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.

Distribution of genes encoding the trypsin-dependent lantibiotic ruminococcin A among bacteria isolated from human fecal microbiota.

Fourteen bacterial strains capable of producing a trypsin-dependent antimicrobial substance active against Clostridium perfringens were isolated from human fecal samples of various origins (from healthy adults and children, as well as from adults with chronic pouchitis). Identification of these strains showed that they belonged to Ruminococcus gnavus, Clostridium nexile, and Ruminococcus hansenii species or to new operational taxonomic units, all from the Clostridium coccoides phylogenetic group. In hybridization experiments with a probe specific for the structural gene encoding the trypsin-dependent lantibiotic ruminococcin A (RumA) produced by R. gnavus, seven strains gave a positive response. All of them harbored three highly conserved copies of rumA-like genes. The deduced peptide sequence was identical to or showed one amino acid difference from the hypothetical precursor of RumA. Our results indicate that the rumA-like genes have been disseminated among R. gnavus and phylogenetically related strains that can make up a significant part of the human fecal microbiota.

Ruminococcin A, a new lantibiotic produced by a Ruminococcus gnavus strain isolated from human feces.

When cultivated in the presence of trypsin, the Ruminococcus gnavus E1 strain, isolated from a human fecal sample, was able to produce an antibacterial substance that accumulated in the supernatant. This substance, called ruminococcin A, was purified to homogeneity by reverse-phase chromatography. It was shown to be a 2,675-Da bacteriocin harboring a lanthionine structure. The utilization of Edman degradation and tandem mass spectrometry techniques, followed by DNA sequencing of part of the structural gene, allowed the identification of 21 amino acid residues. Similarity to other bacteriocins present in sequence libraries strongly suggested that ruminococcin A belonged to class IIA of the lantibiotics. The purified ruminococcin A was active against various pathogenic clostridia and bacteria phylogenetically related to R. gnavus. This is the first report on the characterization of a bacteriocin produced by a strictly anaerobic bacterium from human fecal microbiota.

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.

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.

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.

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.

Antagonistic effect exerted by three strictly anaerobic strains against various strains of Clostridium perfringens in gnotobiotic rodent intestines.

Our purpose was to study bacterial antagonism between a limited number of strictly anaerobic strains and Clostridium perfringens in the intestinal tract of gnotobiotic rodents. Gnotobiotic mice harboring a Bacteroides thetaiotaomicron, a Fusobacterium necrogenes, and a Clostridium sp. strain were protected against pathogenic B, C, and D C. perfringens serotypes. A drastic antagonistic effect of this three-strain association was also observed against a nonpathogenic C. perfringens serotype A (CpA). It was less efficient in gnotobiotic rats than in mice and less efficient in gnotobiotic mice fed an autoclaved diet than in mice fed the same diet sterilized by irradiation. No diffusible inhibitory substances against CpA were detected in feces of gnotobiotic mice harboring the three antagonistic strains, and no nutrient depletion was demonstrated in filtrates prepared from 10-fold diluted feces of these mice. In vitro mixed cultures of the three antagonistic strains failed to inhibit growth of CpA, whereas CpA did not multiply in a 10-fold diluted feces from gnotobiotic mice. A reverse correlation between the initial number of antagonistic strains and the division number of CpA was determined using serially diluted fecal suspensions. Thus, large numbers of viable cells of both antagonistic strains were required to inhibit the target strain in fecal suspensions as was also found in gnotobiotic mice intestines. However, no diffusible inhibitory substance was detectable nor could depletion of growth factors be identified as causing antagonism. Whatever factors that may be responsible for antagonism were found to be influenced by the host and its diet.

[Development of cecal microbial flora following surgical isolation of the large intestine in swine]. / Evolution de la flore microbienne caecale après isolement chirurgical du gros intestin chez le porc.

An ileo-rectal anastomosis was created in growing pigs. The caecum and colon were left in place and their contents slowly emptied through a cannula located in the distal colon; accordingly, no food arrived in this caeco-colic compartment. Animals were slaughtered 14 to 54 days after the surgical operation. Using a quantitative differential analysis technique, the large intestine microflora were studied upon operation and at slaughter. It was observed that the number of strictly anaerobic bacteria did not vary or only slightly increased according to cell population counts. The most marked variations involved disappearance of the Lactobacillus population in all animals and disappearance of some morphological types of bacteria found in the dominant flora at the beginning of the experiment.

[Implantation of a mutant of Escherichia coli requiring diaminopimelic acid in the digestive tract of gnotobiotic mice]. / Implantation d'un mutant de Escherichia coli exigeant en acide diaminopimélique dans le tube digestif de souris gnotoxéniques.

A DAP- auxotroph mutant of Escherichia coli DP50 requiring DAP and thymidine for growth was used as the receptor strain in genetic engineering. It failed to be implanted in axenic mice. However, when an inoculum containing more than 10(7) bacteria/ml was used, the DAP+ reverse mutant devoid of requirement for DAP became implanted. When axenic mice were previously associated with Clostridium difficile containing DAP in the cell wall, the strain DAP- became implanted even when the inoculum was too small to permit implantation in axenic mice. Conversely, C. butyricum and C. perenne, whose cell walls also contain DAP, did not allow the establishment of a DAP- mutant. In animals associated with complex human flora without enterobacteria, neither of the 2 DAP- and DAP+ mutants became implanted.

[Effect of the ingestion of wheat bran on the fecal microbial flora of human donors and of recipient gnotoxenic mice, and on the barrier effects exerted by these flora against various potentially pathogenic microorganisms]. / Effet de l'ingestion de son de blé sur la flore microbienne fécale de donneurs humains et de souris gnotoxéniques receveuses, et sur les effets de barrière exercés par ces flores à l'égard de divers microorganismes potentiellement pathogènes.

The effect of bran ingestion on the flora of the human digestive tract was studied using two methods: quantitative enumeration of various microbial populations of the faecal flora, and a demonstration of the antagonistic effect exerted by the faecal flora against various potentially pathogenic bacteria of the environment. Since this latter study cannot be effected in human subjects, we used a model constituted by axenic mice inoculated with patients' flora. Faecal samples from 3 human donors receiving bran-containing diets were obtained prior to treatment and 30 days thereafter. These faecal samples were inoculated into axenic mice fed a diet with or without bran. The dominant floras of the human donors, before and after bran ingestion, were highly similar. The faecal floras of the gnotoxenic mice resembled those of the donors and no change resulting from the presence of bran in the diet could be observed. The drastic or permissive barrier effects exerted in the gnotoxenic mice by the human donors against Clostridium perfringens, Staphylococcus aureus, Candida albicans and Pseudomonas aeruginosa were not modified by the presence of bran in the diet. The large variability between animals in the barrier effect against Clostridium difficile masked any possible role of the bran. Study of the transit of Bacillus spores in the digestive tract of various mouse groups showed the existence of differences according to the origin of the inoculated floras, but not according to the presence or absence of bran in the diet.

[Effect of age on permissive barrier effect against a strain of enterobacteria exerted by a complex bacterial flora in the digestive tract of mice reared in isolator (author's transl)]. / Influence du vieillissement de l'hôte sur l'effet de barrière permissif à l'égard d'une souche d'entérobactérie, exercé par une flore bactérienne complexe dans le tube digestif de souris maintenues en isolateur.

A permissive barrier effect against Escherichia coli was observed in the faeces of a first group of mice associated with a complex microbial flora. This effect was transmitted to 6 other groups of mice by inoculation of a fecal suspension originated from a mouse of group I. Although mice were kept in isolator in constant biotic and abiotic environmental conditions, the expression of the barrier effect was very variable during several days after inoculation, then became stabilized in every group, but more slowly in group I than in the others. The expression of this barrier effect was compared in male and female mice aged five weeks or 6 months. The barrier effect was significantly more efficient in older than in young mice whatever the sex.

Antagonistic effect of extremely oxygen-sensitive clostridia from the microflora of conventional mice and of Escherichia coli against Shigella flexneri in the digestive tract of gnotobiotic mice.

Two extremely oxygen-sensitive strains of Clostridium sp., designated Clostridium E and P, were obtained from digestive microflora of conventional mice and found to constitute a barrier against Shigella flexneri SF-2 when associated in vivo with Escherichia coli K-12. These and other simplified fractions of the conventional microflora were demonstrated to have an effect comparable to that of the total flora. When K-12 and Clostridium E were established in gnotobiotic mice before the introduction of SF-2, the latter was reduced to a level below detection in the digestive tract. Whe SF-2 was established first, the antagonistic effect exerted by Clostridium E and K-12 was variable and, apparently, related to the rate of establishment of Clostridium E. Mutants of SF-2 resistant to the barrier effect of Clostridium E and K-12 appeared at the end of 3 months when SF-2 was established in gnotobiotic mice alone or with K-12, and after only a week when SF-2 was associated only with Clostridium E. These results suggest that the bacterial antagonsim in this model is related to the production in vivo of an antibiotic substance active against SF-2. It appears that the substance may be produced by Clostridium E, stimulated by K-12.