RESTRICTION ENDONUCLEASE PATTERNS AND MULTIVARIATE-ANALYSIS AS A CLASSIFICATION TOOL FOR LACTOBACILLUS SPP

Three Lactobacillus plantarum and seven Lactobacillus reuteri strains were studied by using restriction endonuclease analysis (REA) combined with principal-component analysis (PCA) and soft independent modeling of class analogy (SIMCA). Chromosomal DNAs from the strains were extracted and cleaved with restriction enzymes, and the DNA fragments were separated according to size by agarose gel electrophoresis. Band patterns were read by using a laser densitometer. The procedure to obtain reproducible digestion patterns with a suitable number of DNA fragments was optimized. Asp 718, ClaI and EcoRI were the most suitable of the 17 restriction enzymes tested, each giving 30 to 50 DNA fragments down to a molecular weight of 4.2 x 106. The digestion patterns of all three enzymes, which gave a data set for 10 strains and 80 variables, were used for classification by PCA and SIMCA. All strains were clearly separated, and the separation within each species was in general accordance with data on DNA-DNA homology found in the literature. A class model was created for L. reuteri 1073, 1068, 1048, and 1044. Closest to this model was L. reuteri DSM 20016(T) (T = type strain), followed by L. reuteri DSM 20015; the most distant strains were the L. plantarum strains. We concluded that restriction endonuclease analysis, combined with PCA and SIMCA, can be used for the classification of Lactobacillus spp.