Pierre Raibaud

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.

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.

Bacteroides sp. Strain D8, the First Cholesterol-Reducing Bacterium Isolated from Human Feces

ABSTRACT The microbial community in the human colon contains bacteria that reduce cholesterol to coprostanol, but the species responsible for this conversion are still unknown. We describe here the first isolation and characterization of a cholesterol-reducing bacterium of human intestinal origin. Strain D8 was isolated from a 10−8 dilution of a fresh stool sample provided by a senior male volunteer with a high capacity to reduce luminal cholesterol to coprostanol. Cholesterol-to-coprostanol conversion by strain D8 started on the third day, while cells were in stationary phase, and was almost complete after 7 days. Intermediate products (4-cholesten-3-one and coprostanone) were occasionally observed, suggesting an indirect pathway for cholesterol-to-coprostanol conversion. Resting-cell assays showed that strain D8 could reduce 1.5 μmol of cholesterol/mg bacterial protein/h. Strain D8 was a gram-negative, non-spore-forming, rod-shaped organism identified as a member of the genus Bacteroides closely related to Bacteroides vulgatus, based on its morphological and biochemical characteristics. The 16S rRNA gene sequence of strain D8 was most similar (>99.5%) to those of two isolates of the recently described species Bacteroides dorei. Phylogenetic tree construction confirmed that Bacteroides sp. strain D8 clustered within an independent clade together with these B. dorei strains. Nevertheless, no cholesterol-reducing activity could be detected in cultures of the B. dorei type strain. Based on Bacteroides group-specific PCR-temporal temperature gradient gel electrophoresis, there was no correlation between the presence of a band comigrating with the band of Bacteroides sp. strain D8 and cholesterol conversion in 11 human fecal samples, indicating that this strain is unlikely to be mainly responsible for cholesterol conversion in the human population.

Inhibition of Clostridium perfringens by an Antibiotic Substance Produced by Bacillus licheniformis in the Digestive Tract of Gnotobiotic Mice: Effect on Other Bacteria from the Digestive Tract

A strain of Bacillus licheniformis, established in the digestive tract of gnotobiotic mice, inhibited the subsequent establishment of a Clostridium perfringens strain ingested by the animals. This inhibitory effect depended on the in vivo production by B. licheniformis of an antibiotic substance having a number of the characteristics of bacitracin. If C. perfringens was the first to become established in the digestive tract of the gnotobiotic mice, B. licheniformis also became established but did not produce any antibiotic. Mutants of C. perfringens resistant to the antibiotic substance were not observed when the antibiotic was produced in situ by B. licheniformis, but were rapidly selected when the Bacillus culture filtrate or bacitracin was administered per os. B. licheniformis was also capable of eliminating from the digestive tract 5 of the 13 additional bacterial strains tested.

Production of an Antibiotic Substance by Bacillus licheniformis Within the Digestive Tract of Gnotobiotic Mice

In monoxenic mice, vegetative cells and spores of Bacillus licheniformis were enumerated and in vivo antibiotic production was measured at various levels of the digestive tract and in the feces. The apparent independence of vegetative cell and spore populations in the cecum and feces, as well as the marked fluctuations of these two populations in the stomach and small intestine, suggested that sporulation of B. licheniformis and production of antibiotic occur only in the upper levels of the digestive tract. Study of dixenic models wherein B. licheniformis was inoculated after Clostridium perfringens or Lactobacillus sp. or before Lactobacillus sp. revealed the simultaneous disappearance of B. licheniformis from the stomach and antibiotic from the feces. This strain persisted in the cecum, but only in the form of vegetative cells. These models demonstrate that the activity of the microflora in the upper segments can affect the equilibrium of the microflora throughout the digestive tract. Images

Translocation of strictly anaerobic bacteria from the intestinal tract to the mesenteric lymph nodes in gnotobiotic rodents

Viable cells of some strictly anaerobic strains belonging to Bacteroides, Clostridium and Fusobacterium genera were present in mesenteric lymph nodes of gnotobiotic rodents harbouring these strains. Various parameters were found to affect the incidence of translocation, including the caecal population level, the length of association with the host and the nature of the strains and host.

Comparaison entre le nombre et la nature des clostridium fécaux et d'autres facteurs de risque impliqués dans la pathologie intestinale des nouveau-nés

Summary One-hundred and fifteen infants aged 1 to 31 days from two intensivecare units were grouped into 6 classes according to clinical criteria (enterocolitis with or without anatomopathological examination and pneumatosis intestinalis, ≪haemorrhagic colitis ≫, acute diarrhoea or absence of intestinal disorders). The total number of viable bacteria, the number of Clostridium and, in some cases, the presence of rota- and/or coronavirus were determined in their stools. The incidence of Clostridium in the stools of infants with enterocolitis (with or without pneumatosis intestinalis) or haemorrhagic colitis was not significantly different from that of infants without intestinal disorders, whereas stools of infants with acute diarrhoea less often contained Clostridium than those of other infants. C. butyricum, C. difficile, C. perfringens, C. tertium, and C. sordellii were identified. Correspondence analysis comparing the variable, ≪clinical profile≫, with 23 other variables, suggested that the variables of gemellity, a birth-weight below 1900 g, a gestational age of less than 35 weeks, respiratory distress, umbilical catheterization and a Clostridium count above 107/g at the onset of clinical signs, i.e. between 8 to 12 days of age, were linked to the clinical profile of necrotizing enterocolitis with pneumatosis intestinalis. Conversely, the absence of gemellity, a high birthweight and gestational age, the absence of respiratory distress or umbilical catheterization, the onset of diarrhoea within 8 days, and the presence of rota- and/or coronavirus in the stools were linked with a clinical profile of acute diarrhoea.

The Use of Germ-Free Mice Associated with Human Fecal Flora as an Animal Model to Study Enteric Bacterial Interactions

In 1964 Dubos and Schaedler first assimilated the complex intestinal microflora and its relationship with its animal host to an ecosystem(12). Composition and functions of the intestinal ecosystem have since been widely studied. The same workers(10) and Freter previously described in vivo antagonisms of intestinal bacteria against enteric pathogens(21, 22). Through the work of Abrams and Bishop(1), Ducluzeau and Raibaud(15), and Van der Waij(49), the existence of microbiological barriers, which enhance resistance to colonization by exogenous bacteria and help to prevent infection, has progressively emerged. Interactions between bacterial species within the lumen of the gut may include reciprocal promotion of a strain by another(43), partial(13) or total(35) inhibition of such or such bacterial population, exchange of genetic information through in vivo DNA transfer(18, 19, 28, 39). These kinds of interactions may be of primary importance in several instances such as : (i): the pathogenesis of local or general infections by organisms originating in the gut, (ii): the chemoprophylaxis of certain forms of infectious diarrhea and of nosocomial infections in surgical and immunocompromised hosts, (iii): the regulation of the spread among bacterial species of pathogenicity and/or resistance genetic determinants.

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 d'un mutant de Escherichia coli exigeant en acide diaminopimélique dans le tube digestif de souris gnotoxéniques

Summary A DAP − auxotroph mutant of Escherichia coli DP 50 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.