Bacterial identification by the 16S rRNA gene

University dissertation from Department of Food Technology, Engineering and Nutrition, Lund University

Abstract: The identification and classification of bacteria was initially performed with morphological methods applied on cultivated bacteria. It was not until the mid 1970s, when sequences of ribosomal RNA were discovered to be useful for bacterial evolution, that a new era started. This method showed to be far more accurate for bacterial identification. Today, identification of bacteria is totally dominated with 16S rRNA genes and the old methods based on morphology have been left behind with the exception of new species definitions. Later, in the mid 1980s the usefulness of the 16S rRNA gene for identification was further improved when the cloning approach was introduced. Cloning makes it possible to detect and identify non-cultivable and dead bacteria from all kinds of ecological nisches. The identification by the 16S rRNA genes from isolated and cloned bacteria from the food hygiene aspect proved to be very important revealing unknown members of the bacterial flora. As demonstrated in paper I, the method does not always result in a certain identification and thus other complementory methods have to be applied. A Yersinia high pathogenicity island was found in a meat isolate most closely related to the type strain of Serratia liquefaciens. However, the identification of the meat isolate was not achieved until the complementary method ribotyping was applied. To evaluate the bacterial composition of fresh and spoiled meat and fish (paper II and III) two complementory approaches cultivation and cloning were conducted in parallel. Evidently, the true composition of the mentioned bacterial floras can not be determined solely by either of the methods. Cloning was applied on human mesenteric lymph nodes to detect translocated cultivable, non-cultivable and dead bacteria. The translocated bacteria (paper IV) was difficult to reveal with cloning hence the low amount of bacteria present. When the bacterial load is small, as in a lymph node, contaminating bacterial DNA from e.g. chemical reagents bias the actual results rendering them uncertain. In conclusion, the present doctoral thesis has investigated identification by the 16S rRNA gene of bacterial isolates and clones from meat, fish and human mesenteric lymph nodes. Occasionally, an identification can not be determined solely with the 16S rRNA gene and therefore other molecular based methods are needed.