Formation of allyl isothiocyanate from sinigrin in the digestive tract of rats monoassociated with a human colonic strain of Bacteroides thetaiotaomicron.

A human digestive strain of Bacteroides thetaiotaomicron was tested for its ability to metabolise sinigrin, a glucosinolate commonly found in Brassica vegetables. Gnotobiotic rats harbouring the bacterial strain were orally dosed with 50 micromol sinigrin. HPLC analysis of the digestive contents showed that sinigrin was degraded in the large bowel, where B. thetaiotaomicron was established at a high level. Concurrently, a hydrolysis product of sinigrin, allyl isothiocyanate, was identified by GC-MS analysis, following headspace solid-phase microextraction of the digestive contents; its production peaked at ca. 200 nmol, 12 h after dosing. This is the first study to demonstrate in vivo the involvement of a human colonic predominant bacterium in the bioconversion of a dietary glucosinolate to a potentially anticarcinogenic isothiocyanate.

Cleavage of L-leucine-containing dipeptides by Clostridium butyricum.

The ability of Clostridium butyricum cultures to hydrolyze three L-leucine-containing dipeptides (Leu-Leu, Leu-Gly and Gly-Leu) in a synthetic minimal medium is demonstrated by using gas chromatography coupled with mass spectrometry. The 13C nuclear magnetic resonance and a labeled dipeptide L-[1-13C]Leu-Gly were used to confirm this activity.

Oligofructose contributes to the protective role of bifidobacteria in experimental necrotising enterocolitis in quails.

Bifidobacteria are dominant in the gut of full-term infants, although colonisation by them is often delayed in preterm neonates. Bifidobacteria are recognised to have beneficial effects on digestive disorders and they might prevent neonatal necrotising enterocolitis (NEC), a gastrointestinal disease that predominantly affects premature infants. They have been shown to protect gnotobiotic quails against NEC-like lesions when the birds were inoculated with faecal flora from preterm infants, decreasing the clostridial population. The present study was designed to investigate whether oligofructose, which stimulates the activity of bifidobacteria, may enhance their protective role. Experiments were done in eight groups of germ-free quails for 28 days. The groups differed as to their bacterial status, diet and environment. Quails were inoculated with one of two flora from premature twins. The first flora included Bifidobacterium pseudo-catenulatum, Escherichia coli and no clostridia. The second flora included clostridial species and was associated with B. infantis-longum. Caecal bacterial population and metabolism changes were investigated with a lactose (6%) diet versus a lactose-oligofructose (3%-3%) diet, either in a gnotobiotic environment or in an ordinary environment permitting post-colonisation by exogenous bacteria. In both environments and with both flora, oligofructose significantly increased the level of bifidobacteria and this was associated with a decrease of E. coli or C. perfringens and C. ramosum. The bacterial changes in the ordinary environment depended on the initial composition of the microflora and the colonisation resistance against exogenous bacteria was more efficient with the flora that included B. pseudo-catenulatum. The changes in caecal pH and short-chain fatty acids were minimal. It was demonstrated that, irrespective of the environmental conditions, the use of oligofructose helped to prevent the overgrowth of bacteria implicated in necrotising enterocolitis in preterm neonates.

Synthesis and polymerization of benzyl (3R,4R)-3-methylmalolactonate via enzymatic preparation of the chiral precursor.

beta-methylaspartate ammonia-lyase, EC 4.3.1.2, (beta-methylaspartase) from Clostridium tetanomorphum was used to produce a 40/60 molar ratio of (2S,3R) and (2S,3S)-3-methylaspartic acids, 2a and 2b, respectively, from mesaconic acid 1 as substrate, on a large scale. To prepare (3R,4R)-3-methyl-4-(benzyloxycarbonyl)-2-oxetanone (benzyl 3-methylmalolactonate) 6, 2a and 2b were transformed, in the first step, into 2-bromo-3-methylsuccinic acids 3a and 3b and separated. After three further steps, (2S,3S)-3a yielded the alpha, beta-substituted beta-lactone (3R,4R) 6 with a very high diastereoisomeric excess (> 95% by chiral gas chromatography). The corresponding crystalline polymer, poly[benzyl beta-(2R,3S)-3-methylmalate] 8, prepared by an anionic ring opening polymerization, was highly isotactic as determined by 13C NMR. Catalytic hydrogenolysis of lactone 6 yielded (3R,4R)-3-methyl-4-carboxy-2-oxetanone (3-methylmalolactonic acid) 7, to which reactive, chiral, or bioactive molecules can be attached through ester bonds leading to polymers with possible therapeutic applications. Because of the ability of beta-methylaspartase to catalyse both syn- and anti-elimination of ammonia from (2S,3RS)-3-methylaspartic acid 2ab at different rates, the (2S,3R)-stereoisomer 2a was retained and isolated for further reactions. These results permit the use of the chemoenzymatic route for the preparation of both optically active and racemic polymers of 3-methylmalic acid with well-defined enantiomeric and diastereoisomeric compositions.

Clostridial pathogenicity in experimental necrotising enterocolitis in gnotobiotic quails and protective role of bifidobacteria.

The pathogenesis of neonatal necrotising enterocolitis (NEC) remains unclear. Gnotobiotic quails fed a lactose diet have been used to investigate the role of clostridial strains originating from faecal specimens of neonates through the intestinal lesions, the changes in microflora balance and the production of bacterial metabolites, i.e., short-chain fatty acids and hydrogen. Bifidobacteria are thought to exert various beneficial effects on host health, including interaction with the colonic microflora. Therefore, it was hypothesised that a protective role could be exercised through bifidobacterial colonisation. A Clostridium butyricum strain (CB 155-3) and a whole faecal flora including three clostridial species (C. butyricum, C. perfringens, C. difficile), each from premature infants suffering from NEC, caused caecal lesions in quails similar to those observed in man, i.e., thickening of the caecal wall with gas cysts, haemorrhagic ulceration and necrotic areas. Conversely, a whole faecal flora including bifidobacteria (identified as Bifidobacterium pseudo-catenulatum) and no clostridia, isolated from a healthy premature infant, was unable to produce NEC-like lesions. When the two clostridial groups were associated with a Bifidobacterium strain (B. infantis-longum, CUETM 89-215, isolated from a healthy infant), bifidobacterial colonisation suppressed all pathological lesions. This study is the first demonstration of a protective role for bifidobacteria against NEC via the inhibition of growth of C. butyricum or the disappearance of C. perfringens. C. difficile was not found to be responsible for the aetiology of the caecal lesions in quails. The main effect of bifidobacteria on lactose fermentation was either a dramatic decrease or a disappearance of butyric acid. The protective role was not associated with changes in H2 production. Therefore, a new step between colonic colonisation and its relevance to NEC is thought to involve the fermentation of unabsorbed lactose into butyric acid at the onset of the disease.

Synthesis of 2-hydroxy acid from 2-amino acid by Clostridium butyricum.

Cultures of Clostridium butyricum type strain in synthetic medium supplemented with various L-2-amino acids revealed the presence of the corresponding 2-hydroxy acid. This metabolite is able to produce the polyester poly(2-hydroxyalkanoic acid). The bioconversion is not stereoselective since D-2-amino acids were also converted. Chiral GC analysis demonstrated that only D-enantiomer is formed from L-leucine.

Thermotoga hypogea sp. nov., a xylanolytic, thermophilic bacterium from an oil-producing well.

A new thermophilic, xylanolytic, strictly anaerobic, rod-shaped bacterium, strain SEBR 7054T, was isolated from an African oil-producing well. Based on the presence of an outer sheath (toga) and 16S rRNA sequence analysis data, this organism was identified as a member of the genus Thermotoga. Strain SEBR 7054T possessed lateral flagella, had a G + C content of 50 mol%, produced traces of ethanol from glucose but no lactate, and grew optimally in the presence of 0 to 0.2% NaCl at 70 degrees C. Its phenotypic and phylogenetic characteristics clearly differed from those reported for the five previously validly described Thermotoga species. Therefore, we propose that strain SEBR 7054T is a member of a new species of the genus Thermotoga, Thermotoga hypogea sp. nov. The type strain of T. hypogea is SEBR 7054 (= DSM 11164).

Thermococcus fumicolans sp. nov., a new hyperthermophilic archaeon isolated from a deep-sea hydrothermal vent in the north Fiji Basin.

An extremely thermophilic archaeon, strain ST557T (T = type strain), was isolated from a deep-sea hydrothermal vent in the North Fiji Basin. This strain is a strictly anaerbic coccus whose cells are about 0.8 to 2 microns in diameter. The optimum temperature, pH and sea salt concentration for growth are 85 degrees C, 8.5, and 20 to 40 g/liter, respectively. Strain ST557T grows preferentially in the presence of elemental sulfur on proteinaceous substrates and on a mixture of 20 amino acids. It grows slowly on pyruvate and maltose. Growth is inhibited by rifampin. The DNA G + C content is 54 to 55 mol%. Sequencing of the 16S rRNA gene revealed that strain ST557T belongs to the genus Thermococcus. We propose that this organism should be placed in a new species, Thermococcus fumicolans.

l-Methionine, a Precursor of Trace Methane in Some Proteolytic Clostridia.

The in vivo formation of methane and of several S-methyl volatile compounds from the terminal S-methyl group of l-methionine is reported for growing cultures of four Clostridium strains (C. hastiforme, C. histolyticum, C. subterminale, and Clostridium sp. strain DSM 1786). After growth in 5 ml of unamended medium, C. hastiforme formed the highest amount of methane (408 nmol per tube in the headspace). When the culture medium was amended with 100 mM l-[S-methyl-H(3)]methionine, the four strains formed [H(3)]methane (proportion in the methane peak, >85%) as well as methanethiol, dimethyl disulfide, dimethyl trisulfide, and S-methyl thioacetate labeled on the methyl moiety. Methanethiol is also a precursor of methane for Clostridium sp. strain DSM 1786. The trace methane formation observed for these four proteolytic, nonglucidolytic Clostridium strains can be of ecological interest, particularly in aquatic sediments and in the gastrointestinal tract of humans and animals. It can explain in part the trace methane formation which cannot be ascribed to methanogens sensu stricto.

Headspace gas chromatographic-mass spectrometric analysis of light hydrocarbons and volatile organosulphur compounds in reduced-pressure cultures of Clostridium.

A static headspace gas chromatographic method for the simultaneous separation of trace light hydrocarbons and volatile organosulphur compounds in gases of nineteen Clostridium cultures at reduced pressure is described. The separation was achieved on n-octane-Porasil C after sampling of the gaseous compounds in a PTFE loop without any pretreatment. Most peaks were identified by gas chromatography-mass spectrometry. The presence of methane and ethylene sulphide among Clostridium volatiles is confirmed and 3-methyl-1-butene, 2-methyl-2-butene, dimethyl trisulphide and S-methyl thioacetate are reported for the first time in the Clostridium group.

Gas chromatographic-mass spectrometric analysis of volatile amines produced by several strains of Clostridium.

A gas chromatographic--mass spectrometric technique is proposed for the analysis of volatile amines which were isolated from Clostridium cultures by vacuum distillation and concentrated as hydrochloride salts. Headspace sampling after alkalinization of the salts under vacuum was the most suitable for subsequent gas chromatographic analysis. With ammonia-loaded helium as carrier gas, methylamines were separated on 4.8% PEG 2OM + 0.3% potassium hydroxide on Carbopack B, and other volatile amines on 28% Pennwalt 223 + 4% potassium hydroxide on Gas-Chrom R. Bacterial volatile amines (dimethylamine, trimethylamine, isobutylamine, 3-methylbutylamine, etc.) were detected with a flame-ionization detector and identified by gas chromatography--mass spectrometry in electron-impact and chemical ionization modes.

[Biosynthesis of toluene in Clostridium aerofoetidum strain WS]. / Biosynthèse de toluène chez Clostridium aerofoetidum souche WS.

Formation of toluene in growing cultures of Clostridium aerofoetidum strain WS was enhanced when the medium was supplemented with phenylacetic acid or with L-phenylalanine and L-methionine together. Evidence for the role of L-phenylalanine was shown by the detection of [2H2]-methyl[2,3,4,5,6-2H5]benzene ("heptadeuterotoluene") in growing cultures with L-[2',3',4',5',6'-2H5]phenyl[2,3-2H3]alanine and L-methionine.

[Neutral and basic compounds present in gases produced by Clostridium sporogenes, Plectridium putrificum and Plectridium glycolicum cultured under vacuum in sodium thioglycolate glucose broth]. / Composés neutres et basiques présents dans les gaz produits par Clostridium sporogenes, Plectridium putrificum et Plectridium glycolicum cultivés sous vide en milieu glucosé au thioglycolate de sodium.

The study of gases produced by Clostridium sporogenes, Plectridium putrificum et P. glycolicum cultured under vacuum in sodium thioglycolate glucose broth makes possible the detection of compounds not described before for these Bacteria, particularly methyl-2 butanal and methyl-3 butanal for P. glycolicum and thiacyclopropane in these 3 Bacteria. Thiacyclopropane is the result of the cyclization of mercapto-2 ethanol produced by the reduction of thioglycolic acid.

[Neutral and basic compounds present in gases produced by Clostridium histolyticum, Clostridium hastiforme and Clostridium ghoni cultured under vacuum in sodium thioglycolate glucose broth]. / Composés neutres et basiques présents dans les gaz produits par Clostridium histolyticum, Clostridium hastiforme et Clostridium ghoni cultivés sous vide en milieu glucosé au thioglycolate de sodium.

The study of gases produced by Clostridium histolyticum, C. hastiforme and C. ghoni cultured under vacuum in sodium thioglycolate glucose broth makes it possible to detect compounds not described before for these Bacteria, and confirms the production of methane in anaerobic spore-forming Bacteria.