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Figure 1 PCR-based approaches to monitor the GI-tract microbiota. The 16S rDNA or rRNA isolated from a GI-tract sample may be amplified by (reverse transcriptase-) PCR using primers that target all or some bacteria. The amplicons may be cloned and sequenced in order to identify the bacteria present in the sample. The 16S rRNA gene comprises highly variable to highly conserved regions, and the differences in sequence are used to determine phylogenetic relationships and distinguish bacteria at different levels from species to domain. The DGGE technique is based on 16S rRNA sequence-specific melting behavior of the PCR products, generated with primers one of which contains a 40-bp GC clamp. Statistical software enables the calculation of similarity indices and cluster analysis to compare the samples. The 16S rRNA sequences may also be used to design new primers specific for bacterial groups or species in order to quantify them in samples by real time PCR. Abbreviations: DGGE, denaturing gradient gel electrophoresis; DNA, deoxyribonucleic acid; GC, guanine cytosin; PCR, polymerase chain reaction; rDNA, ribosomal deoxyribonucleic acid; rRNA, ribosomal ribonucleic acid.

(including Escherichia coli). The large class of Clostridia comprises the Clostridium coccoides-Eubacterium rectale group, and the Clostridium leptum group consists of Ruminococcus species and Faecalibacterium prausnitzii. These analyses indicated that the adult intestinal microbiota constitutes a majority of low and high G + C content Gram-positive bacteria. The latter has been indirectly confirmed by analysis of the metagenome of bacterial viruses recovered from fecal samples that revealed predominantly viral sequences with similarity to genomes of bacteriophages specific for Gram-positive bacteria (24). In fact, this bacterial diversity at the division level relative to other microbial ecosystems is quite low, mainly deriving from the divisions Firmicutes and the Cytophaga-Flavobacterium-Bacteroides (9,19).

Interestingly, molecular inventories based on 16S rDNA clone libraries of microbial communities in inflammatory bowel disease (IBD) patients differed from healthy subjects (25). In several Crohn's disease (CD) patients numerous clones were isolated belonging to phylogenetic groups that are commonly not dominant in adult fecal microbiota of healthy persons, while Bacteroides vulgatus was the only molecular species shared by all patients, and E. coli clones were also detected unlike in healthy persons (25). In another study, 16S rDNA libraries generated from mucosa-associated microbiota of patients with IBD revealed a reduction in diversity due to a loss of normal anaerobic bacteria, especially those belonging to the Bacteroides, Eubacterium and Lactobacillus species. Most of the sequenced clones retrieved (70%) were assigned to known intestinal bacteria, but a significant number of the cloned sequences were affiliated to normal residents of the oral mucosa such as Streptococcus species (26). It was suggested that alteration of the microbiota in mucosal inflammation reflects a metabolic imbalance of the complex microbial ecosystem with severe consequences for the mucosal barrier rather than disrupted defense to single microorganisms (26).

Even though sequencing of cloned 16S rDNA amplicons provides relevant information about the identity of uncultured bacteria, the data are not quantitative. Moreover, PCR and cloning steps are not without bias (27): a recent comparative analysis of clone libraries from a fecal sample pointed out that the number of PCR cycles may affect the diversity of the amplified 16S rDNAs and thus should be minimized (28). More rapid culture-independent options to the cloning procedures include exploring of the complex microbial populations using a variety of fingerprinting methods. See Table 1 for an overview of some current methods used to investigate the intestinal microbiota.

Table 1 Potential and Limitations of Various Methods for Investigating the Diversity of the Human Intestinal Microbiota

Method

Application

Comments

Culturing

Isolation of pure cul-

Not representative for microbiota; insufficient

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