Isolation And Identification Of Lytic Phage Targeting On Duck-derived Salmonella And Evaluation Of The Bacteriostatic Activity For Recombinant Endolysin LysP6

Salmonella is a globally prevalent foodborne pathogen,especially S.Enteritidis and S.Typhimurium,which have caused extremely common adverse events.With the increasing resistance of most bacteria to different types of antimicrobial drugs,human disease prevention and control may encounter an embarrassing situation of no cure in the near future-the“post-antibiotic era”.Bacteriophages are a group of viruses that can specifically infect bacteria.Compared with traditional antibiotics,phages have the advantages of broad distribution,large numbers,host specificity,high proliferation capacity,safety and low cost of research and development.Following the widespread prevalence of multi-drug resistant bacteria,the treatment of bacterial infections with phages has attracted attention.In this study,S.Enteritidis CVCC 3377 was used as the host bacterium to screen for the most effective lytic phages P5 and P6,and their physicochemical properties and biological activities were compared and analyzed.Whole-genome sequencing analysis was performed for phage P6.Gene prediction and functional annotation in P6 were accomplished by biology software to clarify its taxonomic status and genetic characteristics.In order to clone and express the endolysin protein,a recombinant plasmid for the prokaryotic expression of phage P6 endolysin was constructed by homologous recombinant seamless cloning technique.The bacterial inhibitory properties of endolysin protein were comprehensively analyzed by the host profile,biological activity assay and the use of association with EDTA.At last,the lytic activity of phage P6 against multidrug-resistant Salmonella,planktonic Salmonella in vitro and Salmonella biofilm formation was evaluated,which provided scientific reference for the development of phage agents for the prevention and control of Salmonella infection.1.Isolation and identification of duck-derived Salmonella-specific phages and analysis of their biological activityIn this study,13 phage strains were isolated from sewage and fecal samples collected from duck farm environment using S.Enteritidis CVCC 3377 as the host organism.Six phage strains with translucent phage spots were screened for host profiles,and phage P5 and P6were found to be lytic for both S.Enteritidis（P5:60%;P6:90%）and S.Typhimurium（P5:90%;P6:100%）.Biological activity assays showed that phage P5 and P6 maintained high activity at 40-60°C（P5）and 40-70°C（P6）,respectively.Both maintained high activity at p H3-12.The optimal multiplicity of infection（MOI）was 10^-4for both P5 and P6,with a latency period of 10min for both and lysis periods of 80min and 90min for P5 and P6,respectively.The average lysis amounts were approximately 800 PFU/cell（P5）and 1000 PFU/cell（P6）.The stability and lysis activity of phage P6 was found to be better than that of phage P5.Transmission electron microscopy was used to observe the morphology of phage P6,and it has a positive icosahedral head with a diameter of approximately 400nm and a short tail of about 150nm in length.Its morphological classification belongs to the order Phageidae,short-tailed phages.2.Whole genome sequencing analysis of phage P6The whole genome of P6 phage has been sequenced and analyzed.The results indicated that the genome was linear double-stranded DNA,40,811bp in length,with a GC content of47.24%.The base composition consists of A（25.28%）,G（23.17%）,T（24.07%）,and C（27.48%）.There were 63 open reading frames（ORFs）in P6 phage,out of which 30 ORFs known to function.Through mapping the phylogenetic tree of genes encoding the terminal enzyme large subunit（ORF1）and the major coat protein（ORF61）with evolutionary significance,it has been found that phage P6 was closely related to Salmonella phage SW-70（CP051272.1）（98%homology）,belonging to the genus Lederbergvirus;Unclassified Lederbergvirus species.Biological information analysis has revealed the presence of a lysogenic phage-specific repressor protein gene.However,because the gene has an insertion of a base G at position 14 after the start codon.The genome of P6 phage did not contain non-coding RNAs,virulence genes and drug resistance genes,indicating that the phage is biosafe for clinical application.Moreover,the lysis system of phage P6 consisted of an endolysin（ORF10）-perforin（ORF11）-RZ lyase（ORF8）by whole genome sequencing analysis.Endolysin（ORF10）was a key protein in the lysis system with capable of cleaving cell wall peptidoglycan.3.Analysis of the prokaryotic expression and cleavage activity of recombinant endolysin LysP6To analyze the bacterial inhibitory activity of endolysin from P6 paghe,the endolysin protein（34 k Da）was expressed as an inclusion body protein in prokaryotic expression,purified through protein complexation,and subjected to lysis profiling,assessment of biological activity and bacterial inhibition assay in combination with the membrane permeabilizer EDTA.The results showed that the LysP6 was able to lyse various Salmonella strains with different serotypes（S.Enteritidis,S.Typhimurium,S.Kottbus,S.Newlands,S.Muenster,S.Nagoya,S.Newport,S.Bovismorbificans）from ducks and E.coli ATCC 25922after chloroform treatment of the bacterial outer membrane,demonstrating an expanded host spectrum compared to phage P6.The biological activity assay revealed that LysP6 showed the best lytic effect at p H 9 and 10,while almost no lytic activity was observed below p H 5 and at12.The temperature tolerance results showed that LysP6 was more stable below 50°C.Furthermore,when used in combination with the membrane permeabilizer EDTA,LysP6 was effective in inhibiting the growth of Salmonella within 24h.4.Bacteriostatic activity of phage P6 against multidrug-resistant strains,planktonic Salmonella and biofilmsThe double-slab agar plate assay was performed to evaluate the effect of phage P6 on Salmonella of different drug resistance profiles.The results indicated that phage P6 had a lytic effect on 77.78%（28/36）of multidrug-resistant Salmonella,including one S.Enteritidis strain resistant to all seven drugs.To assess the lytic effect of phage P6 on planktonic bacteria,S.Enteritidis（CVCC 3377）was used as the host bacterium.At MOI=10²,phage P6 effectively inhibited the growth of Salmonella within 10 hours.In addition,within 3h,phage P6 reduced the bacterial load of S.Enteritidis CVCC 3377,4,24,and S.Typhimurium 44 by 44%-75%in the Salmonella biofilm.