Characterization And Potential Biocontrol Agents In Food Safety Of Bacteriophage JC01 And JC03,Targeting Cronobacter Sakazakii

Cronobacter is an important food-borne pathogen that can cause severe meningitis,necrotizing enterocolitis and bacteremia in immunocompromised people,especially neonates with a mortality rate as high as 40%to 80%and may cause complications including delayed neurodevelopment,hydrocephalus and permanent nerve damage.Contamination with Cronobacter Sakazakii is common in infant formula milk powder which has become an important factor affecting food public safety worldwide.Cronobacter Sakazakii is classified as a class A pathogen of infant formula powder.Traditional food sterilants have been unable to solve increasingly serious food safety problems.Phages as a specific sterilization method have attracted more and more attention because of their advantages of natural and effectively protection.In this study,two lytic phages,JC01 and JC03 were isolated from hospital sewage targeting Cronobacter Sakazakii.Two phages were highly lytic with narrow host range,large burst size and high stability,however they were significantly different in other respects.Transmission electron microscopy showed that JC01 was classified in the Caudovirales,Siphoviridae and had a single tail fiber like other Chi-like phages,while JC03 had an icosahedral head and a contractile tail and belonged to Caudovirales,Myotaviridae.JC01 could lyse cz-1801,cz-1804 and JC01 could lyse cz-1802,cz-1803.The optimal multiplicity of infection was 0.01 for phage JC01 and 0.1 for JC03,with an incubation period of 20 min and 30 min,respectively,and a lysis period of 40 min for both.Phages JC01 and JC03 were not sensitive to low temperature and could be stored at low temperature,but were more sensitive to high temperature.After incubation at 70°C for 1 h,both phages lost their activity.Phage JC01 was more stable than phage JC03 under acid-base environment.JC01 remained active at p H4-p H11,while JC01 survive at p H4–p H10.JC01 and JC03 were subjected to sequencing and were uploaded to Genbank.Since the gene information of phage JC01 was more novel than that of phage JC03,the genome of JC01 was chosen for in-depth analysis.Thorough functional and genomic analysis of the phage,JC01 was classified as a novel Chi-like phage.Genome of JC01 was 61,736 bp,which consists of 76 open reading frames.JC01 closely resembles to the Chi-like phage genomes in genome size,gene content,and gene order.Comparative genome analysis revealed that the JC01 genome had the highest similarity to the genomes of the Chi-like virus Salmonella phage FSLSP-124,Salmonella phage i EPS5,and Salmonella phage SPN19.Phylogenetic analysis revealed that JC01 is close to Chi-like phages within the family of Siphoviridae.JC01 closely resembles phage Chi and Chi-like phages at the nucleotide and protein levels.In addition,the JC01 genome showed typical gene synteny with the genomes of phage Chi and Enterobacter phage Enc34.They have the same 12bp base sequence at the end of the genome.These data suggested that JC01 is a distant member of the Chi-like phage cluster.It is the first Chi-like phage to infect Cronobacter Sakazakii so far.In this study,we studied the lysis effect of two phages and their application as a phage cocktail to kill Cronobacter Sakazakii.Phage JC01 and JC03 are used as cocktail phage to kill Cronobacter Sakazaki in infant formula milk powder and milk,while also assessing the cocktail phage ability to prevent biofilm formation of Cronobacter Sakazakii in different brands of infant formula milk powder.Both JC01 and JC03 could kill Cronobacter Sakazakii at different MOI(0.01,1,100)within 14 h.The combined-phage preparation(2×10~8pfu/m L)was found to possess a strong bactericidal effect on Cronobacter Sakazakii(?10~4cfu/m L),resulting in a significant reduction in cell numbers,to below the limit of detection(<10 cfu/m L).In the infant formula milk powder,the number of Cronobacter Sakazakii decreased by 4 logs(below the limit of detection)after 2 hours at 4°C and remained 16 hours.While at 25°C,the number of bacteria decreased by 4 logs after 2 hours,but the host bacteria showed regrowth after 12 hours and increased to 0.6±0.02 log CFU/ml at 16 hours,which was still greatly decreased compared with the control group.In liquid milk,the number of bacteria has been below the detection limit after 2hours at 4°C and 25°C.The phage cocktail also demonstrated promise in preventing the establishment of biofilm.In summary,we isolated two specific phage JC01 and JC03,and analyzed their biological characteristics,genome and application,proved that phage JC01 belonged to the distant Chi-like phage cluster.The two phages have great potential in preventing and controlling the contamination of dairy products by Cronobacter Sakazakii.