Culture free DGGE and cloning based monitoring of changes in bacterial communities of salad due to processing.

To assess the possibilities of a culture-independent monitoring of bacterial communities in the food chain, samples of salad from farming sites as well as corresponding, processed products in stores were analysed. The bacterial DNA was extracted using a modified soil extraction protocol. Amplification of 16S rDNA was carried out using primers specific for eubacteria and enterobacteriaceae. Fingerprints of 200/370 bp respectively were obtained by denaturing gradient gel electrophoresis (DGGE) analysis following PCR and nested PCR amplification. In parallel to DGGE analysis, clone libraries containing PCR fragments of the ribosomal gene were constructed and clones were screened by DGGE. DGGE analysis indicated a high diversity of bacterial communities in salad samples. Fingerprints indicated clearly reduced diversity of bacterial communities in processed samples from markets compared to field-grown salads. Surprisingly, primers pointed out in literature as specific for enterobacteriaceae did amplify pseudomonadeceae as well. Therefore, the more specific primers fD2 and rP1 were used subsequently in this study to amplify specific members of the family enterobacteriaceae. A total of 11 different 16S rDNA sequences were obtained and subjected to sequencing and phylogenetic affiliation. Sequences derived from the eubacterial clone library from organically farmed salad were affiliated to the family microbacteriaceae and pseudomonadaceae. In addition, a potential new genus within the family of enterobacteriaceae was detected. Furthermore, a sequence showing 98.9% similarity to Pseudomonas libaniensis (fluorescence subgroup) was found in a processed salad sample but not in the corresponding field samples. This species is generally known as an opportunistic pathogen. Whereas molecular based monitoring of bacterial communities in food still may need more experience and standardisation to detect specific bacteria present, the monitoring strategy presented in this paper, combining DGGE analysis with the construction of clone libraries, is an attractive method for culture-independent monitoring of changes of bacterial communities in the food chain.