Characterization of the Intestinal Microbiota by DNA-Based Methods

University dissertation from Department of Food Technology, Lund University

Abstract: The intestinal flora and the bacteria found in the extraintestinal sites were studied in liver injury rats, with or without Lactobacillus plantarum 299v pre-treatment. The dominating intestinal flora and translocating bacteria were typed by RAPD and identified by 16S rDNA sequencing. It was proved that bacterial strains from the intestine could pass through the intestinal barrier to the extraintestinal sites after liver injury. Bacterial species predominating the intestinal flora were also the ones most frequently found in the liver and the mesenteric lymph nodes, but some bacterial strains isolated from the blood were not found to be part of the dominating flora. Treatment of rats with L. plantarum before induction of liver injury reduced bacterial translocation. Seventeen Brachyspira aalborgi-like clones derived from colonic mucosa of two healthy adults were almost fully sequenced and subjected to phylogenetic analysis together with 11 Brachyspira reference strains. The analysis divided Brachyspira spp. into two lineages, the B. aalborgi and the B. hyodysenteriae lineages. All clones generated in the present study were grouped into the B. aalborgi lineage. Within the lineage of B. aalborgi, three distinct clusters were identified. Cluster 1 included two B. aalborgi reference strains and six clones. Clusters 2 and 3 consisted of nine and two clones respectively. This study provided the first evidence that different phylotypes of B. aalborgi-like organisms are present in the colonic mucosa within and between healthy individuals. The development of fecal bacterial flora in two Swedish infants was monitored by T-RFLP analysis of amplified 16S rRNA genes, and the major changes in the microbiota over time were identified by comparing the T-RFs in the fecal communities with that of corresponding 16S rRNA gene clones. Principal component analysis of the T-RFLP profiles revealed that the fecal flora in both infants was quite stable during the breast-feeding period, and weaning had a major influence on the profiles of bacterial communities. The two infants had different bacterial communities and the fecal flora developed differently between the two infants. T-RFLP in combination with PCA and 16S rRNA gene sequencing was shown to be an effective strategy for comparing fecal microbiota in infants and pointing out the major changes. Bacterial diversity in different regions of the human intestinal tract was compared by analysis of PCR-amplified 16S rDNA clone libraries. The jejunum library was the least diverse, had few or no phylotypes in common with the libraries of distal ileum, ascending colon and rectum, and was dominated (67%) by sequences closely related to the Streptococcus genus. The libraries of distal ileum, ascending colon and rectum were more diverse and dominated by sequences affiliated with Bacteroidetes (27-49%), Clostridium clusters XIVa (20-34%) and IV (7-13%). The results revealed that the microbial community in jejunum is remarkably different from that in distal ileum, ascending colon and rectum, and the major phylogenetic groups are similar from distal ileum to rectum. The colonic microbiota of a patient with ulcerative colitis (UC) was studied by sequence analysis of a 16S rDNA clone library. A total of 163 clones were partially sequenced and assigned to four phylogenetic phyla of domain bacteria: Firmicutes, Bacteroidetes, Proteobacteria, and Acitinobacteria. The most predominant group detected in the UC patient was ã-Proteobacteria (50%), followed by Clostridium clusters IV and XIVa (20 and 10% respectively). Compared with previous studies of healthy subjects, alteration of the dominant bacterial groups in the UC patient was observed. A high proportion of adverse, pro-inflammatory species was present in the UC patient. Even if this alteration is not the cause of the UC in this patient, it most probably enhances of the symptoms of disease.

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