Background: Reuterin produced from glycerol by Lactobacillus reuteri, a normal inhabitant of the human intestine, is a broad-spectrum antimicrobial agent. It has been postulated that reuterin could play a role in the probiotic effects of Lb. reuteri. Reuterin is active toward enteropathogens, yeasts, fungi, protozoa and viruses, but its effect on commensal intestinal bacteria is unknown. Moreover reuterin's mode of action has not yet been elucidated. Glutathione, a powerful antioxidant, which also plays a key role in detoxifying reactive aldehydes, protects certain bacteria from oxidative stress, and could also be implicated in resistance to reuterin.

While the prebiotic effects of fructo-oligosaccharides (FOS), short-chain polymers of fructose, have been thoroughly described after 2-3 wk of ingestion, effects after intake for several months are unknown. We tested the hypothesis that these effects would differ after ingestion for short and long periods in rats. Rats were fed a basal low-fiber diet (Basal) or the same diet containing 9 g/100 g of FOS for 2, 8 or 27 wk, and cecal contents were collected at the end of each time period. Cecal short-chain fatty acid concentration was higher in rats fed FOS than in those fed Basal, and this effect persisted over time: 83.8 +/- 4.1 vs. 62.4 +/- 6.5 micromol/g at 2 wk and 103.5 +/- 5.8 vs. 73.2 +/- 7.4 micromol/g at 27 wk (P < 0.05). The molar butyrate ratio was higher in rats fed FOS regardless of the time period (14.8 +/- 0.6% vs. 6.7 +/- 1.1% at 27 wk, P < 0.05). Lactate concentration in rats fed FOS was elevated after 2 wk and then decreased: 63.5 +/- 21.6 micromol/g at 2 wk vs. 8.8 +/- 3.3 micromol/g at 8 wk (P < 0.05). After 2 wk, FOS increased the concentrations of total lactic acid-producing bacteria, and Lactobacillus sp. (P < 0.05), without modifying total anaerobes. However, most of these effects were abolished after 8 and 27 wk of FOS consumption. In the long term, the FOS-induced increase in intestinal lactic acid-producing bacteria was lost, but the butyrogenic properties of FOS were maintained.

Bacteria isolated from infant feces were immobilized in polysaccharide gel beads (2.5% gellan gum, 0.25% xanthan gum) using a two-phase dispersion process. A 52-day continuous culture was carried out in a single-stage chemostat containing precolonized beads and fed with a medium formulated to approximate the composition of infant chyme. Different dilution rates and pH conditions were tested to simulate the proximal (PCS), transverse (TCS), and distal (DCS) colons. Immobilization preserved all nine bacterial groups tested with survival rates between 3 and 56%. After 1 week fermentation, beads were highly colonized with all populations tested (excepted Staphylococcus spp. present in low numbers), which remained stable throughout the 7.5 weeks of fermentation, with variations below 1 log unit. However, free-cell populations in the circulating liquid medium, produced by immobilized cell growth, cell-release activity from gel beads, and free-cell growth, were altered considerably by culture conditions. Compared to the stabilization period, PCS was characterized by a considerable and rapid increase in Bifidobacterium spp. concentrations (7.4 to 9.6 log CFU/mL), whereas Bifidobacterium spp., Lactobacillus spp., and Clostridium spp. concentrations decreased and Staphylococcus spp. and coliforms increased during TCS and DCS. Under pseudo-steady-state conditions, the community structure developed in the chemostat reflected the relative proportions of viable bacterial numbers and metabolites generally encountered in infant feces. This work showed that a complex microbiota such as infant fecal bacteria can be immobilized and used in a continuous in vitro intestinal fermentation model to reproduce the high bacterial concentration and bacterial diversity of the feces inoculum, at least at the genera level, with a high stability during long-term experiment.

Lactobacillus reuteri ATCC 55730 is a probiotic strain that produces, in the presence of glycerol, reuterin, a broad-spectrum antimicrobial substance. This strain has been shown to prevent intestinal infections in vivo; however, its mechanisms of action, and more specifically whether reuterin production occurs within the intestinal tract, are not known. In this study, the effects of L. reuteri ATCC 55730 on intestinal microbiota and its capacity to secrete reuterin from glycerol in a novel in vitro colonic fermentation model were tested. Two reactors were inoculated with adult immobilized fecal microbiota and the effects of daily addition of L. reuteri into one of the reactors (c.10(8) CFU mL(-1)) without or with glycerol were tested on major bacterial populations and compared with addition of glycerol or reuterin alone. The addition of glycerol alone or with L. reuteri increased numbers of the Lactobacillus-Enterococcus group and decreased Escherichia coli. The addition of reuterin significantly and selectively decreased E. coli without affecting other bacterial populations. The observed decrease in E. coli concentration during the addition of glycerol (in presence or absence of L. reuteri) could be due to in situ reuterin production because 1,3-propanediol, a typical product of glycerol fermentation, was detected during the addition of glycerol.

Species-specific 16S rRNA-targeted, Cy3 (indocarbocyanine)-labeled oligonucleotide probes were designed and validated to quantify different Eubacterium species in human fecal samples. Probes were directed at Eubacterium barkeri, E. biforme, E. contortum, E. cylindroides (two probes), E. dolichum, E. hadrum, E. lentum, E. limosum, E. moniliforme, and E. ventriosum. The specificity of the probes was tested with the type strains and a range of common intestinal bacteria. With one exception, none of the probes showed cross-hybridization under stringent conditions. The species-specific probes were applied to fecal samples obtained from 12 healthy volunteers. E. biforme, E. cylindroides, E. hadrum, E. lentum, and E. ventriosum could be determined. All other Eubacterium species for which probes had been designed were under the detection limit of 10 7 cells g (dry weight) of feces ؊1 . The cell counts obtained are essentially in accordance with the literature data, which are based on colony counts. This shows that whole-cell in situ hybridization with species-specific probes is a valuable tool for the enumeration of Eubacterium species in feces.The genus Eubacterium (41) contains anaerobic, non-sporeforming, gram-positive rods which are distinguished from other genera mainly on the basis of negative metabolic characteristics (37). In the human intestinal tract, Eubacterium is the second most common genus after the genus Bacteroides and is more common than the genus Bifidobacterium (16). The importance of members of the genus Eubacterium has been reported previously (10,24,38,48). Since the identification of Eubacterium species based on phenotypic traits requires experience and is time-consuming (5,15,16,36,44), many studies involving human fecal flora composition have refrained from looking at this genus (13,20,34,42).Considerable effort has been invested in the application of molecular techniques such as PCR (19,27,50) and hybridization (12, 25, 52) for the identification of fecal bacteria. However, the extraction, purification, and amplification of nucleic acids by PCR from fecal samples are often selective and limited. In contrast, whole-cell hybridization with fluorescently labeled, 16S rRNA-targeted oligonucleotide probes allows the determination of the numerical abundance of bacteria, including unculturable strains (39), in various ecosystems such as water (1,26,32), sludge (23), and fecal samples (17,18,29).In contrast to the genus Bifidobacterium, for example, which is a phylogenetically and phenotypically well-defined taxon, Eubacterium species (9, 31) are phylogenetically diverse and thus it is not possible to design a genus-specific probe for Eubacterium spp. Therefore, probes for phylogenetic clusters or species have to be considered. The development and validation of a probe for the detection of a Eubacterium species has been reported recently (45).The purpose of this study was to develop and apply 16S rRNA-targeted oligonucleotide probes to human feces for the detection of numerically dominant fecal Eubact...