Characterize the Role of RpoS Gene on the Survival of C. Sakazakii in Adverse Environmental Stress Conditions

NON-TECHNICAL SUMMARY: Cronobacter sakazakii is an emerging pathogen with a mortality rate of 50-75% in neonates and children. It has been implicated in several infant formula-borne outbreaks of central nervous system infections and necrotizing enterocolitis in many countries, including the U.S. Further, C. sakazakii has been isolated from dry infant formula and has resulted in several recalls of contaminated formula in the U.S. High osmotic pressure is encountered by C. sakazakii during the spray drying step involved in the manufacturing process of powdered formula. C. sakazakii also potentially encounters a variety of unfavorable conditions, including low water activity, fluctuations in temperature and thermal stress during the manufacture and preparation of infant formula. Finally, the pathogen has to successfully overcome the stress due to acidic pH in the gastrointestinal tract, in order to cause foodborne disease. However, very little information is currently available on mechanism of stress survival in C. sakazakii. Regulation of gene expression is a common phenomenon observed in bacteria following exposure to environmental stresses. The gene encoding RpoS or alternate sigma factor plays a critical role in the survival of bacteria exposed to environmental stresses. Although the role of the rpoS gene or gene encoding alternate sigma factors on the survival of adverse environmental stresses has been studied in a number of foodborne bacteria, information on the genetic mechanisms involved in the stress survival of C. sakazakii is scarce. Understanding the knowledge of the molecular mechanisms behind the stress tolerance of C. sakazakii would potentially lead to improved strategies for preventing outbreaks of C. sakazakii infections. <P>

APPROACH: Based on the published rpoS sequence in E. coli appropriate primers will be designed to amplify the corresponding nucleotide sequence from C. sakazakii chromosomal DNA. The entire nucleotide sequence of the fragment will be determined and compared with published sequence of rpoS genes in the NCBI GenBank database. Based on the DNA sequence of the rpoS gene identified in the previous step, an appropriate mutation strategy will be followed to generate a mutant of C. sakazakii in the gene. The mutation in the gene will be confirmed by PCR, southern blotting and sequencing. The survival of the mutant and wild strains, and rpoS gene expression in the wild and mutant strains of C. sakazakii before and after exposure to stresses such as drying, heat shock, acid shock, low water activity, oxidative stress and osmotic stress will be determined and compared. The rpoS gene expression will be determined by Reverse Transcriptase-PCR. The proteins expressed by the wild and mutant strains of C. sakazakii before and after each stress will be analyzed by 2-D gel electrophoresis. Finally, the protein band(s) that are differentially expressed between the wild type and mutant will be identified. The protein sequences will be determined by mass spectrometry and compared with published sequences in nucleotide and protein databases. The viability characteristics of mutant and wild C. sakazakii in reconstituted infant formula will be determined under different conditions.