There is good evidence that the complex microbial flora present in the gastrointestinal tract of all warm-blooded animals is effective in providing resistance to disease. However, the composition of this protective flora can be altered by dietary and environmental influences, making the host animal susceptible to disease and/or reducing its efficiency of food utilization. What we are doing with the probiotic treatments is re-establishing the natural condition which exists in the wild animal but which has been disrupted by modern trends in conditions used for rearing young animals, including human babies, and in modern approaches to nutrition and disease therapy. These are all areas where the gut flora can be altered for the worse and where, by the administration of probiotics, the natural balance of the gut microflora can be restored and the animal returned to its normal nutrition, growth and health status.

https://onlinelibrary.wiley.com/doi/pdf/10.1111/j.1365-2672.1989.tb05105.x

Probiotics in man and animals

Methanogens: reevaluation of a unique biological group.

Prebiotics are non-digestible (by the host) food ingredients that have a beneficial effect through their selective metabolism in the intestinal tract. Key to this is the specificity of microbial changes. The present paper reviews the concept in terms of three criteria: (a) resistance to gastric acidity, hydrolysis by mammalian enzymes and gastrointestinal absorption; (b) fermentation by intestinal microflora; (c) selective stimulation of the growth and/or activity of intestinal bacteria associated with health and wellbeing. The conclusion is that prebiotics that currently fulfil these three criteria are fructo-oligosaccharides, galacto-oligosaccharides and lactulose, although promise does exist with several other dietary carbohydrates. Given the range of food vehicles that may be fortified by prebiotics, their ability to confer positive microflora changes and the health aspects that may accrue, it is important that robust technologies to assay functionality are used. This would include a molecular-based approach to determine flora changes. The future use of prebiotics may allow species-level changes in the microbiota, an extrapolation into genera other than the bifidobacteria and lactobacilli, and allow preferential use in disease-prone areas of the body.

/pdf/dietary-modulation-of-the-human-colonic-microbiota-updating-tz2ksl8b3e.pdf

Dietary modulation of the human colonic microbiota: updating the concept of prebiotics

In this study the microbiota associated with oral endosteal titanium hollow cylinder implants (ITI) was studied using microscopic, immunochemical and cultural methods. Samples from 5 edentulous patients with successfully incorporated implants serving as abutments for overdentures for more than one year were compared with samples from 7 patients with clinically failing implants. Unsuccessful sites were characterized by pocket probing depths of 6 mm or more, suppuration and visible loss of alveolar bone around the implant as visualized on radiographs. These sites harbored a complex microbiota with a large proportion of Gram-negative anaerobic rods. Black-pigmented Bacteroides and Fusobacterium spp. were regularly found. Spirochetes, fusiform bacteria as well as motile and curved rods were a common feature in the darkfield microscopic specimens of these sites. Control sites in the same patients harbored small amounts of bacteria. The predominant morphotype was coccoid cells. Spirochetes were not present, fusiform bacteria, motile and curved rods were found infrequently and in low numbers. The microbiota in control sites in unsuccessful patients and in site in successful patients were very similar. On the basis of these results, it is suggested that “periimplantitis” be regarded as a site specific infection which yields many features in common with chronic adult periodontitis.

The microbiota associated with successful or failing osseointegrated titanium implants.

An anaerobic glove box constructed of clear flexible vinyl plastic is described. It is sufficiently inexpensive and simple in operation to be used not only in research but also in a clinical laboratory by technicians without special training. Conventional bacteriological techniques may be used inside the glove box for culturing and transferring anaerobic bacteria. The box may be heated to 37 C and thus serve as an anaerobic incubator as well, permitting inspection of cultures at any time. Media may be prepared and agar plates may be poured on the laboratory bench in the conventional manner. An overlay of trace amounts of palladium black catalyst over plated agar media reduces the medium to an oxidation-reduction (O-R) potential of - 300 mv within 2 days after introduction into the glove box. In spite of its greater simplicity, the system matched or excelled the roll tube method with respect to all parameters tested, including O-R potential obtainable in the media, O2 concentration in the gas phase, and efficiency in isolating anaerobic bacteria from the mouse cecum. Comparative studies indicate that the conventional anaerobic jar method was inadequate for the isolation of strict anaerobes from human gingival specimens and from the mouse cecum. This was due to the exposure of specimens and media to air during plating on the open laboratory bench. Anaerobic jars were adequate for maintaining the proper conditions for growth of anaerobic bacteria once these had been established in the glove box. Images

Isolation of anaerobic bacteria from human gingiva and mouse cecum by means of a simplified glove box procedure.

A previous study had established that anaerobic continuous-flow (CF) cultures of conventional mouse cecal flora were able to maintain the in vivo ecological balance among the indigenous bacterial species tested. This paper describes experiments designed to determine the mechanisms which control the population sizes of these species in such CF cultures. One strain each of Escherichia coli, Fusobacterium sp., and Eubacterium sp. were studied. Growth of these strains in filtrates of CF cultures was considerably more rapid than in the CF cultures themselves, indicating that the inhibitory activity had been lost in the process of filtration. Growth rates to match those in CF cultures could be obtained, however, by restoring the original levels of H2S in the culture filtrates. The inhibitory effect of H2S in filtrates and in dialysates of CF cultures could be abolished by adding glucose or pyruvate, but not formate or lactate. The fatty acids present in CF cultures matched those in the cecum of conventional mice in both quality and concentration. These acids could not account for the slow rates of growth of the tested strains in CF cultures, but they did cause a marked increase in the initial lag phase of E. coli growth. The results obtained are compatible with the hypothesis that the populations of most indigenous intestinal bacteria are controlled by one or a few nutritional substrates which a given strain can utilize most efficiently in the presence of H2S and at the prevailing conditions of pH and anaerobiosis. This hypothesis consequently implies that the populations of enterobacteria, such as the E. coli strain tested, and those of the predominant anaerobes are controlled by analogous mechanisms.

https://iai.asm.org/content/iai/39/2/676.full.pdf

Mechanisms that control bacterial populations in continuous-flow culture models of mouse large intestinal flora.

Preliminary experiments established that a 0.5-ml inoculum that is introduced directly into the stomach of mice was cleared rapidly into the small intestine. Bicarbonate buffer, but not skim milk, protected such an inoculum from stomach acid until at least 90% of it had entered the small intestine. Passage and survival of various Escherichia coli strains through the mouse gut were tested by introducing a buffered bacterial inoculum directly into the stomach, together with the following two intestinal tracers: Cr(51)Cl(3) and spores of a thermophilic Bacillus sp. Quantitative recovery of excreted bacteria was accomplished by collecting the feces overnight in a refrigerated cage pan. The data show that wild-type E. coli strains and E. coli K-12 are excreted rapidly (98 to 100% within 18 h) in the feces without overall multiplication or death. E. coli varkappa1776 and DP50supF, i.e., strains certified for recombinant DNA experiments underwent rapid death in vivo, such that their excretion in the feces was reduced to approximately 1.1 and 4.7% of the inoculum, respectively. The acidity of the stomach had little bactericidal effect on the E. coli K-12 strain tested, but significantly reduced the survival of more acidsensitive bacteria (Vibrio cholerae) under these conditions. Long-term implantation of E. coli strains into continuous-flow cultures of mouse cecal flora or into conventional mice was difficult to accomplish. In contrast, when the E. coli strain was first inoculated into sterile continuous-flow cultures or into germfree mice, which were subsequently associated with conventional mouse cecal flora, the E. coli strains persisted in a large proportion of the animals at levels resembling E. coli populations in conventional mice. Metabolic adaptation contributed only partially to the success of an E. coli inoculum that was introduced first. A mathematical model is described which explains this phenomenon on the basis of competition for adhesion sites in which an advantage accrues to the bacterium which occupies those sites first. The mathematical model predicts that two or more bacterial strains that compete in the gut for the same limiting nutrient can coexist, if the metabolically less efficient strains have specific adhesion sites available. The specific rate constant of E. coli growth in monoassociated gnotobiotic mice was 2.0 h(-1), whereas the excretion rate in conventional animals was -0.23 h(-1). Consequently, limitation of growth must be regarded as the primary mechanism controlling bacterial populations in the large intestine. The beginnings of a general hypothesis of the ecology of the large intestine are proposed, in which the effects of the competitive metabolic interactions described earlier are modified by the effects of bacterial association with the intestinal wall.

https://iai.asm.org/content/iai/39/2/686.full.pdf

Survival and implantation of Escherichia coli in the intestinal tract.

In vivo loops were prepared in the small intestine of rabbits and injected with mixtures of Vibrio cholerae and polystyrene spheres (1.1-micrometers diameter). The loops were removed and frozen after 15 min and then sectioned in a cryostat. The locations of particles and vibrios were determined microscopically. The vibrio/particle ratio was unity in the lumen of the loops, but increased 10-fold in the deep intervillous spaces, indicating active invasion of the mucus gel by the chemotactic parent strain. Motile nonchemotactic mutants and nonmotile mutants of this strain invaded the mucus at the same rate as inert particles. Similar results were obtained with intestinal loops prepared in germfree mice. When germfree mice were disassociated with mixtures of chemotactic (parent or revertant) and nonchemotactic mutant vibrios in equal proportions, the chemotactic strain rapidly outgrew its nonchemotactic counterpart in the intestine. Nonchemotactic mutants introduced as monoassociates into germfree mice were rapidly overgrown by nonmotile mutants which apparently arose spontaneously in the gut. Motility was therefore beneficial to survival only when it was directed by chemotactic stimuli, whereas it was a liability in the absence of such stimuli. Growth of chemotactic vibrios in small intestinal loops of rabbits paralleled that of nonchemotactic mutants for the first 4 to 6 h. Thereafter, the growth rate of the chemotactic vibrios was significantly faster. This was correlated with a significantly higher degree of association with the mucosa on the part of the chemotactic vibrios.

Role of chemotaxis in the association of motile bacteria with intestinal mucosa: in vivo studies.

This paper elaborates and extends earlier reports from this laboratory on the mechanisms by which coproantibodies protect against enteric bacterial infection. Purified antibody preparations obtained from the lumen of the intestine of orally vaccinated germfree mice, containing very small amounts of S-IgA but no other serologically active immunoglobulins, were found to protect mice against experimental enteric cholera infection. This protection was associated with a reduction in that fraction of the total vibrio population which was adsorbed to the mucosa. Total counts of intestinal vibrio populations were not affected. The mechanism of this process was investigated by using metabolic inhibitors in experimental infections of intestinal loops of rabbits. Iodoacetate reversed the antibody-mediated shift of the vibrio population away from association with the mucosa. This suggests that the mucosal cell may furnish some accessory factor which, in conjunction with antibody, results in an antibacterial mechanism on the mucosal surface. Iodoacetate had no effect on total vibrio population and adsorption of vibrios in nonimmunized intestinal loops. Consequently, a direct effect of iodoacetate on the vibrios, rather than on the mucosal cells, is a less likely explanation for the observed effects.

Rolf Freter

Papers

Isolation of anaerobic bacteria from human gingiva and mouse cecum by means of a simplified glove box procedure.

Mechanisms that control bacterial populations in continuous-flow culture models of mouse large intestinal flora.

Survival and implantation of Escherichia coli in the intestinal tract.

Role of chemotaxis in the association of motile bacteria with intestinal mucosa: in vivo studies.

Protection Against Enteric Bacterial Infection by Secretory IgA Antibodies