Molecular Diversity of Lactobacillus spp. and Other Lactic Acid Bacteria in the Human Intestine as Determined by Specific Amplification of 16S Ribosomal DNA

Abstract

A Lactobacillus group-specific PCR primer, S-G-Lab-0677-a-A-17, was developed to selectively amplify 16S ribosomal DNA (rDNA) from lactobacilli and related lactic acid bacteria, including members of the genera Leuconostoc, Pediococcus, and Weissella. Amplicons generated by PCR from a variety of gastrointestinal (GI) tract samples, including those originating from feces and cecum, resulted predominantly in Lactobacillus-like sequences, of which ca. 28% were most similar to the 16S rDNA of Lactobacillus ruminis. Moreover, four sequences of Leuconostoc species were retrieved that, so far, have only been detected in environments other than the GI tract, such as fermented food products. The validity of the primer was further demonstrated by using Lactobacillus-specific PCR and denaturing gradient gel electrophoresis (DGGE) of the 16S rDNA amplicons of fecal and cecal origin from different age groups. The stability of the GI-tract bacterial community in different age groups over various time periods was studied. The Lactobacillus community in three adults over a 2-year period showed variation in composition and stability depending on the individual, while successional change of the Lactobacillus community was observed during the first 5 months of an infant's life. Furthermore, the specific PCR and DGGE approach was tested to study the retention in fecal samples of a Lactobacillus strain administered during a clinical trial. In conclusion, the combination of specific PCR and DGGE analysis of 16S rDNA amplicons allows the diversity of important groups of bacteria that are present in low numbers in specific ecosystems to be characterized, such as the lactobacilli in the human GI tract.The human gastrointestinal (GI) tract consists of different habitats, in which the entire colon is occupied by mostly obligately anaerobic bacteria (29). The activity of these commensal bacteria in the GI tract has a major impact on the characteristics of the host. The microbiota-associated roles include protection against pathogens, development of the immune system, and positive effects on colonic health and host nutrition (6,14). Although the diversity of the gut microbiota has been investigated extensively by anaerobic culture techniques (7,27), it is receiving renewed interest due to the development and application of molecular techniques, especially those based on the 16S and 23S rRNA genes (45,47,48).Fluorescent in situ hybridization and group-specific hybridization approaches targeting rRNA in combination with advanced microscopy have indicated that the majority of the GI-tract microbial community is not accounted for by cultivation (19, 40, 42, 48). Phylogenetic analysis of rRNA genes, amplified by PCR, has been used as a rapid and efficient strategy to investigate the biodiversity of intestinal bacteria and revealed many novel species (42, 49). Furthermore, fingerprinting methods, such as denaturing or temperature gradient gel electrophoresis (DGGE or TGGE, respectively) of rRNA or ribosomal DNA (rDNA) amplicons, that allow the rap...

Bacteria, phages and pigs: the effects of in-feed antibiotics on the microbiome at different gut locations

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Diversity of the genus Lactobacillus revealed by comparative genomics of five species

“…Those sequences that were not already curated in this database were extracted from the EMBL database using the public Sequence Retrieval System (SRS) server at EBI (Zdobnov et al, 2002). The 16S rRNA sequence of the Bacillus subtilis type strain was added as outgroup, since previous analyses have suggested that enterococci and streptococci are too close to the lactobacilli to use as outgroups for 16S phylogeny (Heilig et al, 2002).…”

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Presence of two Lactobacillus and Bifidobacterium probiotic strains in the neonatal ileum

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