Systematics of Lactobacillus spp. of probiotic potential

University dissertation from Division of Food Technology, Lund University

Abstract: Lactobacillus is a heterogeneous bacterial genus comprising at present 91 species. The high species number often makes classification and identification difficult, especially as the taxonomy to a high degree has been based on phenotypical traits. Members of the genus are of commercial importance, involved in a range of industrial products and applied as probiotics, “live microorganisms which when administered in adequate amounts confer a health benefit on the host”. The development of phylogenetic analysis of the 16S rRNA gene sequence in the 1980s resulted in many changes within the taxonomy of the genus, and showed a discrepancy between the former phenotypic identification and the present molecular-based phylogeny. Therefore, the phylogeny of the genus based on 16S rRNA gene sequence analysis was evaluated in Paper V, concerning the increasing number of species since Schleifer and Ludwig performed such a study in 1995. However, closely related organisms of probiotic interest within Lactobacillus are still under taxonomic discussion. Due to that fact, the present investigation evaluated molecular-based identification methods for differentiation of members of Lactobacillus acidophilus and Lactobacillus casei/paracasei complexes, respectively. At present, these two assemblies contain strains of probiotic applications, e.g. Lactobacillus rhamnosus GG, Lactobacillus casei Shirota, “Lactobacillus paracasei defensis”, Lactobacillus gasseri SBT2055SR, and Lactobacillus acidophilus ADH. As the identity of the conceivable exogenous strain is one of the most important features in finding new probiotics, the identification of members within the complexes is indispensable. In the present study PCR-based methods, REA, RFLP, 16S rRNA gene sequencing and TTGE were used for the differentiation and identification of species and strains within the two complexes of L. acidophilus and L. casei/paracasei. In Paper I and II, the identification possibilities and the classification of the L. casei/paracasei complex were studied. The different members of the group could be differentiated with TTGE. The results indicated that one underlying reason for the difficulties in reaching clear-cut taxa within the complex might be the presence of polymorphisms within the 16S rRNA genes. In addition, a higher degree of variable positions than previously reported was observed in the 16S rRNA gene fragments of the members in the complex. Sequence comparison between the 16S rRNA gene copies of L. casei (CCUG 21451T) and L. zeae (CCUG 35515T) demonstrated that the two species shared almost the same sequences. In Paper III, the composition of the vaginal lactobacilli flora and the L. acidophilus complex were identified with genotypic-based methods. It was shown that the dominating Lactobacillus species in the healthy vagina of Swedish women were Lactobacillus crispatus, Lactobacillus gasseri, Lactobacillus iners and Lactobacillus jensenii. For the first time L. iners was shown as a dominating species in the vaginal ecosystem. With the vaginal lactobacilli flora in mind, we selected strains of human origin for an administration study. Vaginal probiotics are a rather new area of investigation. Consequently, not much is known about the mechanisms, conditions or characteristics needed for vaginal probiotics. In Paper IV, five exogenous strains were re-isolated from vaginal and faecal samples after oral administration. Surprisingly, the most commonly recovered exogenous species was Lactobacillus plantarum 52A, suggesting that this strain possesses the qualities required for establishment in the gastrointestinal tract but also in the vaginal environment. Transmission electron microscopy studies indicated different surface composition of 52A compared to other L. plantarum strains. In conclusion, the present doctoral thesis has investigated the Lactobacillus genus by molecular-based technologies, and selected strains within the genus had been proved to be able to survive and establish in the human gastrointestinal tract and vagina. This could be used as a platform for future investigations of probiotics in general and vaginal ones in particular.

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