Isolation And Identification Of Salmonella Typhimurium Lytic Phage And Analysis Of Its Host Bacterial Resistance Mechanism

Salmonella,a Gram-negative enteric bacillus,is a common foodborne pathogen and a typical human-animal pathogen,mainly causing gastroenteritis,typhoid-like fever,infections,and local septic infections.Among them,Salmonella typhimurium is the major serotype of foodborne Salmonella infections and poses a serious threat to public health.Although antibiotics have certain therapeutic effects on infections caused by this bacterium,the irrational use or even abuse of antibiotics has led to increasing bacterial resistance,and the development of new antibacterial drugs against resistant bacteria is a current hot topic of concern.Phages are viruses that can specifically lyse host bacteria and can be used for the treatment of bacterial infections,especially drug-resistant bacterial infections,with their unique antibacterial mechanism without destroying the normal flora of the organism.However,phage-resistant mutant strains are often generated during the use of phages for the treatment of bacterial infections,which may trigger secondary clinical infections.Therefore,it is important to elucidate the mechanism of bacterial resistance to phage,which can avoid in advance or block the production of phage-resistant bacteria in time for clinical application,to improve the application value of phage therapy.In this study,a broad-spectrum highly efficient lytic phage v B＿Sen S-STP1（STP1）was isolated using Salmonella typhimurium 181016 as the host bacterium,and was systematically studied and analyzed.First,the isolated phage STP1 could form a translucent circular phage spot on the bilayer plate,and a translucent halo would form around the phage spot as the incubation time increased.Biological characterization showed that the optimal multiplicity of infection（MOI）of phage STP1 was 10^-7determined by the double-plate method;the results of one-step growth curve showed that the incubation period of phage STP1 was 15 min,the occultation period was 5 min,the outbreak amount was about 500 PFU/Cell,and the whole lysis cycle lasted 80 min;and phage STP1 also exhibited good temperature and acid-base The test showed that the phage had infective activity between 25-60℃and p H 3-11,indicating that the phage has good tolerance to high temperature and alkaline environment;the lysis spectrum assay showed that the phage had lysis activity against 42 of 52 strains of Salmonella serovar Typhimurium,Abortusequi,Livingstone,Dublin and Pullorum,and its lysis spectrum was relatively wide.Further sequencing of the whole genome of STP1showed that the nucleic acid type of this phage was double-stranded DNA,with 41,435bp in total length and 48%C+G content.The whole genome sequence of this phage was compared with other Salmonella phage sequences in the database,and the highest homology was 97.08%.A total of 60 open reading frames were predicted in this phage whole sequence,and the two proteins with low homology among these open reading frames were ORF 7（91.67%）and ORF 8（96.00%）,which expressed the putative protein FP71＿gp60 and the putative membrane protein,respectively.And annotation of the functions of all open reading frames did not reveal open reading frames with lysogenic,drug resistance or bacterial virulence related gene proteins.In this study,we evaluated the therapeutic and bactericidal effects of phage STP1by in vitro and in vivo assays.In the in vitro bactericidal assay,the host bacterium181016 was mixed with phage STP1 at MOI=0.1 and incubated for 2 h.The number of viable bacteria gradually decreased from the initial 1.0×10⁷ CFU/m L to 3.7×10⁴CFU/m L,showing good in vitro lytic activity.The in vivo therapeutic effect of STPI was then evaluated by establishing a mouse Salmonella infection model.Mice were injected intraperitoneally with 2×MLD（2×10⁷ CFU/mice）Salmonella typhimurium181016 and treated with 1×10⁹PFU of phage STP1 1 h after infection,and the survival rate of infected mice could reach 100%（10/10）.Phage STP1 treatment significantly alleviated histopathological damage and cleared bacteria from mice,and more rapidly cleared bacteria from lungs and kidneys.To investigate the resistance mechanism of the host bacteria,a phage-resistant strain of Salmonella typhimurium,named R13,was successfully induced by co-culturing phage STP1 with the host bacterium 181016.phage STP1 failed to form phage-spots and empty spots on the double plates of R13.A series of characterization and analysis of the biological properties of the resistant bacterium R13 revealed that the resistant bacterium R13 did not differ from the original strain 181016 in microscopic morphology after Gram staining,colony morphology on solid medium and growth curve.The differences between R13 and 181016 at the genetic level were analyzed by whole genome sequencing,and they were also not different.A total of 62 genes were detected to be significantly different at the transcriptomic level,of which 20 genes were significantly up-regulated and 42 genes were significantly down-regulated,with the most up-regulated gene being eut E and the most down-regulated gene being STM0718.It was hypothesized that the difference in expression of different genes is one of the possible reasons for the formation of host bacteria resistance.In summary,in this study,the broad-spectrum and efficient lytic phage STP1 was isolated from Salmonella typhimurium as the host bacterium,its genome sequence and other biological properties were determined in series,and the therapeutic and bactericidal effects of the phage were evaluated in vitro and in vivo,which enriched the phage library of the bacterium,and the resistance mechanism of the artificially induced phage resistant mutant strain was investigated,which initially revealed that Salmonella typhimurium It also investigated the resistance mechanism of the artificially induced phage-resistant mutant strains and revealed the resistance mechanism of Salmonella typhimurium,which laid a solid foundation for the in-depth study of the resistance mechanism of Salmonella phagosomes.