Detection Method And Acid Mechanism Research On Cronobacter Sakazakii

Cronobacter sakazakii is a gram-negative pathogen, which can parasitize the intestines of humans and animals and cause serious diseases, such as neonatal meningitis, bacteremia and necrotizing enterocolitis, with mortality rate ranging from 40% to 80%, hence it draws great attention all over the world. The contaminated milk powder has been found to be the main source of infection. There are defects in the traditional methods for isolating and identifying Cronobacter. It makes a lot of sense for identifying Cronobacter and preventing and treating the infection to develop efficient biotechnology including molecular biological techniques. In addition, the acid tolerance of foodborne pathogens is one of key preconditions of infection. However, the domestic researches on the response mechanism of Cronobacter sakazakii to acid stress were rarely reported.This study established a better method than DFI and mLST for isolating Cronobacter by adding vancomycin, a-glucosidase, cephalosporins and sucrose into the selective media. The sensitivity of this modified method, DFI and mLST was 100%,87.5% and 87.5% respectively, and specificity was 88.8%,77.7% and 87.5% respectively. Then, a specific molecular classification target technology was developed based on restriction fragment length polymorphism analysis of gluB fragment digested by TaqI restriction enzyme, which could rapidly and accurately identifiy Cronobacter and distinguish Cronobacter dublinensis, Cronobacter malonaticus, Cronobacter muytjensii and Cronobacter sakazakii. Finally, the genes OmpW and GrxB were knocked out successfully by bacterial conjugation, and the roles of OmpW and GrxB in the acid resistance of Cronobacter sakazakii were investigated from aspects of acid tolerance, morphology and bio film. The results indicated that the two genes were responsible for acid resistance of this bacterium, which provided reference for the further study on the roles of OmpW and GrxB in pathogenic mechanism of this bacterium.This study laid a good foundation for preventing Cronobacter sakazakii contamination and guaranteeing food security.