Suckling Mice-based Study On Pathogenicity Of Cronobacter Sakazakii

Cronobacter sakazakii(formerly Enterobacter sakazakii)is an opportunistic pathogen that causes meningitis,sepsis and necrotizing enterocolitis in neonates and infants through consumption of contaminated milk-based foods.At present,the domestic research on Cronobacter sakazakii is mainly focused on biological characteristics,rapid detection and biological control,and the virulence factor and pathogenicity of Cronobacter sakazakii were not much reported.The pathogenesis of Cronobacter sakazakii is still inconclusive.In this study,the 18 Cronobacter sakazakii strains which were subtyped by three molecular typing methods,pulsed-field gel electrophoresis(PFGE),multi-locus typing(MLST)and Enterobacterial Repetitive Intergenic Consensus PCR(ERIC-PCR),were used to perform the screening test of suckling mice according to intestinal corpse ratio.Besides,by combining the source information(food brand,food type)and MLST(ST type),three Cronobacter sakazakii strains were finally screened for suckling mice experiment.In the experiment of suckling mice,we observed the characteristics of the disease that could be caused by the infection of Cronobacter sakazakii,and analyzed the histopathological changes that happened to suckling mice.Combining the detection of bacteria and cytokines in blood,the pathogenicity of Cronobacter sakazakii was demonstrated.By whole genome resequencing,the eight strains of Cronobacter sakazakii were compared with the sequence of standard strain ATCC BAA894,which provided the basis for better revealing the pathogenicity of Cronobacter sakazakii.The main findings are as follows:(1)The results of the virulence screening for 18 Cronobacter sakazakii strains showed that:according to the intestinal corpse ratio,strain 12-12(2),14-15(1)and 36-8-1 were selected as the strongest virulence group,strain 6-16(2)and 5-9(1)were selected as the general virulence group,strain 2-9-1,3-11(1)and 7-17(1)were selected as the weakest virulence group.Combining the source information(food brand,food type)and MLST(ST type),three Cronobacter sakazakii strains were finally screened for suckling mice experiment:2-9-1(ST4),14-15-(1)(ST93)and 36-8-1(ST4).(2)The results of suckling mice invasion experiment showed that:the mice were inoculated with Cronobacter sakazakii strains at the concentration of 10gcfu/mL,and both suckling mice in the experimental group and the control group were alive after seven-day experiment.In the experimental group,the movements of the suckling mice are normal,but the growth and development were slower than the control group.The mice in the same age group had smaller body size.After suckling mice were sacrificed,the intestinal tissue,liver,kidney,spleen and brain tissue of the experimental group had significant difference with the control group.The pathogenicity of the strain 36-8-1 was the more obvious than the others.The relationship between different strains and pathological changes showed that the stronger virulence that the strain had,the more obvious pathological changes the organism got.(3)The results of blood analysis showed that:there was no colony formation on the cultured blood plate.(4)The results of TNF-a,IL-1 and LDH viability showed that:the contents of the experimental group were significantly different from the control group.The levels of TNF-a(538.030 ng/L),IL-1(148.895pg/mL)and LDH(2765.32U/L)in strain 36-8-1(ST4)were the highest among the tested strains.And its TNF-a and LDH had significant difference with the other two strains.(5)The results of whole genome resequencing showed that:By comparing with the reference sequence standard strain ATCC BAA894,there were significant difference between strain 7-17(1),strain 5-9(1)and the other tested strains in SNP.For strain 5-9-1,the insertion mutations,deletion mutations were most in InDel(insertion-deletion)results.All the indexes were the highest among the other strains.However,strongest virulence strain 36-8-1(ST4),which assumed to be have an outstanding performance,showing a disappointing result.The insertion mutation,the deletion mutation and the InDel of CDS region were relatively low when compared with the other strains.In a word,Cronobacter sakazakii 14-15-(1)(ST93)and 36-8-1(ST4)were strongest virulence strains and revealed strongest pathogenicity in suckling mice experiment.Cronobacter sakazakii 2-9-1(ST4)was weakest virulence strain and revealed weakest pathogenicity in suckling mice experiment.