The Experimental Treatment Of Salmonella Phage And Study On The Mechanism Of Phage Resistance

Salmonella is an important pathogen for public health due to food poisoning and acute intestinal disease by zoonotic trait.As its antibiotic resistance increases and the resistance spectrum widens,it is increasingly difficult to prevent and treat Salmonella infection with conventional antibiotics.We established a mouse model of oral Salmonella infection;and evaluated therapeutic effect of phage SP1 on mouse model of oral Salmonella infection;and also studied the mechanism of Salmonella resistance to phages.To investigate genetic and pathogenic traits of S.enterica QH,its genome comparison and the infection model in mice were performed in this study.Compared with other Salmonella strains,several large sequences containing prophages and genomic islands were inserted into S.enterica QH genome.Furthermore,nucleotide and synonymous codon usage patterns display mutation pressure and natural selection serving as drivers for the evolutionary trend of S.enterica QH at gene level.The unique codon usage pattern of S.enterica QH probably contributes to adaptation to environmental/host niches and to pathogenicity.In an early oral S.enterica QH infection,the levels of CD4~+and CD8~+lymphocytes significantly reduce in peripheral blood of mice,but the increasing transcription levels of some cytokines(IFN-?1,IFN-?and CXCL10)might have pleiotypic immune effects against S.enterica QH infection.Of note,IL10 displays significant enhancement at levels of transcription and translation,suggesting that immunosuppressive effects mediated by IL10 may function as an early oral S.enterica QH infection.Here,we isolated four lytic Salmonella phages from different geographic regions in China and find that they are all belong to Myoviridae and have similar biological characteristics.The four phages share the similar evolutionary patterns with two strains derived from China and two strains derived from Chile.Based on the background of AT content>GC content in 4 phage genomes,information entropy analysis indicates that the overall nucleotide usage biases are shaped in gene population of 4 phages.The nucleotide usage biases at different codon positions are stronger than the overall nucleotide usage bias for gene population(p<0.001).This genetic feature directly contributes to synonymous codons tending to A/T end.The interplay between nucleotide composition constraint from bacteriophage itself and natural selection force4 phages into the similar evolutionary trends at the overall codon usage patterns.The nucleotide composition of phages constraint which plays an important role in shaping their synonymous codon usage patterns include keto skew at the first codon position,pyrimidine skew at the second position and AT skew at the third position.The phage SP1 showed rapid adsorption and efficient lysis on S.enterica QH.However,the therapeutic effect of phage SP1 on infected mice model is poor,oral phage SP1 relieved the clinical symptoms of infected mice,but during the continuous administration of phage SP1 treatment,the spleen bacteria capacity of mice still up to3.1×10~5 cfu/mL.We speculate that the delay of phage SP1 treatment and the phage resistance of S.enterica QH may be main reasons.We screened a mutant named QHM that resistance to phage SP1 from S.enterica QH,and SP1 could not adsorb on the surface of mutant QHM.The LPS of S.enterica QH is receptor of phage SP1,and the LPS structure of mutant strain QHM was incomplete.The base G mutated to base T at the 721 bp in the rfaJ gene that related to LPS synthesis in the mutant QHM genome,it caused the codon GAG that encoding glutamic acid mutate to the stop codon TAG,which resulted in the synthesis of glycosyl transferase failed,and the structure of the lipopolysaccharide formed incomplete.In order to identification the function of rfaJ gene,we constructed the deletion strain QH?rfaJ,and found that the rfaJ gene knockout will cause the incomplete LPS structure of the S.enterica QH,and the phage SP1 cannot absorb and infect the deletion strain QH?rfaJ.Finally,by analyzing the growth relationship between S.enterica QH,mutant QHM and phage SP1 and the fluctuation experiment,we found that the resistance mechanism is produced by natural mutation,and have no connection with phage SP1.In this study,a comprehensive and systematic study on the biological and genetic characteristics of S.enterica QH and its phages are performed.And through the infection model of mice and the phage treatment experiments,the immune response caused by foodborne Salmonella infection in mice and the therapeutic effect of phage were systematically evaluated.And the mechanism of Salmonella resistance to phage was explained.Our research provides theoretical basis for the study of phage therapy and phage resistance mechanism after Salmonella infection.