The Isolation And Identification Of Pseudomonas Aeruginosa Lytic Phage YH6and Its Biological Characteristics

Pseudomonas aeruginosa is a common opportunistic pathogen that is widelypresent in the environment. The treatment of the infection caused by P. aeruginosahas become even more difficult because of the emergence of multidrug-resistantstrains. Phage therapy is expected to be useful as an alternative therapeutic measurefor treating multidrug-resistant P. aeruginosa infections.In this study, we used P. aeruginosa D9isolated from diseased mink in laboratoryearly as host strain and isolated a new phage from sewage, named YH6. Thebiological characteristics of YH6were studied, which indicated that the phage isspecific cleavage of P. aeruginosa, the plaque of YH6was clear, bright and no halo.The phage belongs to Podoviridae in Caudovirales, which has an isometricallyhexagonal head of about65nm in diameter and a about25nm short tail. The curve ofYH6showed a short latency period of about30min and a rise period of60min. Thephage titers were highest when the MOI was0.01~0.001, reaching approximately1010PFU/mL. YH6was resistant to chloroform and ether. YH6was relatively stable at40°C and50°C. The infectivity ability of YH6was relatively stable at pH6.0~10.0.Host spectrum analysis showed that the YH6has very broad spectrum, lysing82.61%of P. aeruginosa strains.The sequencing and analysis show that the complete genome of YH6is made of73,050bp of double-stranded DNA containing90open reading frames (ORFs). PhageYH6has an overall G+C content of54.88%and the gene-coding potential of theglobal genome is92.64%. YH6showed similar morphology with several other P.aeruginosa phages, such as LUZ7, vB_PaeP_C2-10_Ab09, PA26and LIT1. Moreimportantly, there is no bacterial-virulence or lysogenesis-related ORF in the YH6genome, which made it eligible for use in phage therapy. The genome of phage YH6was sequenced and the endolysin gene (LysYH6) of YH6was identified by blast analysis, then LysYH6was expressed and purified. LysYH6showed a significantinhibitory activity against P. aeruginosa at the exist of EDTA (25mM/mL).Animal experiments showed that using a mink source of P. aeruginosa D9(2×107CFU) infection in mice could also cause pneumonia in the model. A singleadministration intranasal of YH6(2×107PFU, MOI=0.1) was sufficient to protect themice from fatal P. aeruginosa infection. The ability of YH6to reduce bacterial countsin the blood, spleen, and lung was investigated. The mice that were treated with YH62h after the inoculation demonstrated a drop of1.5log units by10h after YH6treatment and ultimately became undetectable within2days. By contrast, the bacterialcounts in untreated mice showed approximately4log units increasement, reaching to108CFU/mL, and caused death within2days. Moreover, YH6greatly improvedpathological manifestations of lung injury from pneumonia. In view of the effectiveprotective efficacy in mice, phage YH6might be an adjuvant or alternative treatmentstrategy for infections caused by multidrug-resistant P. aeruginosa.In summary, phage YH6showed potential application for controlling thehemorrhagic pneumonia of mouse caused by P. aeruginosa infections. This study laida solid foundation for the follow-up application of phage therapy in mink model.