Virulence Comparison And Pathogenic Factors Screening Of Cronobacter Sakazakii

Cronobacter sakazakii, a non-spore facultative anaerobes and Gram-negative, is an opportunistic foodborne bacterial pathogen that can cause serious illnesses such as bacteremia, septicemia, meningitis, and death in at-risk infants who are orally fed contaminated infant formulas. More seriously, C. sakazakii can lead to death with the average mortality rate of40%to80%in infants. Recent years, C. sakazakii has attracted worldwide concern and researches focused on its hazards have been widely carried out. However, very limited information is available regarding the characteristics of these diseases and the specific virulence factors of this bacterial species.Based on the experiments with neonatal SD rat and detection results of brain, blood and ratio of gut weight to remaining carcass weight, virulence of43C. sakazakii isolates were examined. The results showed that, isolates numbered7,23,36,38,50and52are more virulent than the others, and isolates numbered17,18,22,24,26,27,28,31,32,47,48and49exhibited lower virulence level. In order to investigate the virulence factors, we combined Box-PCR typing results, chose No.36as the higher virulence strain and No.17as the lower virulence strain to perform a comparison experiment. Two-dimensional gel electrophoresis （2-DE） was used to discriminate the different proteins in the two isolates and matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry （MALDI-TOF/TOF-MS） was performed to identify the proteins. More than108different protein spots could be observed clearly on the2-DE gels. Among these spots,101proteins were successfully identified and15proteins were speculated as potential virulence-related proteins including GroEL, HtpG, DegP, ClpB, OmpW, Flagellin, TerE, TerZ, TerD, TerX, TerA, VirB4, and BipA.In addition, uncharacterized protein yxxBOS and MLBr01669with no function presented.And in order to investigate the pathogenic mechanism of C. sakazakii, three kinds of hypothetical protein genes, a biofilm related gene （ESA02170）, an outer membrane protein gene （ESA02516） and a virulence-related gene （ESA02736） were selected to carry out gene knocking out experiments. Primers were designed according gene sequence and were used to amplify fragments upstream and downstream to the target genes, as well as the chloramphenicol resistant gene fragment. Then, recombinant DNA technology was employed to ligate the three fragments into pMD18-T vector which was used to obtain fragments containing long flanking regions that can be used to improve nocking efficiency. The length of upsteam fragment and downsteam fragment of ESA02170were672bp and615bp respectively, and the length of whole targeting fragment was2283bp. The upstream region and downstream region of ESA02516were791bp and632bp in length, and the whole knocking fragment was3508bp in length. The upstream region, downstream region, and whole fragment for ESA₀2736knocking out were661bp,652bp and2117bp respectively. The vector pKD46was electroporated into competent cells of strain ATCC BAA-894and the competent cells of ATCC BAA-894strain containing plasmid pKD46were prepared. The long fragment constructed previously was electroporated into the competent cells containing pKD46plasmid to produce specific mutants. The results of mutant selection using PCR method indicated that the colonies contained chloramphenicol resistant genes. But further studies should be performed to certificate the proposed mutants.Our research laid a good foundation on the research of virulence factor identification and mechanism for C. sakazakii.