Production of an antibacterial substance in the digestive tract involved in colonization-resistance against Clostridium perfringens.

Ruminococcus gnavus E1, Bacteroides thetaiotaomicron LEMF4, Clostridium hathewayi LEMC7, and Clostridium orbiscindens LEMH9 were isolated from ex germ-free mice inoculated with a human faecal microbiota. When initially germ-free mice who were previously inoculated with the strain E1 alone, or a four-strain consortium [E1, LEMF4, LEMC7, and LEMH9], were then challenged with 108 counts of Clostridium perfringens; the target strain was rapidly eliminated from the digestive tract of the animals (<10² cfu g⁻¹ of faeces). R. gnavus E1 was able to produce a diffusible anti-C. perfringens substance that accumulated in the faeces of monoassociated animals, but failed to be detected in the faeces of mice associated with the four-strain consortium. The capability to produce the antibacterial substance was transferred in the digestive tract of gnotobiotic mice to a Dorea longicatena strain. Further experiments realized with the D. longicatena wild type strain and the transconjugant support the assumption that the diffusible antibacterial substance was necessary for obtaining the antagonistic effect against C. perfringens, but that it acted as a precursor in the mechanism of interaction of the four-strain consortia.

Influence of oral inoculation with plasmid-free human Escherichia coli on the frequency of diarrhea during the first year of life in human newborns.

BACKGROUND: This study was carried out to determine whether early inoculation of the plasmid-free human Escherichia coli into human newborns would reduce the frequency of acute diarrhea during a 1-year period. The plasmid-free E. coli strain isolated from the fecal microbiota of a healthy adult was nontoxigenic in vivo and in vitro and sensitive to all usual antibiotics. METHODS: In the experimental group, 51 healthy newborns were inoculated orally with 106 viable cells of the bacteria within 2 hours after birth. In the control group, the same number of newborns received the heat-killed bacteria. The clinical trial was double blind, and the newborns were randomly assigned to the experimental and control groups. RESULTS: Six months and 1 year after bacterial inoculation, infants in the experimental group showed a higher mean body weight (7.59 +/- 1.15 kg and 9.88 +/- 1.31 kg, respectively; P < 0.05) when compared with the control group (7.03 +/- 1.09 kg and 8.92 +/- 1.38 kg, respectively). At the end of the clinical trial, 48% (23/48) of the infants in the experimental group had shown at least one diarrhea episode during the 1-year period, as opposed to 71% (34/48) in the control group. These values were significantly different (P = 0.037), showing a 32.3% protective effect of inoculation. CONCLUSIONS: The present study shows that protection against diarrhea was obtained by oral inoculation with a single dose of plasmid-free human E. coli soon after birth.

Effects of racecadotril and loperamide on bacterial proliferation and on the central nervous system of the newborn gnotobiotic piglet.

METHODS: The effects of 4 days of oral administration of different doses of two drugs, an enkephalinase inhibitor (the antisecretory agent, racecadotril) and a mu-receptor agonist (loperamide), on intestinal growth of a bacterial nonpathogenic strain (Escherichia coli E 404) and on the central nervous system (CNS) were compared in newborn gnotobiotic piglets. RESULTS: The E. coli content of the proximal jejunum (segment S1) and the E. coli ratio of stomach:segment S1 were similar in the racecadotril (20 mg/kg b.d., n = 5) and control groups. In contrast, in the loperamide group (1 mg/kg b.d., n = 4), the E. coli content of segment S1 and the E. coli ratio stomach:S1 were both significantly higher than with racecadotril or control (P = 0.04 and 0.005, respectively, for E. coli content; P = 0.05 and 0.03, respectively, for stomach:S1). There were no clinical signs of neurotoxicity and no deaths with racecadotril given orally at a high dose of 130 mg/kg b.d. (n = 5)--nearly 60 times the paediatric dosage. In contrast, an equivalent high dose of loperamide (5 mg/kg b.d.) resulted in death in three out of four piglets. CONCLUSIONS: In contrast to loperamide, racecadotril did not induce bacterial overgrowth and did not produce central neurotoxicity.

Isolation and characterization of a plasmid from Lactobacillus fermentum conferring erythromycin resistance.

Lactobacillus fermentum is a lactic acid bacterial species commonly found in the digestive tracts of pigs and rodents and also present in man. We characterized a 5.7-kb plasmid, pLEM3, conferring erythromycin resistance, which was isolated from a porcine strain of L. fermentum. Plasmid pLEM3 established efficiently in L. fermentum, conferred high-level erythromycin resistance (MIC > 1 mg/ml), and was segregationally stable. A deletion derivative of pLEM3, called pLEM5, was constructed and found to be as genetically stable as the parent. A multiple cloning site was inserted into pLEM5, generating plasmid pLEM7. Nucleotide sequence determination of pLEM5 revealed similarities with known genes. The replicon itself is a member of the pC194 family of rolling circle plasmids. The region responsible for erythromycin resistance was 98.2% identical to the erm gene of conjugative transposon Tn1545.

Inactivation of tryptic activity by a human-derived strain of Bacteroides distasonis in the large intestines of gnotobiotic rats and mice.

Tryptic activity disappeared and trypsin was no longer detected with an antitrypsin antiserum in the large intestines of gnotobiotic rats and mice monoassociated with a human-derived strain of Bacteroides distasonis, whereas tryptic activity was not modified in the small intestines. This function was shown to be strain specific.

Relationship between intestinal colonization of Bifidobacterium bifidum in infants and the presence of exogenous and endogenous growth-promoting factors in their stools.

The relationship between the intestinal colonization of a test strain of Bifidobacterium bifidum requiring human milk growth-promoting factors in vitro and the presence of growth-promoting factors either in the stools of human neonates or in their diet was investigated. Thirty-one infants were inoculated with a single dose of this strain within the first 8 d of life. Spores of a strictly thermophilic Bacillus admixed with the B. bifidum inoculum were used as transit marker, and the fecal population levels of both strain B. bifidum and the transit marker were recorded within 6 d after inoculation. Strain B. bifidum was found in the predominant flora of six neonates. It was eliminated more quickly than the transit marker from the stools of 17 neonates. Its population remained at a low level in the remaining eight neonates. Amounts of B. bifidum growth-promoting factor in the infant stools were not significantly different whether they harbored strain B. bifidum at a high population level or not. Although these amounts were significantly higher in infants fed human milk containing B. bifidum growth-promoting factor than in infants fed formula milk without B. bifidum growth-promoting factor, strain B. bifidum became established in one of the 18 infants fed human milk and in five of the 13 formula-fed infants. No relationship could be found between the population levels of strain B. bifidum and those of facultatively anaerobic streptococci and enterobacteria already present on d 0 and 1. These results clearly show that no relationship exists between the intestinal colonization of B. bifidum and the amounts of exogenous or endogenous growth-promoting factors found in stools.

Trypsin-dependent production of an antibacterial substance by a human Peptostreptococcus strain in gnotobiotic rats and in vitro.

An antibacterial substance appeared within 1 day in feces of gnotobiotic rats harboring a human intestinal Peptostreptococcus strain. It disappeared when the rat bile-pancreatic duct was ligatured or when the rats ingested a trypsin inhibitor. Anaerobic cultures of the Peptostreptococcus strain in a medium supplemented with trypsin also exhibited an antibacterial activity, which was also inhibited by the trypsin inhibitor. In vitro the antibacterial substance from both feces and culture medium was active against several gram-positive bacteria, including other Peptostreptococcus spp., potentially pathogenic Clostridium spp. such as C. perfringens, C. difficile, C. butyricum, C. septicum, and C. sordellii, Eubacterium spp., Bifidobacterium spp., and Bacillus spp. Whatever the order of inoculation of the strains, a sensitive strain of C. perfringens was eliminated within 1 day from the intestine of rats monoassociated with the Peptostreptococcus strain. These findings demonstrate for the first time that very potent antibacterial substances can be produced through a mechanism involving intestinal bacteria and exocrine pancreatic secretions.

Lithostathine, an inhibitor of CaCO3 crystal growth in pancreatic juice, induces bacterial aggregation.

Lithostathine is a pancreatic secretory protein which controls CaCO3 crystal growth in pancreatic juice. Trypsin hydrolysis of the molecule generates two fragments of 11 and 133 amino acids. The N-terminal undecapeptide bears the inhibitory activity for crystal growth. We demonstrate that the C-terminal part of the molecule, which is structurally related to Ca(2+)-dependent lectins, can induce bacterial aggregation. Ca(2+)- and pH-dependent aggregation was obtained for Escherichia coli strain KH 802 and 9 of 19 strains isolated from the predominant flora of human feces. Aggregation of E. coli could be reversed by dilution and bacteria could resume normal growth. Lithostathine is apparently the only component of normal pancreatic juice displaying such activity. Lithostathine is therefore a bifunctional protein which might be involved in the control of the bacterial ecosystem in the intestine.

Rape-seed meal toxicity in gnotobiotic rats: influence of a whole human faecal flora or single human strains of Escherichia coli and Bacteroides vulgatus.

Gnotobiotic growing rats harbouring either a whole human faecal flora or single human strains of Escherichia coli (EM0) or Bacteroides vulgatus (BV8H1) were fed for 7 weeks on semi-synthetic diets in which the protein source was either soya-bean meal (SM) or rape-seed meal (RM). For each bacterial status the RM-diet group was compared with the control group fed on the SM diet. The association of human faecal flora with the RM diet was responsible for reduced feed intake and reduced weight gain, an enlargement of the liver and thyroid and a decrease in both thyroxine and triiodothyronine plasma levels. The association of the B. vulgatus BV8H1 strain with the RM diet reproduced all these effects, except that triiodothyronine plasma levels were not significantly modified. Rats inoculated with the E. coli EM0 strain and fed on the RM diet exhibited a goitre and lowered thyroxine and triiodothyronine plasma levels. These results show that the human intestinal microflora may be involved in glucosinolate metabolism when cruciferous vegetables are consumed by man. The specificity of the symptoms observed according to the rat bacterial status supports the hypothesis that bacteria yield specific toxic glucosinolate derivatives according to their enzymic potential.

Presence of PAF-acether in stool of patients with pouch ileoanal anastomosis and pouchitis.

Platelet-activating factor is an endogenous phospholipid produced by a wide variety of inflammatory cells. Platelet-activating factor induces severe pathological changes in various organs and, among numerous potent effects, causes bowel necrosis. Pouchitis is a poorly understood complication of ileoanal pouch anastomosis which occurs in patients who undergo surgery for ulcerative colitis. The aim of this study was to measure ileal or fecal platelet-activating factor and lyso platelet-activating factor contents in normal volunteers (n = 12), in patients with terminal ileostomy (n = 7), and in patients with ileoanal anastomosis (n = 15) (8 patients have pouchitis defined by the presence of ulcerations on the reservoir). Fecal samples were processed and assessed for platelet-activating factor by platelet aggregation assay. The aggregating material was further characterized as platelet-activating factor by the following: inhibition of the platelet aggregation it induced by specific platelet-activating factor receptor antagonist (BN 52021; IHB, Le Plessis Robinson, France); abolition of platelet aggregation after incubation with phospholipase A2 but not with lipase A1; and retention time on high-performance liquid chromatography. Stool platelet-activating factor content (in nanograms per gram of stool, mean +/- 1SD) was significantly increased in patients with pouchitis (22.2 +/- 16 ng/g) compared with patients with normal reservoir (1.59 +/- 0.63 ng/g, P less than 0.01), terminal ileostomy (0.59 +/- 0.43 ng/g, P less than 0.01), and healthy controls (0 +/- 0 ng/g of stool, P less than 0.001). Lyso platelet-activating factor (nanograms per gram of stool) was increased in patients with pouchitis (10,704 +/- 5499 ng/g) compared with patients with normal reservoir (4721 +/- 4549 ng/g of stool, P less than 0.05), terminal ileostomy (3042 +/- 4019 ng/g, P less than 0.02), and healthy volunteers (128 +/- 107 ng/g, P less than 0.001). In patients with ileoanal anastomosis and pouchitis, increased platelet-activating factor production could be implicated in the inflammation and ulcerations observed in the reservoir.

Identification and properties of an alpha-amylase from a strain of Eubacterium sp. isolated from the rat intestinal tract.

1. A bacterial amylase was isolated from the intestinal content of monoxenic rats inoculated with Eubacterium sp. B86. 2. Affinity chromatography on cross-linked starch allowed its separation from rat endogenous amylases. 3. The bacterial enzyme was characterized by its pI, molecular weight and action pattern. It behaves as a typical endo-amylase (alpha-amylase).

Modulation of gut wall paf-acether and precursors by intestinal microflora.

Comparison between holoxenic and axenic mice led to the conclusion that the presence of an intestinal microflora produced a decrease in wall paf in conventional mouse caecum, whereas an increase in wall lyso-paf and alkyl-acyl-glycerophosphocholine (A-A-GPC) content was noticed. By contrast, the presence of flora had no significant incidence on wall paf, lyso-paf and A-A-GPC content of conventional mouse jejunum. Thus, the modulation of gut wall phospholipid composition by intestinal microflora is evidenced for the first time.

Glucosinolates toxicity in growing rats: interactions with the hepatic detoxification system.

1. Glucosinolate-rich diet (RM) in growing rats increased liver (a), kidneys (b), and thyroid (c) weights and depleted feed intake (d), growth curve (e) and T4 and T3 plasma levels (f). 2. Oral administration of phenobarbital enhanced the toxic effect of RM on (b), (d) and (e) and did not modify the toxic effect of RM on (a), (c) and (f). 3. RM had a depleting effect on hepatic microsomal P-450 specific activity. 4. RM had an enhancing effect on hepatic glutathione S-transferase and UDP-glucuronyltransferase specific activities. 5. These results indicate that some glucosinolate derivatives released by gut microflora metabolism are further metabolized by the hepatic detoxification system, and that they could play the role of co-toxic or co-detoxic molecules.

Experimental cecitis in gnotobiotic quails monoassociated with Clostridium butyricum strains isolated from patients with neonatal necrotizing enterocolitis and from healthy newborns.

Using axenic quails fed a diet containing lactose, we have investigated the potentially pathogenic roles of six Clostridium butyricum strains of human origin. Three strains (CB155-3, CB1002, and CB203-1) isolated from neonatal necrotizing enterocolitis patients and two of three strains (CB19-1 and CB25-2) isolated from healthy newborns led to cecal or crop lesions or both similar to those observed in human neonatal necrotizing enterocolitis: thickening of the cecal wall with gas cysts, hemorrhagic ulcerations, and necrotic areas. The lactose-negative strain (CB46-1) did not develop any lesions. The neuraminidase-producing strain (CB155-3) caused lesions in all monoassociated quails, whereas the other strains caused lesions in 28 to 85% of animals. Removal of dietary lactose suppressed all pathological incidence. These results show that lactose fermentation is a prerequisite in these pathological changes and stress the roles played by both the strain and the host in the expression of C. butyricum enteropathogenicity.

Antagonism exerted by an association of a Bacteroides thetaiotaomicron strain and a Fusobacterium necrogenes strain against Clostridium perfringens in gnotobiotic mice and in fecal suspensions incubated in vitro.

Antagonism between an association of Bacteroides thetaiotaomicron and Fusobacterium necrogenes strains and two strains of Clostridium perfringens was evidenced both in vivo in gnotobiotic mice and ex vivo in fecal suspensions incubated for 22 h at 37 degrees C. Several features of this antagonism were similar in and ex vivo. (i) An obligate and continuous synergy between B. thetaiotaomicron and F. necrogenes was required; (ii) the two C. perfringens strains did not respond to the same extent to this antagonism; and (iii) expression of the antagonism was host and diet dependent. Neither diffusible nor soluble inhibitory substances were detectable in feces of gnotobiotic mice, nor could depletion of nutrients be identified as causing antagonism in both in and ex vivo experiments. Our findings support the hypothesis that a reversible bacteriostasis induced by the inhibitory strains acting together continuously, and hindering the target strain from utilizing available nutrients, was responsible for this antagonism.