Restriction Fragment Length Polymorphism of Lactobacillus reuteri and Lactobacillus fermentum, Originating from Intestinal Mucosa, Based on 16S rRNA Genes

Abstract

Restriction Fragment Length Polymorphisms (RFLPs) of the 16S rRNA gene were determined for fourteen field strains originally labelled Lactobacillus reuteri , four Lactobacillus fermentum strains, and eight type and reference strains of Lactobacillus . The strains were mainly originating from intestinal mucosa. The probe was a 420 bp internal fragment of a Lactobacillus reuteri 16S rRNA gene, amplified by Polymerase Chain Reaction (PCR). The patterns were obtained by cleavage of the chromosomal DNAs by the restriction endonucleases E co RI and Hind III separately. Unweighted Pair Group Method, using arithmetic Averages (UPGMA) based on Jaccard coefficients (S J ) of the ribopatterns resolved five clusters at the 30% similarity level. The L. reuteri field strains formed two clusters according to their source of isolation; strains originating from rat formed one cluster while those from pig and human strains formed the other cluster. These strains could not be affiliated to the type strain of L. reuteri . However, two field strains, classified by phenotypically means as L. fermentum , clustered together with the type strain of this species. The numbers of 16S rRNA genes of the L. reuteri strains were, depending on the strain, determined to be at least five through seven. The ribopatterns were compared to the results of DNA:DNA hybridization and partial sequencing of the 16S rDNA genes. All but one of the L. reuteri field strains had more than 70% DNA similarity and identical 16S rDNA sequence to the type strain L. reuteri DSM 20016 T . Two of the L. fermentum field strains had identical 16S rDNA sequence and more than 70% DNA similarity to L. fermentum ATCC 14931 T . Ribotyping appears not to be a means to identify L. reuteri at the species level but eventually this is possible with L. fermentum .