| Pseudomonas aeruginosa is a gram-negative opportunistic pathogen widely distributed in nature,which can cause serious infection in human or animal tissues when the body’s resistance is reduced or the immune system is damaged.Dog pyoderma is a kind of common skin diseases,veterinary clinical pseudomonas aeruginosa is one of the common pathogenic microorganism dog pyoderma,due to irrational use of antibiotics in clinical antibiotics,lead to the emergence of multi-resistant bacteria,which makes dogs pyoderma treatment more difficult,and phage as a can be a potent weapon against drug-resistant bacteria,will become a new choice for the treatment of bacterial infections.This study from the Shen Zhen a purulent skin at the animal hospital patients in the dog skin swab separation into a multiple drug-resistant strains of pseudomonas aeruginosa,as bacteria to host bacteria separation to a virulent phage strains,and the biological characteristics and phage genome of exploration,and then through the animal experiment contrast and phage therapy with combined use of antibiotics in dog pyoderma treatment effect.A pseudomonas aeruginosa strain named SZPA-VT01 was isolated from skin swabs of dogs with pyoderma.SZPA-VT01 has strong antibiotic resistance and is multiple drug resistance bacteria.The total drug resistance rate of 22 drugs detected reached 63.6%.It has high drug resistance rate to fluoroquinolones,macrolides,chloramphenicol and tetracycline,and is not resistant to carbapenem and aminoglycoside drugs.Based on the drug susceptibility results,the genes of related drug resistance types are tested.The active efflux pump system Mex CD-Opr J family(Mex C3,Mex D1,Opr J1)mediates tetracycline,chloramphenicol,fluoroquinolone resistance,the active efflux pump regulatory gene Mex R,the outer pore membrane protein Opr D(Opr D2)that affects membrane permeability,and the β-endogen that mediates β-lactam drug resistance were selected The amide-inducing enzyme Amp C(Amp C1)and aminoglycoside modifying enzyme Acc family(Acc3-ⅰ,Acc3-ⅱ)were amplified,and six drug resistance genes Mex C3,Mex D1,Opr J1,Mex R,Opr D2 and Amp C were amplified.The amplification results of drug resistance genes were positively correlated with drug sensitivity test results.The virulence gene detection results of plc H,apr A,exo T and exo U were positive,while the virulence gene detection results of pil B,exo S and alg D were negative,and the virulence gene detection rate was 57%(4/7).A virulent phage strain was isolated from SZPA-VT01 as the host bacteria,and its biological characteristics and whole genome were analyzed.The results showed that after purification,the phage plaque was clear and transparent,with a clear boundary and a diameter of 1-2mm.Through observation under transmission electron microscopy,the head diameter of the phage was about55 nm and the tail length was about 75 nm.The phage is a double-strand linear DNA phage with a total genome length of 92114 bp,GC content of 49.20%,and CDSs of 173.According to online comparison system blastn in NCBI,it is a member of Pakpunavirus of Myoviridae family.Among the 173 CDS,38 CDS had known functions and 135 CDS had putative proteins with unknown functions,which did not carry virulence genes or antibiotic resistance genes.The phage was named v B＿Pae M＿PPA1 according to the latest ICTV virus classification system.The best MIO for PPA1 was 1.And the pyrolysis period was 50 min,and the pyrolysis volume was 52PFU/cell.Host range analysis showed that it could lyse one escherichia coli,which proved that it had the ability to lyse across species range.It has good alkali resistance and stability,and can maintain relatively high valence activity under the condition of ph5-9.It can maintain a high titer at 50℃ and has good thermal stability.The optimal long-term storage condition is-20℃,but the long-term storage at room temperature can also be relatively stable at the high efficiency level;In vitro,the higher the potency,the better the bacteriostatic effect.In vitro and animal experiments,pseudomonas aeruginosa SZPA-VT01 was used as host bacteria,and phage PPA1 was used in combination with enrofloxacin,and the efficacy of phage alone was compared.In vitro experiment combined effect is obviously better than that of phage used alone,in4 hours,phage gradually began to unable to restrain the growth of the bacteria,bacteria concentration gradually increased,and a combination of bacterial concentration can be smoothly to suppress at a very low level,phage and grace,have synergy,characters and treatment in vitro,to reduce resistance.In animal experiments,pseudomonas aeruginosa SZPA-VT01 was used to establish canine pyoderma model.Through the skin smear cytology,building the skin pathological and 16 s r RNA sequencing assess the effect of building,by itching rating scale,cytology bacteria counting scale as well as the superficial skin lesions scale assessment animal experiment treatment effect,and before the start of the building,before treatment and after the experiment,both fasting blood samples collected three experimental dogs,Blood routine examination(CBC),C-reactive protein(CRP),and 22 biochemical results were measured to evaluate the safety of the experiment.The bacteriostatic effect of phage PPA1 and enrofloxacin was better than that of phage alone in vitro.In the animal experiment,the combination therapy was slightly better than the single phage group,the bacteriostatic effect of phage PPA1 and enrofloxacin was not significantly different from the bacteriostatic effect of phage alone,and the whole treatment process had no effect on the liver,kidney,pancreas and spleen functions of animals. |
