Isolation And Biological Characterization Of Pseudomonas Aeruginosa Virulent Phages

The world is on the cusp of a “post-antibiotic era”.The emergence and expanding of multidrug-resistant,extensively drug-resistant,and pandrug-resistant pathogens pose a huge challenge to public health.As the ubiquitous opportunistic pathogen,P.aeruginosa can cause life-threatening infections in patients suffering from cystic fibrosis,severe burns,AIDS or those with immunocompromising conditions,leading to considerable morbidity and mortality.On February 27,2017,WHO listed carbapenem-resistant Pseudomonas aeruginosa as a priority pathogen for which new antibiotics are urgently needed.Bacteriophages,viruses that infect and replicate within bacteria,have shown several incomparable advantages over antibiotics,including their abundance,specificity,short development periods,low cost,safety and effectiveness,which is a promising strategy to combat bacterial pathogens.In this study,four virulent phages were isolated from environmental sample using Pseudomonas aeruginosa standard strains PAO1 and ATCC 9027 as the hosts,named as v B_Pae M_SCUT-S1,v B_Pae M_SCUT-S2,v B_Pae S_SCUT-S3 and v B_Pae S_SCUT-S4,respectively.Their biological characteristics were studied,including phage morphology,host range,genomic analysis,structural protein identification,stability,one-step growth curve,lysis kinetics,and biofilm eradication.Phages S1 and S2 belong to Myoviridae,and the member of Pbunavirus and Pakpunavirus genus,respectively.Phages S3 and S4 belong to Siphoviridae,and the member of Septima3 virus genus.Among the 7 tested Pseudomonas aeruginosa strains,phage S2 could infect all,S1 infectd 6 strains,while S3 and S4 merely infected 3 strains.The genome size of S1 is 66089 bp,encoding 94 ORFs.The genome size of S2 is 94434 bp,encoding 179 ORFs and 11 t RNAs.The genome size of S3 is 42633 bp,encoding 62 ORFs.The genome size of S4 is 42929 bp,encoding 59 ORFs.No integrase,excisionase,and repressor genes,which are considered indicative of potential for a lysogenic cycle,were found in their genomes,demonstrating that all of them are virulent phages.Besides,no antibiotic-resistant and virulence genes were found either.Phages S1–S4 are stable over a wide range of p H and temperature,indicting their adaptive ability to changing conditions.The one-step growth curve showed that the burst sizes of S1–S4 were 134 pfu/infected cell,40 pfu/infected cell,95 pfu/infected cell and 20 pfu/infected cell,respectively.Phages S1 and S2 could inhibit the growth of PAO1 in vitro,but the bacteria developed resistance after 12-h exposure.Similarly,phages S3 and S4 could inhibit the growth of ATCC 9027 initially,but the bacteria resistance occurred after 6-h exposure.Phage cocktails did not show an obvious synergistic effect and the curves of low MOI fell behind that of high MOI.The biomass of 24-h-grown biofilm was effectively reduced by the treatment of phages S1–S4,and the 24-h clearance were 44%,69%,74% and 82%,respectively.The drug resistance of P.aeruginosa among China is grim now.Bacteriophages show great potential as alternatives to antibiotics.This work presents a detailed analysis of P.aeruginosa phages to enhance our understanding of phage diversity and evolution.Also,phages S1–S4 may be used as reserve resources to pave the way for phage modification and the clinical treatment of P.aeruginosa infection.