CLASSIFICATION OF LACTOBACILLUS-REUTERI BY RESTRICTION-ENDONUCLEASE ANALYSIS OF CHROMOSOMAL DNA

A total of 14 field strains originally labeled Lactobacillus reuteri, 4 field strains of Lactobacillus fermentum, and 12 Lactobacillus type strains, as well as four additional reference strains, we classified by performing a restriction endonuclease analysis of chromosomal DNAs digested with Asp 718, ClaI, and EcoRI. The patterns were analyzed by using (i) Jaccard coefficients and the unweighted pair group algorithm with arithmetic averages and (ii) principal-component analysis (PCA) and soft independent modeling of class analogy (SIMCA). Grouping by using the Jaccard coefficients and the unweighted pair group algorithm with arithmetic averages resulted in six clusters, defined at a similarity level of 69%. Cluster 1 comprised 10 field strains of L. reuteri, Lactobacillus sp. strain DSM 20056, and L. reuteri DSM 20016T (T = type strain) and could be divided into four subclusters. Cluster 2 included two L. reuteri field strains. Cluster 3 included one field strain labeled L. reuteri and one field strain labeled L. fermentum. Cluster 4 could be divided into three subclusters and included two field strains labeled L. fermentum, one reference strain and the type strain of L. fermentum, and four additional type strains. Cluster 5 contained two Lactobacillus plantarum strains. Cluster 6 included two type strains. The two numerical methods gave the same general results, but the PCA-SIMCA method resulted in a more complete view of the relationships. The SIMCA analysis grouped L. reuteri DSM 20016T , Lactobacillus Sp. strain DSM 20056, and, with one exception, all of the L. reuteri field strains as a ''class.'' Lactobacillus helveticus DSM 20075T and Lactobacillus casei subsp. pseudoplantarum DSM 20008T were the type strains most closely related to the L. reuteri SIMCA class. The L. reuteri strains isolated from rats could be separated from the L. reuteri strains isolated from humans and pigs by the PCA. DNA-DNA-hybridization showed that strains classified as L. reuteri on the basis of the results of the restriction enzyme analysis also exhibited >70% DNA-DNA-homology to the type strain of L. reuteri. The phenotype of L. reuteri (ability to ferment carbohydrates) coincided in most cases with the genotype; this seemed not to be the case for L. fermentum strains.