| Cronobacter spp.is a group of facultative anaerobic and gram-negative pathogen that can parasitize in the intestinal tract of humans and animals.The genus Cronobacter have found that can cause life-threatening infection in neonates and infants with necrotizing enterocolitis,bacteraemia and meningitis with case fatality rates ranging between 40 and 80% through the consumption of infant formula.However,the source and route of contamination of Cronobacter spp.in food,and its molecular mechanism for survival in acid conditions have not been clarified.In this thesis,comprehensive analysis of the phenotype and genotype characteristics among strains based on the contamination investigation and risk identification of Cronobacter spp.in Chinse vegetables.API20 E,drug sensitivity test,ERIC-PCR,molecular serotype,MLST typing and genome sequencing technology were adopted into this analysis.On the basis of this,more acid-resistant strain was successfully screened from food-borne isolates of Cronobacter spp.Then,conjoint analysis between transcriptome and proteome was conducted to reveal the overall regulatory process of Cronobacter sakazakii under acid stress.Finally,the acid resistance mechanism of candidate genes was further investigated.The specific contents are as follows:1?A total of 122 positive samples of Cronobacter spp.were detected from 403 vegetable samples collected at national scale,with a contamination rate of 30.27%.The highest pollution rate was 35.21% in the South China of the seven different geographical regions.The contamination level of Cronobacter spp.in 16.39% of positive samples was higher than 110 MPN/g,and the contamination level of Cronobacter spp.in 11.48% of positive samples was 10?MPN < 110.Results of drug sensitivity test showed that,59.77 % of Cronobacter spp.isolates were resistant to cefalotin,and two strains showed multiple drug resistance.Results of the molecular serotype showed that,C.sakazakii O1 serotype was dominant serotype(23.98%).ERIC-PCR and MLST results reflected that distinct genetic diversity of vegetable isolates,and many new loci and new ST types were discovered.The results of MLST analysis showed that clinically relevant 15 STs were detected from isolates,and ST60 was the dominant ST in this study(8 strains).2?RNA-seq and iTRAQ techniques were applied to construct the transcription and protein expression profiles of C.sakazakii at pH 3.6,pH 4.5 and pH 5.5,respectively.There were 181 differentially expressed genes in the strain treated with pH 5.5,among which 76 genes were up-regulated and 105 genes were down-regulated.There were 946 differentially expressed genes in the strain treated with pH 4.5,248 of which were up-regulated and 698 were down-regulated.There were 717 differentially expressed genes in the strain treated with pH 3.6,among which 439 genes were up-regulated and 278 genes were down-regulated.Transcriptome data indicated that physiological activities,such as flagella,amino acid synthesis and metabolism,sugar metabolism and fatty acid metabolism,played an important role in the acid-resistant process of C.sakazakii exposed to acid stress.The proteome data revealed that 164 proteins were differentially expressed in the acid stress group at pH 5.5 treatment,among which 79 genes were up-regulated and 85 genes were down-regulated.322 genes were differentially expressed in the acid stress group at pH 4.5 treatment,including 150 up-regulated genes and 172 down-regulated genes.Under pH 3.6 treatment,770 genes were differentially expressed in the acid stress group compared with the control group,of which 439 genes were up-regulated and 278 genes were down-regulated.The protein levels of chemotaxis,flagellum assembly,sugar metabolism pathway and amino acid metabolic pathways of C.sakazakii were significantly changed under acid stress.A good consistency existed in GO,KO and other functional enrichment between transcriptome and proteome.However,the correspondence between gene and protein expression is poor.3?Based on WGCNA of transcriptome,it was found that there is a high correlation between two functional pathways(the phosphorylase system and nucleic acid synthesis and transport)and the acid stress response.Expressions of the 23 candidate genes were verified by qPCR,and the results of qPCR were compared with the corresponding RNA-seq data.The results showed that the expression trends of most genes were consistent between qPCR and RNA-seq data,indicating that RAN-seq data were reliable.The expressions of genes 0576,1324,1325 and 1326 are high,and 1324,1325 and 1326 demonstrated high homology with fructose-PTS operon fruKBA.Therefore,we constructed experimental system for gene knockout and complement in C.sakazakii,and knockouted and completed of fructose-PTS 1324,1325 and 1326 to compare their acid resistance.The results showed that 1325 gene seemed to have a more obvious acid resistance in C.sakazakii.However,1324 gene only contributed to acid resistance when pH 3.6,and negative regulation might be presented in this mutant when pH 5.5 and pH 4.5.4?According to the preliminary study of our group,the candidate gene grxB was knocked out and differences of phenotype and acid tolerance between wild type and mutant were compared.The phenotype including lower survival rates,more serious damage of biomass,higher leakage of ATP in the cell under acid condition were presented in ?grxB,indicating that grxB involved in C.sakazakii acid response process.This may be due to the fact that grxB,as an important part of Grx system,can consume protons through glutathione metabolism pathway to improve its acid resistance.Moreover,cross-protection against acid-oxidation stress might be involed in positive response to acid resistance in C.sakazakii,as grxB is an important protein maintaining the intracellular REDOX state system.grxB played an important positive regulatory role in CSH and autoagglutination,revealing the necessity of grxB in C.sakazakii adhesion process.In addition,although motor ability wasn't influenced by the absence of grxB,the formation of biofilm was affected to some extent.There is a certain correlation between acid stress response and biofilm formation ability in C.sakazakii.To sum up,based on the investigation of Cronobacter spp.contamination in vegetables in China,resource database and risk identification database of food-borne Cronobacter spp.with Chinese characteristics were established;According to the analysis of transcriptome and proteomics data,the expression profiles of C.sakazakii under stresses were revealed;Moreover,effect of fructose-PTS system and grxB gene on acid stress tolerance in C.sakazakii was preliminarily demonstrated.The conclusions of this thesis provided theoretical basis and data support for effective prevention and control on hazards caused by Cronobacter spp. |
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