Characterisation of Food Associated Bacteria by DNA based Methods, with Special Reference to Enterobacteriaceae

Abstract: The presence of genes in food, encoding some virulence factors, was studied by PCR, and species of Enterobacteriaceae, associated with food, were studied by the DNA-based methods of TTGE, ribotyping and sequencing. The flora of fresh and chill-stored pork were analysed by a culture-independent approach, using specific amplification of 16S rRNA genes followed by cloning and sequencing. No evidence for the presence of toxin coding genes related to E. coli was found in meat and fish but a strain of Serratia liquefaciens positive for the Yersinia HPI, was isolated from a sample of beef. Up to now, this is the first Serratia found to be HPI positive but the significance, from a food hygiene point of view, is not clear and S. liquefaciens is only occasionally isolated from clinical specimens. For the primary purpose of evaluating the TTGE method for identification of Enterobacteriaceae associated with food, strains isolated from meat, fish and milk and human isolates of Klebsiella, a genus that is frequently found in food, were included in the study. TTGE proved to be a tool for differentiating E. coli, E. vulneris, E. cloacae, E. amylovora, H. alvei, K. terrigena, P. agglomerans, R. aquatilis, and distinguished between the clinically important strains of K. pneumoniae and K. oxytoca. In addition, it was possible to identify R. aquatilis genomospecies (GS) 1 and 2 and presumably one additional GS group of this species using TTGE. The closely related species of K. planticola, K. ornithinolytica and E. aerogens were not separated by TTGE. Furthermore, the C. freundii and Y. ruckeri type strains could not be distinguished from each other and S. liquefaciens and S. proteamaculans could not be identified due to diffuse or lack of band on the gel. Ribotyping was useful to subdivide Rahnella aquatilis into genomic subtypes and for confirmation of the TTGE grouping. The strains of H. alvei and S. liquefaciens were genomically defined by ribotyping. Sequencing of 16S rRNA genes was useful for the verification of the identity of Klebsiella isolates and the different Rahnella genomospecies. In addition a protocol was developed for rapid identification of Klebsiella spp. By picking a colony from an agar plate, preparing the DNA for PCR and running TTGE over night, identification was possible within 24 hours. A culture-independent analysis of the flora of fresh and chill-stored pork gave an overall picture, of the bacterial composition, which was in agreement with cultivation-dependent methods previously used. The initial flora turned out to be diverse in terms of the numbers of genera present, and a change occurred during refrigerated storage into a gram-negative psychrotrophic flora, dominated by Pseudomonas. The method used here also indicated the possibility of detecting bacterial species such as Eperythrozoon, that do not grow on ordinary media.