Isolation,Screening And Characterization Of Cronobacter Sakazakii Phage And Its Application

Cronobacter sakazakii is an important foodborne pathogen that can cause severe septicemia,meningitis,necrotizing enterocolitis and other diseases in newborn.High temperature sterilization is a conventional method to control this bacteria but would damage some nutrients of the food.Chemical disinfectants is another popular way to use but with the challenges of multiple resistant to drug strains.To overcome those defects,it is necessary to find an alternative effective control method.Phage is considered as a new effective bacteriostatic way due to its specificity,non-drug resistance,less side effects and low cost.The objectives of this thesis were to isolate naturally the specific phages,to screen and characterize them,and to investigate their effects on the pathogens in the milk products.The main results are as follows:1.Phage TBC-1,TBC-2 and TBC-3,which were isolated from river with Cronobacter sakazakii as the host,had good lysis ability to the host.The plaque of TBC-1 and TBC-2 was clear and bright,and the phage of TBC-3 had irregular edge and fuzzy plaque.TBC-1(100%),TBC-3(77.8%)and TBC-2(55.6%)showed strong lysis ability to sakazaki strains(n=9)in laboratory.2.Phage TBC-1 and TBC-3 had different characteristics.The capsid of TBC-1was icosahedron with a contractible tail.The optimal MOI was 0.001.The one-step growth curve showed that the latent time was 20 min and the outbreak period was 50min and the number of outbreak was 100.The phage kept its high stability at 40-50?and p H=4-10.It had an effective lysis ability to the host and the inhibition is higher with the increased MOI.TBC-1 could remain stable for 7 months in skimmed freeze-dried milk powder,glycerin and LB broth at 4?.TBC-3 was an icosahedron with a long non-contractible tail,and its optimal MOI was 0.1.It was hardly to get its growth curve.Based on the morphology of the plaque and electron microscopy,it was concluded that the phage was a temperate phage.3.Phage TBC-1 belongs to Myoviridae,Ounavirinae subfamily and Kolesnikvirus genus.It can be cut into 8 bands by Eco R I.The nucleic acid type is ds DNA.The result of sequencing showed that the total length of the genome is 85313bp.It had 118 open reading frames(ORFs),29 functional reading frames and the rest are hypothetical protein.The average GC content is 43.88%.The known functional modules are divided into structure,packaging,DNA replication and repair,nucleotide metabolismlysis and other proteins.There was no lysogen gene.The phylogenetic tree shows that it belongs to Myoviridae,Ounavirinae subfamily and Kolesnikvirus genus.4.Phage TBC-1 had significant bacteriostatic effect on Cronobacter sakazakii in milk products.TBC-1 can kill ATCCBAA894 effectively by using different MOI and the inhibition is higher with increased MOI.ATCC12868 just could be killed at MOI=1000.TBC-1 had higher inhibition at 25?,compared with 37?in milk and milk powder,TBC-1 at MOI=10~4 had higher inhibition than that at MOI=10~2.TBC-1at MOI=10~6 could inhibit the growth of ATCCBAA894 in milk at 25?,but it could not kill the host in the milk powder,which shows food matrices can effect the lysis effenciency.In some conditions(MOI=10~6 at 37?in milk and MOI=10~4 at 25?in milk powder),the inhibition effect of TBC-1 on ATCCBAA894 is higher than that on indicate bacteria.Different host show the different lysis ability.The results showed that phage TBC-1 isolated from river had great bacteriostatic effect.It could kill host effectively in the milk and can be applied in the contaminated milk and other food products.TBC-1 can be further developed into a new bacteriostatic method for drug-resistant bacteria.