Molecular identification and characterisation of bifidobacteria and lactobacilli in the human gastrointestinal tract: Dissertation

Bifidobacteria and lactobacilli are considered to be members of the beneficial microbiota in the human gastrointestinal (GI) -tract. The present study describes the development and validation of new molecular methods for the detection and analysis of bifidobacteria and lactobacilli and the application of new techniques to study Bifidobacterium and Lactobacillus populations in the human intestine. A method based on genus-specific PCR of 16S rDNA and denaturing gradient gel electrophoresis (DGGE) was developed and validated for profiling Bifidobacterium populations in human faeces. The PCR-DGGE method is a qualitative tool for assessing species/strain composition of complex communities by a single PCR reaction and subsequent resolution of the amplification products by DGGE in a sequence-dependent manner. The approach greatly facilitates the monitoring of faecal samples from large numbers of subjects to reveal bifidobacterial diversity and shifts occurring in it. The identification of DGGE fragments can be done by subsequent cloning and sequencing of the PCR products. Genotypic methods were developed and evaluated for the identification and characterisation of Lactobacillus casei -group lactobacilli (L. casei, L. paracasei, L. rhamnosus, and L. zeae). L. rhamnosus species-specific PCR was developed and validated. The discriminatory power of the three fingerprinting techniques, pulsed field gel electrophoresis (PFGE), randomly amplified polymorphic DNA (RAPD) and ribotyping, was compared. All three techniques were highly effective in differentiating strains below the species level and they can be placed in the following order with respect to their discriminatory power: PFGE > ribotyping > RAPD. Newly developed molecular methods were used to trace ingested probiotic strains L. rhamnosus GG (LGG) and B. lactis Bb12 in the GI-tract. The identity of LGG colonies was verified using a species-specific PCR and Bb12 was detected using the PCR-DGGE method. Both probiotic strains colonised the gut transiently and they were no longer detected in the faeces one week after the end of the administration in most subjects. The synbiotic approach with galactooligosaccharide (GOS) did not prolong the persistence of Bb12. Furthermore, LGG was found to attach in vivo to colonic mucosae and, although the attchment was temporary, to remain for more than a week after discontinuation of LGG administration. PCR-DGGE method was used to monitor qualitative changes in adult faecal Bifidobacterium populations in response to B. lactis Bb12 and/or GOS administration. In most subjects two weeks administration of Bb12 and/or GOS did not affect the qualitative composition of indigenous bifidobacterial populations, while Bb12 transiently colonised the gut. Qualitative molecular analysis was used to study the bacterial, bifidobacterial and lactobacilli populations in faeces of breast-fed and formula-fed infants before and after weaning. Genus and group-specific PCRs combined with DGGE and subsequent sequencing of the amplified 16S rDNA fragments revealed no difference in the prevalence or species distribution of Bifidobacterium and Lactobacillus between the two groups of infants. In general, DGGE patterns of 16S rDNA showed equal complexity of bacterial communities in breast-fed and formula-fed infants. Equally intensive changes occurred in the faecal microbiota in infants of both groups due to weaning.