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.

Remanent effect of some dietary regimens on the establishment of two Clostridium strains in the digestive tract of gnotobiotic mice.

Axenic mice and rats fed different diets were associated only with two strains of Clostridium. The first one identified as Clostridium perenne was isolated from the fecal dominant flora of an adult rat. The second one belonging to the group I of Clostridium was isolated from the dominant flora of the feces from a piglet. Both strains were capable of becoming established in the digestive tract of animals fed dietary regimens called "permissive", while they did not become established in animals fed "nonpermissive" diets. However, when these bacterial strains had become established in animals fed a permissive diet, they persisted in the dominant flora even if the permissive diet was replaced by the nonpermissive one. This phenomenon was called remanent effect of the diet. It disappeared after 103 days for C. perenne, but not for the other Clostridium strain. This effect was not due to a selection of a genetic variant from the original strain or to a durable change in some characteristics of the host-animal associated with the bacterial strain.

Fatty acid composition of lipids in the maternal diet and establishment of a Lactobacillus sp. strain in the digestive tract of suckling gnotobiotic mice and rats.

Adult gnotobiotic mice and rats, monoassociated with a homofermentative strain of Lactobacillus sp. of intestinal origin, were fed either a commercial rodent chow A or a semisynthetic diet B. Similar numbers of lactobacilli were established in their gastrointestinal tract whatever diet they ate. The lactobacilli were established in the digestive tract of the newborn of A mothers at 2 days but were hardly established in mouse or rat pups of A mothers than in their mouse homologues. Comparative analysis of milk lipids in the A and B series showed a linoleic acid (18:2 n-6) content which was three times higher in the B than in the A series. Two diets S and H differing only by their lipid fractions, which, respectively, presented the same fatty acid compositions as lipids from diets A and B were then given to two others lots of Lactobacillus monoassociated mice. The establishment kinetics of the strain were the same in the mouse pups of these two lots precedently observed in the B series. The difference observed in the establishment kinetics of the Lactobacillus strain in the digestive tract of suckling gnotobiotic mice was thus attributed to other dietary factors than the fatty acid composition of the maternal diet.

Implantation of bacteria from the digestive tract of man and various animals into gnotobiotic mice.

Fourteen microbial strains isolated from conventional rats were inoculated into axenic rats and mice receiving identical diets. The populations of these organisms which became established in the feces of gnotobiotic adult recipient rats and mice were quite similar. The only major difference was that one strain, belonging to the genus Clostridium, disappeared from the feces of gnotobiotic mice, whereas this strain became established in gnotobiotic rats. Most of the strictly anaerobic strains were absent or present only in small numbers before weaning in young rats and mice. A clear-cut barrier effect against Salmonella typhimurium was found in adult gnotobiotic mice colonized with a complex flora derived from a conventional chicken. The microflora established in these recipient mice exerted the same barrier effect when further transferred into axenic chickens. Inoculation of feces from a human donor into adult gnotobiotic recipient mice produced colonization by several strains from the donor, whereas other strains, belonging to the genera Bifidobacterium, Lactobacillus, and Clostridium were present in the donor, but did not persist in recipient mice. In these mice, nonetheless, the colonizing human fecal flora exerted an effective barrier against a toxigenic strain of Clostridium difficile. This barrier effect spontaneously disappeared several weeks later. Administration of clindamycin to the recipient mice led to large variations in the number of viable cells of C. difficile.

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.

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.

Measurement of fecal bile acid excretion in gnotobiotic rats: comparison of gas-liquid chromatography and [4(-14C)] cholesterol isotopic equilibrium.

Gas-liquid chromatography (G.L.C.) and the method of [4(-14C)] cholesterol isotopic equilibrium (C.I.E.) were used to determine the fecal bile acid excretion in gnotobiotic rats. The same samples were submitted to both methods. In these conditions, it was observed that the fecal bile acid excretions determined by G.L.C. were 38% of lower than when determined by C.I.E. In thin-layer chromatographic analyses (T.L.C.) of the bile acid extracts obtained from rats in which a [4(-14C)] cholesterol isotopic equilibrium was established, 33 to 35% of the radioactivity of this fraction was not observed in the rat primary bile acids. No bile acids could be observed in G.L.C. made with eluates obtained from the T.L.C. areas containing this radioactivity. It therefore appears that the difference observed in the results obtained by G.L.C. and C.I.E. is due to the fact that chemical species which are not measured by the former method can be determined by the latter one. T.L.C. analyses of bile acid extracts from axenic rats in which either a [26(-14C)] cholesterol or a [2,4(-3H)] cholic acid and [24(-14C)] chenodeoxycholic acid equilibrium were established, lead to the conclusion that the chemical composition of these undetermined substances is complex: part of these substances comes from the transformation of bile acids; another part is made of molecules which maintain the 26(-14C) of cholesterol.

Interaction between Escherichia coli and Shigella flexneri in the digestive tract of "gnotobiotic" mice.

When a strain of Shigella flexneri and then a strain of Escherichia coli were implanted at an interval of 1 day in the digestive tract of germfree mice, the S. flexneri was eliminated in 8 days. However, when S. flexneri was mono-associated with germfree mice in the absence of E. coli, a population of S. flexneri appeared which was resistant to the antagonistic effects of E. coli in vivo. The emergence of this population appeared to be a stepwise process, extending over a period of 3 months. The interaction between S. flexneri and E. coli in vivo and in vitro was significantly different in that the Shigella capable of coexisting with E. coli in the digestive tract was suppressed by that organism in vitro.

[The microbial flora in the digestive tract of the monogastric and its effect on the nutritional metabolism of the host]. / La flore microbienne du tube digestif chez le monogastrique et son influence sur les métabolismes nutritionnels de l'hôte

The purpose of the present article is to give a survey of present knowledge of the ecological relationships between the various bacterial populations composing the microflora of the digestive tract of the host animal. Analysis of the relationships can be made with the aid of recent techniques: utilization of axenic animals (germ-free) and of gnotoxenic animals inoculated with known bacterial populations; perfecting of bacteriological techniques for the quantitative differential analysis of the various bacterial populations in the digestive tract, especially in the case of strictly anaerobic species. Some characteristics of the equilibrium between the various bacterial populations in the different segments of the gastro-intestinal tract have been described as well as some of the mechanisms which regulate this equilibrium. These mechanisms either imply interactions between bacteria or the action of the host animal and the environment on the microflora. Lastly, various examples concerning the action of the microflora in the digestive tract on the nutritional physiology of the host animal are given; action on the anatomy of the digestive tract and on the metabolism of carbohydrates, proteins, lipids and vitamins.

[Interest in gnotoxenic systems for the study of host-microbial flora of the digestive tract]. / Intérêt des systèmes gnotoxéniques pour l'étude des relations hôte-flore microbienne du tube digestif.

It is necessary to use experimental models in all studies of interactions between a host, its digestive tract microbial flora and the environment because these interactions are complex. The gnotoxenic animal, reared in an isolator as the axenic animal and harbouring a known microbial flora, constitutes either an analytic or mimetic experimental model. The gnotoxenic animal may be considered as an analytic model when used to determine which biotic or abiotic environmental factor of the host-animal plays a role in the intestinal ecosystem. The potential metabolic or immunologic role of a given bacterial strain in the intestinal ecosystem may be determined when the axenic animal is associated with this strain. The variation of the expression of the potential role of this strain in relation to the environment can be ascertained by diversifying the diet of the host or by introducing other bacterial strains into the intestinal ecosystem. The role of an association of strains in host physiology and host protection against potentially pathogenic target strains can also be studied using this analytic model. An analytic model is created by associating either isolated strains of the holoxenic or heated or diluted suspensions of holoxenic digestive tract flora. Axenic mice associated with these simplified flora are called meroxenic. The gnotoxenic animal is considered to be a mimetic model when it harbours a microbial flora isolated from an animal of a different species. The advantage of the mimetic model is that it provides an easy-to-use biological tool, i.e. gnotoxenic mice, to first determine the potential role of these microbial strains. The validity of the mimetic model is then tested by comparing gnotoxenic mice and gnotoxenic piglets or chickens. When all the gnotoxenic animals are given the same diet, this comparison permits an estimation of the animal-host role in the expression of the potential activities of microbial strains. The mimetic model, composed of gnotoxenic animals harbouring microbial strains of human origin, is the only experimental model which can be used to study the role of microorganisms in the intestinal ecosystem of man.

beta-Glucuronidase activities of intestinal bacteria determined both in vitro and in vivo in gnotobiotic rats.

The beta-glucuronidase activities of bacterial strains isolated from the rat intestinal tract were studied both in vitro in culture media and in vivo in the intestinal contents of gnotobiotic rats. Only 50 of 407 strains tested were found to be positive in vitro. They belonged to the three genera Clostridium, Peptostreptococcus, and Staphylococcus. The in vitro-negative strains were also negative in vivo. The beta-glucuronidase activities of the beta-glucuronidase activities of the positive strains were generally greater in vivo than in vitro. The highest in vivo activities were found in the intact bacterial cells and in the soluble fractions prepared from disrupted pellets. There was a discrepancy between the activities obtained from both conventional and gnotobiotic rats harboring selected positive strains, suggesting that the main beta-glucuronidase-positive strains have not yet been isolated from the intestines of conventional rats.

Ability of two Clostridium difficile strains from man and hare to produce cytotoxin in vitro and in gnotobiotic rodent intestines.

Cytotoxin production by human (VP1) and hare (FD) strains of Clostridium difficile were compared both in vitro in a broth culture and in vivo in intestinal contents of gnotobiotic rodents. Strain VP1 produced about 1,000 times more cytotoxin than the FD strain, both in vitro and in vivo, although the population levels of the two strains were not significantly different either in vitro or in vivo. Ninety percent of gnotobiotic rats and 100% of gnotobiotic mice established with the VP1 strain died within 3 days, whereas no mortality in rats or mice was observed with the FD strain. The cytotoxin titre increased during the 3 weeks following establishment of the FD strain in mice, decreasing thereafter. Mice previously established with the FD strain were protected from VP1 strain challenge. Cytotoxin production was greatly decreased in diassociated mice, although the population levels of the two strains did not differ to a great extent.

[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.

[Pathogenicity of various toxinogenic types of Clostridium perfringens administered by mouth to axenic and holoxenic mice]. / Pouvoir pathogène de différents types toxinogènes de Clostridium perfringens inoculés per os à des souris axéniques et holoxéniques.

Axenic mice died with signs of enterotoxaemia after oral ingestion of Clostridium perfringens type C or D. Under the same conditions, C. perfringens type B was less pathogenic, and types A and E showed no pathogenicity. The microflora of conventional mice prevented the establishment of C. perfringens types B, C and D in the digestive tract and protected them against the pathogenicity of these strains. Toxins produced in the caecum of monoxenic mice harbouring C. perfringens type C were not neutralized by the anti-C. perfringens type C antiserum. This suggests that the toxins produced in vivo by this strain were different from those produced in vitro.

[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.