors were detected in the three phage genomes.In order to verify the therapeutic effect of the phage cocktail,firstly,a dose of 1.5×10~7/mouse multidrug-resistant P.aeruginosa B49 was intranasally administered.Obvious clinical symptoms of pneumonia,such as dyspnea,depression,and hair disturbance,were observed in the mice,and all the mice died within 48h.Pathological sections showed obvious bleeding and inflammatory cell infiltration in lung tissue.After 12h of infection,the bacterial load of lung and blood reached 5×10~8 CFU/m L and 1.8×104 CFU/m L,respectively.These results indicate that the model of hemorrhagic pneumonia has been successfully constructed.The phages cocktail was prepared by mixing phages P-B8,P-B31 and P-B55,and then used in the treatment of hemorrhagic pneumonia model in mice.45 BALB/c mice were randomly divided into 3 groups with 15 mice in each group,which were PBS treatment group,phage cocktail treatment group and blank control group.Three mice were randomly selected from each group at different time periods,and after HE staining,the lung tissue pathologically changes of each group were observed,and then the bacterial load in blood and lung and the number of phages in lung were measured.The levels of cytokines IL-6,IL-1βand TNF-αin lung homogenate were determined by ELISA kit.The results showed that compared with PBS treatment group,the survival rate of mice in the phage cocktail treatment group reached 100%,and the lung tissue injury was significantly reduced.The phage cocktail treatment significantly decreased the number of bacterial colonies in lung tissue(3×10~7 CFU/m L to 2×10~4 CFU/m L).After phage cocktail treatment,the level of inflammatory factors in lung tissue of mice was significantly reduced.With the extension of infection time,there was almost no difference between them and the blank control group.In summary,in this study,several strains of lytic phages against multi-drug resistant P.aeruginosa were isolated,and three of them were selected to determine their biological characteristics.They were then used to construct a phage cocktail,which showed good therapeutic effect on the mouse model of hemorrhagic pneumonia caused by multidrug-resistant P.aeruginosa B49 infection.To provide backup antibacterial agents for the prevention and control of human and animal infections caused by multidrug-resistant P.aeruginosa. |
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