Escherichia coli strains colonising the gastrointestinal tract protect germfree mice against Salmonella typhimurium infection.

BACKGROUND: Escherichia coli is part of the normal gastrointestinal microflora which exerts a barrier effect against enteropathogens. Several E coli strains develop a protective effect against other Enterobacteriaceae. AIMS: Two E coli strains, EM0, a human faecal strain, and JM105 K-12 were tested for their ability to prevent in vivo and in vitro infection by Salmonella typhimurium C5. METHODS: Inhibition of C5 cell invasion by E coli was investigated in vitro using Caco-2/TC7 cells. The protective effect of E coli was examined in vivo in germfree or conventional C3H/He/Oujco mice orally infected by the lethal strain C5. RESULTS: EMO expresses haemolysin and cytotoxic necrotising factor in vitro. In vitro, the two strains did not prevent the growth of C5 by secreted microcins or modified cell invasion of C5. In vivo, establishment of EM0 or JM105 in the gut of germfree mice resulted in a significant increase in the number of surviving mice: 11/12 and 9/12, respectively, at 58 days after infection (2x10(6)/mouse) versus 0/12 in control germfree group at 13 days after infection. Colonisation level and translocation rate of C5 were significantly reduced during the three days after infection. In contrast, no reduction in faecal C5 excretion was observed in C5 infected conventional mice (1x10(8)/mouse) receiving the EM0 or JM105 cultures daily. CONCLUSIONS: Establishment of E coli strains, which do not display antimicrobial activity, protects germfree mice against infection and delays the establishment of C5 in the gut. Possible mechanisms of defence are discussed.

Bifidobacterium strains from resident infant human gastrointestinal microflora exert antimicrobial activity.

BACKGROUND AND AIMS: The gastrointestinal microflora exerts a barrier effect against enteropathogens. The aim of this study was to examine if bifidobacteria, a major species of the human colonic microflora, participates in the barrier effect by developing antimicrobial activity against enterovirulent bacteria. METHODS: Antibacterial activity was examined in vitro against a wide range of Gram negative and Gram positive pathogens. Inhibition of Salmonella typhimurium SL1334 cell association and cell invasion was investigated in vitro using Caco-2 cells. Colonisation of the gastrointestinal tract in vivo by bifidobacteria was examined in axenic C3/He/Oujco mice. Antimicrobial activity was examined in vivo in axenic C3/He/Oujco mice infected by the lethal S typhimurium C5 strain. RESULTS: Fourteen human bifidobacterium strains isolated from infant stools were examined for antimicrobial activity. Two strains (CA1 and F9) expressed antagonistic activity against pathogens in vitro, inhibited cell entry, and killed intracellular S typhimurium SL1344 in Caco-2 cells. An antibacterial component(s) produced by CA1 and F9 was found to be a lipophilic molecule(s) with a molecular weight of less than 3500. In the axenic C3/He/Oujco mice, CA1 and F9 strains colonised the intestinal tract and protected mice against S typhimurium C5 lethal infection. CONCLUSION: Several bifidobacterium strains from resident infant human gastrointestinal microflora exert antimicrobial activity, suggesting that they could participate in the "barrier effect" produced by the indigenous microflora.

The human Lactobacillus acidophilus strain LA1 secretes a nonbacteriocin antibacterial substance(s) active in vitro and in vivo.

The adhering human Lactobacillus acidophilus strain LA1 inhibits the cell association and cell invasion of enteropathogens in cultured human intestinal Caco-2 cells (M. F. Bernet, D. Brassard, J. R. Neeser, and A. L. Servin, Gut 35:483-489, 1994). Here, we demonstrate that strain LA1 developed its antibacterial activity in conventional or germ-free mouse models orally infected by Salmonella typhimurium. We present evidence that the spent culture supernatant of strain LA1 (LA1-SCS) contained antibacterial components active against S. typhimurium infecting the cultured human intestinal Caco-2 cells. The LA1-SCS antibacterial activity was observed in vitro against a wide range of gram-negative and gram-positive pathogens, such as Staphylococcus aureus, Listeria monocytogenes, S. typhimurium, Shigella flexneri, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Enterobacter cloacae. By contrast, no activity was observed against species of the normal gut flora, such as lactobacilli and bifidobacteria. The LA1-SCS antibacterial activity was insensitive to proteases and independent of lactic acid production.

Antibacterial effect of the adhering human Lactobacillus acidophilus strain LB.

The spent culture supernatant of the human Lactobacillus acidophilus strain LB produces an antibacterial activity against a wide range of gram-negative and gram-positive pathogens. It decreased the in vitro viability of Staphylococcus aureus, Listeria monocytogenes, Salmonella typhimurium, Shigella flexneri, Escherichia coli, Klebsiella pneumoniae, Bacillus cereus, Pseudomonas aeruginosa, and Enterobacter spp. In contrast, it did not inhibit lactobacilli and bifidobacteria. The activity was heat stable and relatively sensitive to enzymatic treatments and developed under acidic conditions. The antimicrobial activity was independent of lactic acid production. Activity against S. typhimurium SL1344 infecting human cultured intestinal Caco-2 cells was observed as it was in the conventional C3H/He/oujco mouse model with S. typhimurium C5 infection and oral treatment with the LB spent culture supernatant.

Antagonistic activity exerted in vitro and in vivo by Lactobacillus casei (strain GG) against Salmonella typhimurium C5 infection.

The aim of this study was to compare the antagonistic properties of Lactobacillus casei GG exerted in vitro against Salmonella typhimurium C5 in a cellular model, cultured enterocyte-like Caco-2 cells, to those exerted in vivo in an animal model, C3H/He/Oujco mice. Our results show that a 1-h contact between the invading strain C5 and either the culture or the supernatant of L. casei GG impeded the invasion by the Salmonella strain in Caco-2 cells, without modifying the viability of the strain. After neutralization at pH 7, no inhibition of the invasion by C5 was observed. The antagonistic activity of L. casei GG was examined in C3H/He/Oujco mice orally infected with C5 as follows: (i) L. casei GG was given daily to conventional animals as a probiotic, and (ii) it was given once to germ-free animals in order to study the effect of the population of L. casei GG established in the different segments of the gut. In vivo experiments show that after a single challenge with C5, this strain survives and persists at a higher level in the feces of the untreated conventional mice than in those of the treated group. In L. casei GG germ-free mice, establishment of L. casei GG in the gut significantly delayed the occurrence of 100% mortality of the animals (15 days after C5 challenge versus 9 days in germ-free mice [P < 0.01]). Cecal colonization level and translocation rate of C5 to the mesenteric lymph nodes, spleen, and liver were significantly reduced during the first 2 days post-C5 challenge, although the L. casei GG population level in the gut dramatically decreased in these animals.

Differentiation-associated antimicrobial functions in human colon adenocarcinoma cell lines.

We report that the enterocytic cells of the HT-29 glc-/+ cell subpopulation strongly expressed two antimicrobial enzymes: the lysozyme and alpha1-antitrypsin. Moreover, we found that 20 to 30% of these cells expressed positive immunoreactivity using the mAbs directed against the gut porcine PR-39 and cecropin P1 antimicrobial peptides, but did not express immunreactivity against the human antimicrobial polymorphonucleated neutrophil-associated HNP 1-3 defensin and the Xenopus skin magainin. The HT-29 glc-/+ cell subpopulation develops bacteriolytic activity against the enterovirulent diffusely adhering C1845 Escherichia coli characterized by dramatic alterations of the bacterial cell, suggesting lysis, and bacterial death. In contrast, no expression of immunoreactivity against the antimicrobial peptides and no C1845 bacterial alteration were found in the cultured human embryonic undifferentiated INT407 cells and the colon adenocarcinoma T84 crypt cells. The development of the bacterial alteration and the expression of the antimicrobial components were examined as a function of the cell differentiation using the Caco-2 cell line which spontaneously differentiates in culture. We found that the bacterial alteration and the expression of the PR-39 immunoreactivity are differentiation-associated events. Altogether, our results suggest that in the intestine the enterocytes could develop antimicrobial defenses participating in the protection of the gut epithelium against enterovirulent microorganisms.

Pathogenicity of the diffusely adhering strain Escherichia coli C1845: F1845 adhesin-decay accelerating factor interaction, brush border microvillus injury, and actin disassembly in cultured human intestinal epithelial cells.

The diffusely adhering Escherichia coli strain C1845 harboring the fimbrial F1845 adhesin can infect cultured human intestinal epithelial cells. The mechanism by which E. coli C1845 induces diarrheal illness remains unknown. This study investigated the injuries of cultured human intestinal cells promoted by E. coli C1845. Membrane-associated decay accelerating factor was identified as the intestinal receptor for the F1845 fimbrial adhesin of the E. coli C1845 strain by using purified F1845 adhesin, antibody directed against the F1845 adhesin, and monoclonal antibodies directed against the decay accelerating factor. Using monolayers of Caco-2 cells apically infected with E. coli C1845 and examined by scanning and transmission electron microscopy, we observed that strain C1845 induced injury to microvilli (MV) characterized by elongation and nucleation of the MV. We observed that infection of T84 and Caco-2 cells by E. coli C1845 was followed by disassembly of the actin network in the apical and basal cell domains. MV injury was differentiation related: E. coli C1845 promoted MV injury only when the cells were fully differentiated. The disassembly of the actin network occurred in poorly differentiated and fully differentiated Caco-2 cells but not in undifferentiated cells. Moreover, apical actin disassembly was observed in fully differentiated Caco-2 cells infected with the laboratory strain E. coli HB101(pSSS1) expressing the F1845 adhesin. In conclusion, E. coli C1845 promotes MV lesion in human epithelial intestinal cells, resulting from disassembly of the actin network.

Differential expression of complement proteins and regulatory decay accelerating factor in relation to differentiation of cultured human colon adenocarcinoma cell lines.

Self protection of host cells against inadvertent injury resulting from attack by autologous complement proteins is well reported for vascular epithelium. In intestinal epithelium, the expression of C complement proteins and regulatory proteins remains currently poorly reported. This study looked at the distribution of C complement proteins and regulatory decay accelerating factor (DAF) in four cultured human intestinal cell lines of embryogenic or colon cancer origins. C3 and C4 proteins and DAF were widely present in human colon adenocarcinoma T84, HT-29 glc-/+ cells compared with human embryonic INT407 cells. In contrast, no expression of C5, C5b-9, and CR1 was seen for any of the cell lines. Taking advantage of the Caco-2 cells, which spontaneously differentiate in culture, it was seen that the C3, C4, and DAF were present in undifferentiated cells and that their expression increased as a function of the cell differentiation. These results, taken together with other reports on the presence of C complement proteins and DAF in the intestinal cells infer that the expression of regulatory C complement proteins develops in parallel with the expression of C proteins to protect these cells against the potential injury resulting from the activation of these local C proteins. Moreover, the finding that the pathogenic C1845 Escherichia coli binds to the membrane bound DAF in the cultured human intestinal cells synthetising locally C proteins and regulatory C proteins supports the hypothesis that E coli could promote inflammatory disorders by blocking local regulatory protein function.

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.

Efficiency of various bacterial suspensions derived from cecal floras of conventional chickens in reducing the population level of Salmonella typhimurium in gnotobiotic mice and chicken intestines.

The antagonistic effect exerted towards Salmonella typhimurium by the flora issued from conventional chickens was studied in gnotobiotic animals. In germfree chickens and mice inoculated with S. typhimurium, the highest bacterial counts were observed in ceca, and were not significantly different in either host. The protection afforded by the inoculation of cecal flora issued from a conventional chicken was more effective when this flora was inoculated first into germfree chickens than when it was given only after inoculation with S. typhimurium. Administration of a cecal flora from a 15-day-old chick to gnotobiotic mice and chicken resulted in the inhibition of a further intestinal colonization by S. typhimurium in both hosts. Sixteen strains were isolated among the predominant populations of the fecal flora from chicken flora recipient mice. Association of 14 strains of strictly anaerobic bacteria with 2 strains of Escherichia coli and Streptococcus faecium only decreased the number of S. typhimurium in the ileum of gnotobiotic mice, but not in their cecum. Anaerobe cultures were obtained from 10(-6) and 10(-8) dilutions prepared from the fecal flora of gnotobiotic recipient mice. Antagonistic bacteria were present only in cultures from the 10(-6) dilution. Cecal concentrations of volatile fatty acids were shown not to be the sole factor implicated in the antagonistic effect against S. typhimurium.

[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.

[Antagonistic effect against "Clostridium perfringens" exerted in the digestive tract of gnotobiotic mice by "Clostridium" strains isolated from the microflora of conventional mice (author's transl)]. / Effet antagoniste à l'égard de Clostridium perfringens exercé par des souches de Clostridium isolées de la microflore de souris holoxéniques dans le tube digestif de souris gonotoxéniques.

Bacterial strains isolated from the digestive tract of conventional mice produced a barrier effect against a strain of Clostridium perfringens type A (strain CP) in the gastrointestinal tract of gnotobiotic mice. This barrier effect was observed when mice, monoassociated with Escherichia coli K12, were inoculated with a mixed culture of fusiform-shaped clostridia. The collection of fusiforms (collection D) was obtained from a single colony chosen from among the 26 types studied. Collection D and E. coli K12 together exerted a barrier effect against all strains of C. perfringens type A tested. No effect was observed against a C. perfringens type C strain. The expression of the barrier effect depended on the order in which the strains were used to inoculate the mice. Thus strain CP was eliminated from the digestive tract when the mice had previously been associated with collection D and E. coli K12. If the mice were inoculated with strain CP first, however, the barrier effect was only partial. We have also been able to use two strains of clostridia (C1, C3) instead of E. coli K12 to produce a drastic barrier effect against strain CP. The order in which the strains were used to inoculate the mice did not, in this case, influence the production of a barrier effect. Three strains (Da, Db, Dc) isolated from collection D, produced only a partial barrier effect against strain CP in mice previously associated with E. coli K12. These results illustrate the difficulties encountered in determining the minimal number of bacterial strains involved in the production of a barrier effect.

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.

[Elimination from the digestive tract of a "gnotoxenic" child of a "Lactobacillus casei" strain, isolated from a commercial preparation: antagonistic effect of an "Escherichia coli" strain of human origin, demonstrated in "gnotoxenic" mice (author's transl)]. / Elimination du tube digestif d'un enfant "gnotoxénique" d'une souche de Lactobacillus casei, issue d'une préparation commerciale: démonstration chez des souris "gnotoxéniques" du rôle antagoniste d'une souche de Escherichia coli d'origine humaine

A strain of Lactobacillus casei from a pharmaceutical preparation became established in the digestive tract of an axenic child. It was eliminated by the first bacteria occurring after inoculation of the child's digestive tract with a human flora, before the child was taken out of the isolator. Using "gnotoxenic" mice, we found that the elimination of L. casei was due to a bactericidal effect of the Escherichia coli of human origin, within the digestive tract.