Characterization And Biological Features Of Virulent Bacteriophage φPA-HF17Infecting Pseudomonas Aeruginosa

Pseudomonas aeruginosa, an important member of thepseudomonad and non-fermentation, gram-negative rod, is widelydistributed in soil and water. Similarly, it is also present in moistenvironments in hospitals. P. aeruginosa is a common opportunisticpathogen that causes ventilator-acquired pneumonia, acute lowerrespiratory tract infections, chronic respiratory infections in cystic fibrosispatients, catheter-associated urinary tract infections, skin and woundinfections. P. aeruginosa has become one of key pathogens associatedwith nosocomial infections because of its strong adaptability. Additionally,the increasingly emergence of multi-drug resistant P. aeruginosa and theformation of biofilms by P. aeruginosa bring great difficulties for clinicaltreatments. Bacteriophages (phages) are viruses that specifically infectbacteria and are found in soil, ocean, lakes and animals. Now, faced withthe dire circumstances of bacterial resistance, Western researchers andsome international organizations are giving phage therapy a serious look.In this study,27clinical strains of P. aeruginosa were used for phageisolation, and16virulent bacteriophages were isolated from sewage watersamples obtained from resident area and local hospitals using double-agarmethod. Four virulent phages (named φ PA-HF7, φPA-HF12, φPA-HF17and φPA-HF20) of16isolated phages were chosen for morphologicalanalysis using Transmission electron microscopy (TEM). According tothe TEM micrograph, phage φPA-HF7belongs to the Siphoviridae family, phages φPA-HF12was classified as Myoviridae family, while φPA-HF17and φPA-HF20were assigned to the Podoviridae family. Furthermore,several aspects of biological characteristics of phage φPA-HF17wereinvestigated. Phage φPA-HF17had a head of about50nm in diameterwith a short tail of5-10nm in length. Restriction analysis indicated thatφPA-HF17was a dsDNAvirus with an approximate genome size of38kb,and phage DNA was sensitive to Nde I, Xho I, Xba I, Pst I, Hind III andEcoR I. Bacteriophages φPA-HF17exhibited a higher lytic activityagainst P. aeruginosa strains, lysing12strains of P. aeruginosa isolates(44.0%of strains tested). Multiplicity of infection of φ PA-HF17was0.01(MOI＝0.01), and phage φ PA-HF17had a latent period of10min,a burst period of60min and a burst size of218PFU/cell. Adsorption testshowed that over90%of phage φ PA-HF17adhered to and penetratedinto host cells in4min when MOI＝0.01. SDS-PAGE analysis revealedthree major bands and eight minor bands, and the most abundant one withmolecular weight of40kDa was most likely the capsid protein of φPA-HF17. Stability test demonstrated that φ PA-HF17was stable over awide pH range (4to10) and at60℃for30min. The whole genomesequencing revealed that the genome of the phage φ PA-HF17wascomposed of39208bases, and the content of GC reached49.68%, a totalof46of the protein coding genes, do not have the tRNA and rRNA.Based on our studies, a conclusion may be drawn as followings:1. Four newly isolated P. aeruginosa phages named φPA-HF7,φPA-HF12, φPA-HF17and φPA-HF20are virulent, and phage φPA-HF7belongs to the Siphoviridae family, while phages φPA-HF12wasMyoviridae family, φPA-HF17and φPA-HF20were assigned to thePodoviridae family. 2.Phage φPA-HF7genome size for39208base, GC contentpercentage was49.6%.3. Phage φPA-HF7exhibits a short latent period, strong lytic activity,relatively broad lytic spectrum, wide pH stability and heat resistance.These characteristics suggest that phage φPA-HF7has a high potential forphage therapy to control drug-resistant P. aeruginosa.