Novel Lytic Phages Protect Cells And Mice Against Pseudomonas Aeruginosa Infection

Objective: Our aim to a new phage drug for anti-P.aeruginosa and afford the theoretical and experimental proof for phage therapy,novel lytic phages against P.aeruginosa were isolated and purified from various environmental settings and systematically determined for their critical biological characteristic,such as genetic traits,morphology structures,host ranges,and antibacterial capacity,etc.Thus,phage candidates that could be potentially used in therapeutic tests were screened and carefully assessed.Indeed,acute and chronic bronchopulmonary infection mouse models through airway delivery of P.aeruginosa were employed to evaluate antibacterial effect of phages in vivo.Methods: P.aeruginosa laboratory strains PAO1,ATCC27853 and PA14 were used as hosts to isolate,purify and verify lytic phages by double agar plate methods.The purified phages were concentrated by polyethylene glycol 8000(PEG8000),and large amounts of endotoxin were removed from the phage solution by cesium chloride(Cs Cl)density gradient centrifugation.The phenotypic characteristics of plaques formation on different bacterial lawns were observed by spot tests,and the host ranges was determined.Then,after negative staining with 2%(w/v)potassium phosphotungstate,the micromorphology of phages was viewed using a transmission electron microscopy(TEM).Indeed,the phage genomic DNA was precipitated by adding anhydrous ethanol and global genome sequencing was performed by a biological technological company using Illumina Nova Seq.Furthermore,in vitro bacteriolytic activity of phages was determined by monitoring bacterial growth dynamics.Finally,acute and chronic bronchopulmonary infection mouse models were established through airway delivery of P.aeruginosa.Two strains were employed to establish acute pneumonia including laboratorial strain PAO1,and W19,a multi-drug resistance(MDR)clinical isolate.The cell aggregates used for development of chronic pneumonia are based on agar beads laden with clinical isolate FRD1,a mucoid phenotype strain isolated from the sputum of a cystic fibrosis(CF)patient.To evaluate the efficacy of therapeutic phages in vivo,the colony-forming units(CFU)in infected-lungs,cytokines(interleukin-6(IL-6),tumor necrosis factoralpha(TNF-α))in bronchoalveolar lavage fluid(BALF),as well as histopathological analysis of lung tissues were detected in this experiment.Results: Using PAO1 and ATCC27853 as host strains,we successfully isolated several phages,named HX1,MYY9,MYY16 and TH15.First,the plaques morphologies of HX1 and MYY9 were circular,clear and transparent,whereas the MYY16 plaque was different in size and irregular in shape,and turbid in edges and clear in interiors,which showed less transparent than the other three phages.And the TH15 plaque was relatively small in size.Second,TEM showed that TH15 had a contractile tail,suggesting that it belongs to the order Caudovirales and Myoviridae family.Other phages,MYY9,HX1 and MYY16,possessed an uncontractile short tail,belonging to the order Caudovirales and Podoviridae family according to the International Committee on Taxonomy of Viruses(ICTV).The results of host-range specificity test showed that the infectivity(%)of HX1 and MYY9 against clinical isolates and laboratory strains were 87.10%,similarly.MYY16 and TH15 were 70.97% and 58.06%,respectively.Third,MYY9 and HX1,belonging to same family,shared highly similar features: 97.84% identical sequences and they may be new members of the “phi KMV-like” phages genus by primary amino acid sequencing analysis.According to the amino acid sequences and homology to functional domains aligned with known phages,the putative products of coding sequences(CDS)were predicted.Critically,no CDS that are associated with drug resistance,human virulence genes or lysogenization,such as site-specific integrases or repressors,were noticed,suggesting our newly identified phages may be suitable for drug development.Then,bactericidal effects in vitro indicated that compared to MYY16,MYY9 and HX1 exhibited stronger ability to modulate host cells growth.Remarkably,our results showed that even at MOI =0.000001,both HX1 and MYY9 could effectively inhibit the growth of PAO1.In contrast,when the MOI <1,MYY16 had no overt impact on the growth of PAO1.And the inhibitory effect of HX1 and MYY9 against W19 and FRD1 revealed strong potential to impede the bacterial growth,despite to different extent.Finally,intratracheal instillation of P.aeruginosa PAO1 and W19 was performed to induce mouse acute pneumonia,while chronic pneumonia was established using agar beads laden with clinical isolate FRD1,a mucoid phenotype strain that was noted for high mucus yield with muc A mutation.Our experiments indicated that phages therapy reduced bacterial loads in lungs of pneumonia mice,alleviated the synthesis and release of pro-inflammatory cytokines,TNF-α and IL-6,as well as emerged milder inflammatory responses with less cell infiltration and weaker tissue damage signs by histopathological analysis.Conclusion: Lytic phages targeting to P.aeruginosa isolated in this experiment exhibits an antibacterial effect both in vivo and in vitro,reducing the colonization of infected-lung,alleviating lung inflammatory cell responses,identifying these phages as potential alternative therapeutics for MDR strainscaused bacterial infections.