The human intestinal tract contains a complex ecosystem consisting mainly of obligatory anaerobic bacteria, usually referred to as the normal flora. The composition of this flora plays an important role in human health and disease. To measure the effect of treatments aimed at modulating the normal flora, we have developed fluorescent 16S rRNA targeted oligonucleotide probes for numerically important groups of bacteria in the human intestine. These probes have been used for whole cell fluorescent in situ hybridization (FISH) to study the composition of the human intestinal flora over time. To facilitate detection and enumeration, we have combined FISH with automated microscopic image analysis. Measurement of 48 samples can be done overnight. In this study we have focused on the number of bifidobacteria present in faecal samples of different age groups. The percentage bifidobacteria in faeces ranges from 0 to 78.9 % depending on the age group. In each age group large variations were found. In faecal samples from formula-fed newborn infants we found individual differences ranging from 0 to 54.1% bifidobacteria and in faecal samples from elderly (77-91 yr) individual differences ranged from 0.2 to 41.3%.
The current status of fluorescence in situ hybridization (FISH) is critically examined ten years after the initial application of fluorescently labeled, rRNA-targeted oligonucleotide probes as “phylogenetic stains” by Edward DeLong and coworkers in 1989 for the in situ identification of whole fixed bacterial cells in natural samples. The method has in the meanwhile found numerous applications including the identification and enumeration of bacteria in human feces. Still, however, the principal problems that need to be solved before a FISH assay is successfully applied have remained the same. These include e.g.: (i) Permeabilization of the cell envelope for free probe diffusion to the intracellular target molecules, mostly 16S rRNA, by a fixation protocol that preferentially also maintains the cell morphology.(ii) A selection of the probe target site that takes into consideration that not all rRNA sites are equally accessible. In this respect, the predictive power of a complete in situ accessibility map of 16S rRNA of Escherichia coli will be discussed.(iii) Low cellular ribosome content as found in many environmental samples automatically results in weak probe-conferred staining. Methods to increase the signal strength will be discussed together with issues of instrumentation and automation. Depending on the samples of interest and the questions to be addressed a high quality epifluorescence microscope with optional image analysis, a confocal laser scanning microscope or a flow cytometer may be the instruments of choice.
The gastrointestinal tract (GIT) of mammals harbours a complex community of obligate and facultative anaerobic bacteria. The composition of the GIT microbiota is dependent on the physiological condition, age, genetics, and diet of the host. During the past 5 years a number of molecular fingerprinting methods have been developed to characterise complex communities based on 16S rRNA sequence diversity. This paper describes the use of temperature and denaturing gradient gel electrophoresis (T/DGGE) of bacterial 16S rRNA/DNA in faecal samples from humans, in which special attention was given to the quantification of the sequence diversity. After birth the GIT community develops into a relatively simple community consisting of 1-8 major types within three months. Adults show more complex, but remarkably constant patterns. These patterns are hardly affected by changes in diet. Significant differences were observed between different individuals, particularly between genetically unrelated persons. In general, bacterial communities of faecal samples from genetically related adults (i.e. twins, brothers, sisters) show higher similarity of DGGE banding patterns than those from genetically unrelated individuals, although occasionally all persons in one family show highly similar profiles. The DGGE banding patterns of humans are significantly different from those of other mammals, such as pigs, gorillas and cats, indicating that genetic factors of the host affect the composition of the GI-tract flora. The major bacterial groups were identified by cloning and sequencing of the dominant 16S rDNA molecules. At least three sequences with highest homology to Ruminococcus obeum and Eubacterium halii and Fusobacterium prausnitzii were present in all human subjects and are likely to play a universal role in the GI-tract. Other sequences were found in variable ratios in different individuals.
A number of health benefits have been claimed for probiotic bacteria such as Lactobacillus acidophilus, bifidobacteria, and Lactobacillus casei. Because of the potential health benefits, these organisms are increasingly incorporated into dairy foods. A number of health benefits have been claimed including antimicrobial, antimutagenic and anticarcinogenic properties, reduction in serum cholesterol, improvement in lactose tolerance in lactose intolerant people and adherence to intestinal cells. This review will cover some health benefits of probiotic bacteria.
Thorotrast is a colloidal solution containing 20% thorium (232Th), and used to be used primarily in diagnostic radiology for visualization. Once thorotrast is infused into the body, it is not eliminated easily. Over 90% of thorotrast forms deposits. It emits α-rays, and thorotrast-injected patients are semipermanently irradiated from α-particles deposited in the body. Thorotrast gives rise to malignant tumors by delayed effect of deposition, and especially liver cancer is highly associated with thorotrastosis. Deposition of thorotrast as an exogenous substance inhibits phagocytosis by suppressing the reticuloendothelial system (RES) as well and bringing about a Shwartzman preparatory state (preshock state) in patients. A majority of thorotrast-injected patients died abruptly. This short natural history of thorotrastosis is thought to have some relation to RES suppression by long-term thorotrast deposition resulting in vulnerability to shock and infection.