Isolation And Screening Of C.sakazakii Phage And Study Of Its Bacteriostatic Effect

Cronobacter.sakazakii is a sporless,facultative and anaerobic Gram-negative Enterobacter.As a conditioned pathogen,it can cause severe meningitis,necrotizing colitis,bacteremia and other diseases in neonates,leading to a high mortality rate(40%?80%)and severe sequelae of nervous system injury,as well as developmental disorders.It is necessary to strictly control the contamination of C.sakazakii in food because of its widespread existence in all kinds of food and food processing environment.Phages are host-specific,targeting one or more host bacteria in most cases,effectively killing specific strains without damaging beneficial bacteria in the food or intestines.In addition,phage can effectively lyse cells existing in bacterial biofilms,which supports their use as an effective means to prevent and control biofilm-associated infections.Thus,phages and their derivatives have emerged as a novel,viable and safe option for the prevention or eradication of a variety of contaminants in a variety of foods and their processing environments.In this study,C.sakazakii ATCC 29544 was used as the host bacterium to isolate phage in the native environment of C.sakazakii,and its morphological characterization and biological characteristics were studied.The phage JK004 with the best bacteriostatic activity was selected by evaluating the phage growth inhibition effect on host bacteria.The genomic information of phage JK004 was analyzed by whole genome sequencing technology,and the bacteriostatic activity of phage JK004 was evaluated by bacteriostatic application test.Through the above research,the following results are obtained.(1)A total of 5 strains of C.sakazakii phages were isolated.Five strains of C.sakazakii phages,named JK001,JK002,JK003,JK004 and JK005,were isolated by sampling the finished and semifinished products in the flour factory,using C.sakazakii ATCC 29544 as host bacteria.The phage plaques of the five strains were round,translucent in the middle,without halo ring,consistent with the characteristics of strong phage,with a diameter of about 1-2.2 mm.Through transmission electron microscope observation,5 strains of phages all have typical icosahedral heads and contractable tails,belonging to the Caudovirales order and the Myoviridae family.(2)The biological characteristics of 5 strains of phages were evaluated.The phage host range results showed that all the 5 strains could lyse C.sakazakii ATCC 29544.The optimal infection complex number of 5 strains of phages was 0.1,0.01,0.1,0.01,0.1,respectively.The adsorption rates of host bacteria were 82%,89%,90%,95% and 81%.One-step growth curve showed that the incubation periods of 5 phages were 10 min,10 min,20 min,20 min,10 min,respectively.The outbreak periods were 50 min,40 min,50 min,40 min,20 min,respectively.Stable periods were60 min,70 min,50 min,60 min,90 min,respectively.The cleavage quantities were 16.5 pfu/infected cell,208 pfu/infected cell,134 pfu/infected cell,138 pfu/infected cell and 15.3 pfu/infected cell,respectively.(3)5 strains of phages has good heat resistance,acid and alkali resistance and salt tolerance.The effects of different physicochemical conditions on the activity of phages showed that the potency of 5 strains remained basically unchanged at 30?-40?,continued to decrease at 50?-70?,and was completely inactivated at 80?.When the p H value is 6-8,the aging value is relatively stable.When the acid or alkali is partial,the potency gradually decreases.When p H?2 or ?10,it is completely inactivated.Four of the five phages could maintain a titer of more than 4 log after 72 h treatment in 10% Na Cl solution.(4)Phage JK004 has high antibacterial activity.The results showed that the growth inhibition duration of 5 strains of phages was 3,4,3,5 and 3 h,respectively.The reduction of OD600 was0.063,0.112,0.099,0.164,0.083.Phage JK004 had the longest bacteriostatic duration and the largest reduction of OD600,and the bacteriostatic effect was significantly higher than that of other phages.Therefore,phage JK004 was selected as the object of subsequent study.(5)The genomic information of phage JK004 was clarified.Using the whole genome sequencing technology,the genetic material of phage JK004 was double stranded DNA,the length of which was 93825 bp,the average GC content was 32.86%,and the genes constituted a loop.A total of 125 coding genes were predicted,and the length of coding genes was 84840 bp,and the average length of genes was 679 bp,accounting for 90.42% of the whole genome.The genomic annotation results showed that there were no known virulence genes or transgenic genes in the phage JK004 gene,which indicated that the phage JK004 had potential safety and practical application value,and it was inferred to be Punavirus genus by collinearity analysis.(6)Phage JK004 has a good bacteriostatic effect.Phage JK004 can effectively inhibit the formation of biofilm of C.sakazakii,and the biofilm inhibition rate reached 87% after 48 h.C.sakazakii could be effectively removed from polyethylene,rubber and stainless steel materials.After6 h treatment,C.sakazakii on different materials were reduced by 3.33 log,3.32 log and 2.79 log,respectively.It can effectively inhibit the growth of C.sakazakii in rehydrated infant formula milk powder.After treatment at 4? for 16 h,the number of host bacteria decreased significantly.After treatment at 37? for 2-4 h,C.sakazakii could not be detected within 12 h.