Characterization And Preliminary Application Of Bacteriophage CT01 And CT02,Targeting Shigella Flexneri

Shigella is the pathogen of human bacillary dysentery.Shigella flexneri is the main epidemic serotype in developing countries,and its mortality rate is higher than other Shigella.Shigella contamination is common in dairy products,chicken,fruit and bread,and it has been listed as a key pathogen.Traditional food disinfectants can no longer meet the increasingly serious food safety problems,and phage is gradually accepted as a safe and effective bactericide.In this study,two lytic phages,named CT01 and CT02 against Shigella flexneri,were isolated from sewage.The transmission electron microscope results showed the two phages are different in morphology.The heads of them were icosahedral,and phage CT01 had a contractible tail;Phage CT02 had a short tail.The optimal multiplicity of infection was 0.001 for phage CT01 and 0.1 for CT02,with an incubation period of 30 min and 15 min,and a lysis period of 40 min and 25 min.The two phages were not sensitive to low temperature and could be stored at low temperature;They were more sensitive to high temperature,inactivated at 70℃ and80℃,respectively.Phage CT01 was more tolerant to acid and alkali.It was still active in the range of p H2-12,while phage CT02 was inactivated at p H12.Phage CT01 and CT02 were sequenced and uploaded to Genbank,and their Accession Numbers were OK018184 and OP917825,respectively.The full-length sequence of phage CT01 was 168362 bp,and the GC content was 35.9%.It was predicted that there are 273 ORFs with 17 t RNA;.The full-length sequence of phage CT02 was 38876 bp,and the GC content was 51%.It contained 47 ORFs without t RNA.Cluster analysis was carried out on the two bacteriophages.The results of phylogenetic tree showed that phage CT01 is a member of Tequatrovirus genus,belonging to Straboviridae family and Tevenvirinae subfamily.Comparative genomics analysis showed that the average nucleotide identity of phage CT01 with other Tequatrovirus members was more than 80%.Phage CT02 is a member of Teetrevirus genus of Autographiviridae family.Comparative genomics analysis showed that the average nucleotide identity of phage CT02 with other members of Teetrevirus was > 80% and < 95%,which strongly indicated that phage CT02 might be a new member of this genus.In this study,the bactericidal effect model of single phage on contaminated chicken breast,the experimental model of phage cocktail on contaminated chicken breast and the experimental model of cocktail phage on Shigella flexneri prevention in chicken breast were established.The results showed that in the single bacteriophage sterilization experiment,both bacteriophage CT01 and bacteriophage CT02 showed good bactericidal effect.In the treatment experiment,phage cocktail could reduce the number of Shigella flexneri,and the sterilization effect on cooked chicken breast was better than that on raw chicken breast.In the preventive experiment,phage cocktail has played an inhibitory role on Shigella flexneri,and the inhibitory effect is more obvious in cooked chicken breast.In summary,the two bacteriophages CT01 and CT02 have certain potential in controlling and preventing the contamination of Shigella flexneri in food.