| Pseudomonas aeruginosa is a widely distributed opportunistic pathogen that can infect many mammals,poultry,reptiles and fish,causing mastitis in cattle,abortion,otitis externs in dogs,corneal ulcer and metritis in horses,dead embryos in poultry,rotten wool in sheep,hemorrhagic pneumonia in mink and other diseases,which have adverse effects on human health,livestock production and life.Because Pseudomonas aeruginosa has strong biofilm formation ability,it is easy to develop resistance to commonly used antibiotics,the development of new antibiotics is slow,and China is trying its best to promote the implementation of the strategy of "reducing resistance,limiting resistance and prohibiting resistance",there is an urgent need for new green anti-agent.Bacteriophage can lyse host bacteria specifically.Compared with antibiotics,bacteriophage is not easy to produce resistant bacteria,does not cause bacterial imbalance,has low immunogenicity,abundant quantity,safety and other advantages,and has become a bacteriostatic weapon in the "post-antibiotic era".In this study,a total of 25 isolates of Pseudomonas aeruginosa were isolated from2020 to 2023,of which only 1 strain was multidrug-resistant from human origin and the rest were sensitive to 10 commonly used clinical antibiotics.Phenotypic analysis showed that 7 strains were resistant to amtronam,four strains were resistant to ceftazidime,the cephalosporin antibiotic,two strains were resistant to tobramycin,the aminoglycoside,and one strain was resistant to meropenem,the carbapenem antibiotic.The 9 whole genome sequenced bacteria were divided into 7 ST types,which were closely related to each other and belonged to the same large evolutionary clade,but were significantly different from the model strain PAO1 of Pseudomonas aeruginosa.None of the genomes carried drug-resistant plasmid,but contained 6-12 unequal CRISPR sequences,6-10 unequal gene islands,and 13-15 unequal gene clusters.Except for human origin 3102-3a T,the genomes of the other 8 strains all contained 2-7 unequal phage genes.In this study,a total of 16 phages were isolated from sewage samples and purified for whole genome sequencing.Their genome sizes varied greatly,among which the maximum size of pPA.m1308.2Dr.1 was 28 7641 bp and the minimum size of pPA.N3180.3AT.3.According to the morphological classification of phage,the sequenced phages included 10 long-tailed phages,4 myotailed phages and 1 short-tailed phage.The sequencing phage genome mainly consists of four gene cluster modules:cleavage module,DNA packaging module,structural assembly module,nucleic acid replication and regulation module.In addition,some functional genes are unknown.pPA-3099-2aT.2 belongs to Caudoviricetes,and the genome of Pakpun2 genus is ds DNA,with a length of 93 031 bp and a GC content of 49.4%.It contains holin,Nacetylmuramidase,Cell Wall Hydrolase and other cleavage related proteins.The analysis of biological characteristics showed that the diameter of the head of pPA-3099-2aT.2 was about 76.686 nm,with a flexible tail sheath,and the length of the stretch was123.886 nm.Good stability,can be maintained in-20℃-60℃ and p H value of 4-10 environment,the optimal p H value is 6,the optimal temperature is 37℃;The incubation period was less than 10 min,the lysing period lasted to 100 min,and the lysing volume was about 178 PFU/cell.8 strains of Pseudomonas aeruginosa from different sources including ST198,ST274,ST3683,ST3875 and ST3216 were lysed.The bacteriostatic effect of pPA-3099-2aT.2 was good in vivo and in vitro,and the bacteriostatic effect of phages cocktail was better than that of single phage.The bacteria that obtained phage pPA-3099-2aT.2 resistance were more sensitive to a variety of antibiotics,cefepime changed from intermediate to sensitive phenotype,and amtrinam changed from resistant to intermediate phenotype.The MIC for amtronam antibiotics was 32 μg/m L for wild strain(WT P.aeruginosa 3099-2aT)and 16 μg/m L for phage-resistant bacteria,suggesting that the combination of phages and antibiotics could reduce the antibiotic dosage.The experimental results showed that the combined application of phages could reduce the dosage of amtronam from 16 μg/m L to 0.025μg/m L.pPA-3099-2aT.2 could not only reduce the biofilm formation of Pseudomonas aeruginosa,but also effectively eliminate the biofilm formation.Phage pPA-3099-2aT.2 spray is easy to use and has good environmental bacteriostatic effect.In summary,25 strains of Pseudomonas aeruginosa and 16 strains of phage were isolated in this study.The biological characteristics and genome of P.aeruginosa 3099-2aT and pPA-3099-2aT.2 were analyzed.pPA-3099-2aT.2 belongs to the member of Pakpunavirus genus of Caudoviricetes,has good stability,can identify multiple ST subtypes of host bacteria,has strong lysing ability and in vitro and in vivo antibacterial ability,and is widely used,convenient and effective.This discovery can provide a new way for the classification of phages and the development of antibacterial drugs of Pseudomonas,and has a wide range of potential applications in the prevention and treatment of Pseudomonas aeruginosa infection. |
PDF Full Text Request