Campylobacter jejuni, the cause of acute bacterial enteritis, is a Gram-negative, microaerophilic, spiral bacterium. The single polar flagellum of the organism has an important role inc ausing disease. In the published genome a nnotation of C. j ejuni some of the key regulatory flagellar genes are missing. Therefore the regulatory control of flagellar biogenesis is still not clear in the organism. FliK, the protein that controls the length of the flagellar hook and is also involved in switching of secretory pathway specificity in Salmonella, is among the regulatory proteins missing in the organism. We have identified the missingjliK gene in C. jejuni, which is Cj0041, and the homologue in its close relative Helicobacter pylori, HP0906. Although the protein encoded by Cj0041 is not readily identified by BLAST search against the major databases, a BLAST query of the C. jejuni genome database (Sanger Institute) with the FliK sequence from Aeromonas hydrophila identifies the Cj0041 gene product at E=0.056. Cj0041 knock-out mutants in two strains (NCTC11168 and 81116) 0 f C. jejuni were non-motile and showed a polyhook phenotype, which is in line with that of FliK mutants in Salmonella. SDS PAGE and mass spectrometry analysis with 'flagellar' filament proteins from Cj0041 mutants revealed increased production of FlgE hook protein in mutants compared to wild-type and indicated the synthesis of these polyhooks with mainly FlgE proteins. Microarray analysis of these mutants, verified by real-time PCR, showed overexpression of flagellar and other regulatory genes dependent on cr54 , one of the sigma factors involved in flagellar biogenesis, and provided a clear delineation of the cr54-dependent regulon. These results established the importance of Cj0041 or jliK in regulation of flagellar biogenesis in C. jejuni. We also have established by two-hybrid analysis the roles of Cj 1464 as jlgM and Cj 1465 asjlgN, two other key missing genes in the gene annotation of the organism. The roles of other flagellar genes such asjliS,jliD and Cj0848c were also studied to get a clear picture of flagellar biogenesis in C. jejuni. This study established that the organism shares most of the key regulatory flagellar genes found in model organisms like Salmonella and made it possible to define the regulation of flagellar biogenesis in C. jejuni more clearly than it has been described to date.

Enterobacter sakazakii is a bacterial contaminant of powdered infant formula milk that has been associated with necrotising enterocolitis, bacteraemia and a rare form of infant meningitis. The presence, persistence and growth of the organism in infant formula milk needs to be better understood to limit the occurrence of infection, and improved isolation methods need to be developed in order for companies to implement appropriate food safety management systems. A collection of E. sakazakii isolates from diverse clinical, food and environmental sources was compiled. Isolates identified biochemically as E. sakazakii formed four genomic clusters when housekeeping gene sequences (165 rONA and hsp60 loci) were compared. The reliability of presumptive isolate identification using commercial biochemical galleries was investigated in comparison to identification by 165 sequencing. The Biolog GN2 system appeared to be the most reliable identification gallery. A novel chromogenic medium, based on the a-glucosidase reaction, was developed to improve the efficiency of E. sakazakii isolation methods and is commercially available as Chromogenic Enterobacter sakazakii medium, Oruggan-Forsythe-Iversen formulation (OFI), CM1055, Oxoid ltd. The sensitivity and specificity of the OFI medium was compared with other proposed media. Also 486 food samples were tested for the presence of E. sakazakii. The organism was isolated from 67 samples using the OFI medium compared with only 19 using the conventional method. A novel enrichment medium was also investigated to improve recovery of E. sakazakii. Preliminary investigation of factors that may be associated with increased risk of acquiring E. sakazakii infection from contaminated infant formula indicated that E. sakazakii strains are able to survive in a desiccated state for over 6 months. They are also able to form biofilms on infant feeding equipment, can attach and invade human epithelial (CaCO-2) cells in vitro and can survive in human serum. Some strains may persist in macrophages, and many produce exopolysaccharide capsules which enhance biofilm formation and may contribute to evasion of host immune defences.

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