Classification of Lactobacillus plantarum by Restriction Endonuclease Analysis of Total Chromosomal DNA Using Conventional Agarose Gel Electrophoresis

Abstract

A total of 17 Lactobacillus plantarum strains that originated from different environments and 24 reference strains were classified by performing a restriction endonuclease analysis of total chromosomal DNAs digested with Eco RI, Hind III, and Cla I, and the resulting patterns were visualized after the fragments were separated according to size by agarose gel electrophoresis. The patterns were analyzed by using the Pearson product moment correlation coefficient and the unweighted pair group algorithm with arithmetic averages. All but two l. plantarum strains formed a cluster that was separated from the reference strains at a similarity level of 29% (cluster 1). The two remaining L. plantarum strains (cluster 2) merged with cluster 1 at a level of similarity of 28%. The reference strains formed four additional clusters, and four reference strains were stragglers. Cluster 3 (three Lactobacillus pentosus strains) and cluster 4 (including Pediococcus acidilacti CCUG 32235T [T = type strain] and Lactobacillus fermentum ATCC 14931T) merged with cluster 1 at a level of similarity of 25%. Cluster 5 comprised and Lactobacillus reuteri strains, and cluster 6 contained the type strains of Lactobacillus amylovorus, Lactobacillus gasseri, and Lactobacillus vaginalis. Cluster 1 (L. plantarum) was divided into three subclusters, and this subdivision reflected to some extent certain phenotypic features of presumed ecological significance, including the ability to adhere to intestinal mucosa (subcluster 1c) and starch and glycogen degradation (subcluster la). A principal-component analysis significantly distinguished the strains belonging to different species. Also, the subcluster 1c strains could be separated from the rest of the L. plantarum strains. The results of restriction endonuclease analysis of total chromosomal DNA were found to be reproducible, and this method can be used to (i) differentiate between similar strains belonging to the same Lactobacillus species and group strains within a species, (ii) distinguish between strains of different Lactobacillus species, and (iii) place strains in specific Lactobacillus species.