Persistence of Coxsackievirus B4 in pancreatic ductal-like cells results in cellular and viral changes.

INTRODUCTION: Although known as cytolytic viruses, group B coxackieviruses (CVB) are able to establish a persistent infection in vitro and in vivo. Viral persistence has been reported as a key mechanism in the pathogenesis of CVB-associated chronic diseases such as type 1 diabetes (T1D). The impact of CVB4 persistence on human pancreas ductal-like cells was investigated. METHODS: A persistent CVB4 infection was established in ductal-like cells. PDX-1 expression, resistance to CVB4-induced lysis and CAR expression were evaluated. The profile of cellular microRNAs (miRNAs) was investigated through miRNA-sequencing. Viral phenotypic changes were examined, and genomic modifications were assessed by sequencing of the viral genome. RESULTS: The CVB4 persistence in ductal-like cells was productive, with continuous release of infectious particles. Persistently infected cells displayed a resistance to CVB4-induced lysis upon superinfection and expression of PDX-1 and CAR was decreased. These changes were maintained even after virus clearance. The patterns of cellular miRNA expression in mock-infected and in CVB4-persistently infected ductal-like cells were clearly different. The persistent infection-derived virus (PIDV) was still able to induce cytopathic effect but its plaques were smaller than the parental virus. Several mutations appeared in various PIDV genome regions, but amino acid substitutions did not affect the predicted site of interaction with CAR. CONCLUSION: Cellular and viral changes occur during persistent infection of human pancreas ductal-like cells with CVB4. The persistence of cellular changes even after virus clearance supports the hypothesis of a long-lasting impact of persistent CVB infection on the cells.

Human milk can neutralize Coxsackievirus B4 in vitro.

The role of enteroviruses in type 1 diabetes has long been suspected. A lower risk of type 1 diabetes is associated with breastfeeding, which could be due to a protective effect against enteroviruses. The neutralizing activity of breast milk against CVB4, a representative of enteroviruses was investigated in this study in vitro. Breast milk was cytotoxic to Hep-2 cells up to a dilution of 1/32, whereas the aqueous fraction obtained after centrifugation was not cytotoxic; although it inhibited the cytopathic effect of CVB4 on Hep-2 cell monolayers. The anti-CVB4 neutralizing activity of aqueous fractions of breast milk from 49 donors living in Northern France and 15 donors living in Congo, where enteroviral infections are more prevalent, were determined. The levels of colostrum activity expressed as titre ranged from <2 to 32 in 36% of the donors from France whereas they were >128 in every donor from Congo. Pasteurized colostrum had a lower anti-CVB4 activity compared to fresh samples (P < 0.0001, n = 49). The treatment of colostrum samples with jacalin-coated beads that bind specifically to human IgA, showed that IgA plays a role in anti-CVB4 activity. There was no correlation between the neutralizing activities of breast milk and serum (P = 0.37, n = 25). The current study showed that the variations in anti-CVB4 activity in breast milk can be attributed to environmental and living conditions. Whether a low protective activity of breast milk against enteroviruses expose newborns to a higher risk of type 1 diabetes deserves further investigation.

Modulation of bacterial translocation in mice mediated through lactose and human milk oligosaccharides.

Massive resection of the small intestine in infants is imposed to the regulation of several intestinal pathological situations, as intestinal adaptation cannot be relied upon. Many nutritional disturbances are occurring following surgery procedure. In this vein, long-term parenteral feeding is adopt to improve prognosis not always successfully. Clostridia and more specifically Clostridium perfringens, are suspected to participate in the physiopathology of the rising situation. In order to investigate the effect of lactose and human milk neutral oligosaccharides (HMNOs) on Clostridia, germfree mice were inoculated either with enterotoxigenic C.perfringens strain isolated from a patient with NEC, or with a human microbiota harboring C.clostridioforme group(HF). In this vein, different doses of lactose were administrated during 2 weeks in adult mice on an attempt to evaluate the lactase activity. Intake of lactose (70 g/L) and HMNOs (7 g/L) in C.perfringens monoassociated mice induced mortality within a week. In HF mice, no mortality was observed. An increase in Clostridia occurrence was observed in the median ileum after intake of 7 g lactose (p = 0.017). Higher clostridial numbers occurred in caecum following intake of 70 g lactose (p < 0.05) and HMNOs (p < 0.025). Bifidobacteria were found increased from distal ileum to colon following 70 g of lactose intake, whereas they decreased in the caecum of mice drinking lower lactose concentrations. Finally, bacteremia was more frequent in 70 g lactose/L mice (p < 0.02), whereas at lower doses of lactose bifidobacterial translocation was observed. As a result, human milk oligosaccharides could favor clostridial population when reaching the lower intestine. The shortness of the small intestine in infants underwent massive intestinal resection seems to be associated to an incomplete breakdown of lactose. Enteral feeds formulas deprived in lactose would be more suitable in enteral feeding of infants.

Selective decontamination by antimicrobials during long term treatment: perspectives for saving host indigenous microbiota.

Short bowel syndrome (SBS) occurs in patients fed total parenteral nutrition (TPN) after massive intestinal resection. TPN weaning is often associated with occlusion or sepsis. In the present study the intestinal biotope was investigated in young patients (n = 14) with massive intestinal resection and recurrent symptoms of sepsis or occlusion during enteral food introduction. They were treated by aminosides for a long term period. Ileal effluents were collected for enumerating bacteria. In some case, blood and rectal specimen were also collected. A few patients developed bacterial overgrowth (1), occlusion (1), sepsis (4), osteoarthritis (1) or pneumonia (1) during the survey. A drastic drop of bifidobacteria that was not prevented by human milk feeding was observed prior occlusion or respiratory infection. Detection of clostridial vegetative forms preceded sepsis and decrease in clostridia parallelled recovery. In conclusion, onset of symptoms was related with extreme imbalance of the ileal flora. Supplementation with bifidobacterial compounds that were well tolerated in two patients could be of interest in children with recurrent symptoms.

Coxsackievirus B4 and type 1 diabetes pathogenesis: contribution of animal models.

The role of enteroviruses, in particular type B coxsackieviruses (CV-B), in type 1 diabetes (T1D) pathogenesis is supported by epidemiological, clinical and experimental observations.The investigation of T1D pathogenesis benefits from the contribution of animal models called spontaneously diabetic. Among these animals the non-obese diabetic (NOD) mouse and the bio-breeding diabetes-prone (BBDP) rat present a genetic susceptibility manifested by the expression of an autoimmune diabetes similar to the pathology observed in human beings. Other models whose genetic predisposition is less known are of considerable contribution as well. Numerous major observations relative to several aspects of T1D pathogenesis in the context of CV-B infections, such as susceptibility, diabetogenicity, pancreatotropism, mechanisms of beta cells destruction and others, have been deduced thanks to investigations with animal models. Despite their limits, these models are necessary in improving our knowledge of the role of enteroviruses, like CV-B4, in the pathogenesis of T1D, and the recent advances ensuing from their contribution may have important therapeutic and preventive spin-offs.

[D-lactic acidosis in a child with short bowel syndrome]. / Acidose d-lactique chez un enfant présentant un syndrome de grêle court.

INTRODUCTION: d-lactic acidosis is a rare and severe complication of short bowel syndrome in children that may result from important ileal bacterial overgrowth by lactobacilli. Intestinal flora (Lactobacilli) is responsible for the production of d-lactic acid after fermentation of food carbohydrates. OBSERVATION: We report on the case of a 6-year-old child with a short bowel syndrome treated with both home enteral and parenteral nutrition. The patient suddenly presented with acute neurological symptoms including dysarthria and disorientation. Biological analysis revealed metabolic acidosis, increased plasma d-lactic acid assessed by organic acid chromatography analysis and a very important increase in expired hydrogen during glucose breath test. Lactobacillus fermentum (known to produce d and L isomers of lactic acid) was isolated in the gastric liquid and rectal swabs. Clinical and biological evolution was rapidly favourable after treatment with intravenous sodium bicarbonate, antibiotic therapy and interruption of enteral nutrition. CONCLUSION: d-lactic acidosis should be suspected when neurological symptoms occur in a child with short bowel syndrome. They can be prevented by treating intestinal bacterial overgrowth.

Establishment and follow-up of bifidobacterial species in the gut of healthy bottle-fed infants of 1-4 months age.

Twenty-one healthy bottle-fed infants were screened monthly (1-4 months) for bifidobacteria in their stools. Bifidobacteria were detected by culture and isolates specified by PCR. Alternatively, direct PCR in undiluted fecal suspensions was carried out for detection of bifidobacteria under the cultural detection level. All infants harbored cultivable bifidobacteria throughout the study period. Beerens medium was shown to permit a better recovery of bifidobacteria than MRS and horse blood Columbia agar. Direct PCR detection proved valuable in detecting species for which no cultural isolate could be recovered since the species were under the cultural detection level. B. bifidum, B. longum-infantis and B. breve were confirmed as dominant and stable species in infant stools while B. adolescentis and B. catenulatum group exhibited unstable colonization profiles. A trend towards B. breve decrease began at month 3 while carriage of the B. catenulatum group and B. adolescentis was rising. This observation warrants further analysis to assess a possible switch occurring at month 3 in bottle-fed infants, between so-called infant and adult bifidobacterial species.

[Intestinal bacterial colonisation and translocation in C3H/HeJ conventional mice fed a unique dose of live or dead Bifidobacterium breve C50]. / Colonisation intestinale et translocation bactérienne chez la souris conventionnelle C3H/HeJ apres ingestion d'une dose unique de Bifidobacterium breve C50 vivantes ou mortes.

The efficiency and safety of use of Bifidobacterium breve C50 (BbC50), a potential probiotic, was assessed as regards intestinal microbial colonisation and bacterial translocation. A suspension of BbC50, containing 1-5 to 107-108 live bacteria, was fed to C3H/HeJ mice. The passage of live BbC50 was not demonstrated by culture either in the intestine or extra-intestinal organs. However, mice receiving the highest dose of live bacteria harbored more lactobacilli and less Bacteroides fragilis group in the cecum and colon when compared to control mice. Translocation of lactobacilli observed in the control group was not regulated by Bb50 feeding. Indeed, the spleen was significantly more frequently contaminated in mice fed BbC50, whatever the dose of live bacteria. The kidneys were also significantly more contaminated with lactobacilli in mice fed the highest dose of live Bb50. Moreover, higher dose of live BbC50 was associated with greater number of extra-intestinal contaminated organs. To conclude, BbC50 feeding induced a favorable balance in the mouse intestinal flora and was never found translocating, demonstrating its efficiency and safety of use. However, BbC50 seemed to interfere with the ability of lymphoid organs (e.g. the spleen) to eliminate translocating lactobacilli.

Partial characterization of bifidobacterium breve C50 cell-free whey compounds inducing modifications to the intestinal microflora.

Cell-free whey obtained from milk fermented with Bifidobacterium breve C50 (Bb C50) has been shown to modify the intestinal flora in humans and mice. Previous work showed that no antibiotic-like or barrier effect due to overgrowing bifidobacteria was implied in the microbial modifications. The present study was focused on characterizing the compounds and mechanisms involved. Protein, sugar, and enzymatic profiles of Bb C50 whey were therefore determined and compared to those of a whey unable to modify the intestinal flora of humans and mice. No remarkable difference was noted except for a higher lactosidase activity in Bb C50 whey. Various physical treatments were then applied to fractions of Bb C50 whey. Activity was assessed in C3H mice by analyzing changes in the intestinal flora balance throughout a 15-d administration of each treated whey. Heating at 80 degrees C and aerobic storage for 2 wk completely abolished Bb C50 whey activities. In contrast, the addition of a reducing agent (cysteine hydrochloride), either at the beginning of a 15-d aerobic storage or prior to administration, as well as preserved these activities. Susceptibility to heating and oxidation suggested that an enzyme might play a role in the induced microbial changes. Since the Bb C50 lactosidase was partly inactivated by the oxidative treatment, it could support the in vivo activity. The enzyme might reach the intestinal lumen and partly degrade substrates, such as mucins, usually used by the intestinal flora. The released molecules might then favor the development of a new microbial balance.

The effects of mesenteric ischemia on ileal colonization, intestinal integrity, and bacterial translocation in newborn piglets.

The effects of mesenteric ischemia on ileal colonization, intestinal integrity, and bacterial translocation (BT) in newborn piglets were investigated in 362-day-old Pietrain piglets. Group I, controls were not operated upon; group II underwent a sham laparotomy; and group III underwent ligation of the mesenteric vessels in the distal ileum. After 3 days, the kidneys, spleens, livers, and ileal segments were harvested for microbial and histologic analyses. Two piglets in the ischemic group died; microscopic examination showed severe histologic lesions of the ischemic area. Escherichia coli counts were increased in the ischemic segment compared to the upper loop (P < 0.05). Ischemia favoured staphylococcal colonization, whereas in the sham group a drastic reduction of these organisms was observed (P < 0.005). BT to the kidneys, spleen, and liver occurred normally in the control group. Ischemia significantly increased the total microflora in the spleen and liver (P < 0.05) and furthered dissemination of Clostridium perfringens in the kidneys (P < 0.05); 50% of ischemic animals had proteolytic clostridia in this organ (P < 0.05). Moreover, the incidence of E. coli in the kidneys, spleen, and liver was significantly higher in the sham and ischemic groups than in the controls (P < 0.05). Ileal ischemia thus induced significant histologic lesions, and surgery rather than gut microflora controls translocation.

The regulatory effects of whey retentate from bifidobacteria fermented milk on the microbiota of the Simulator of the Human Intestinal Microbial Ecosystem (SHIME).

AIMS: To investigate the effects of whey retentate from Bifidobacteria fermented milk. METHODS AND RESULTS: The simulator of the human intestinal microbial ecosystem (SHIME) was used. The composition of the microbiota and its metabolic activities were analysed. Changes in the microbial composition became apparent within 15 days of the treatment in the vessels representing the ileum and the large intestine. The whey retentate favoured the growth of endogenous bifidobacteria and induced a decrease in Bacteroides fragilis and in sulphite-reducing clostridia, especially Clostridium perfringens. After the administration was stopped, these populations tended to revert to their original levels, except for the streptococci and the staphylococci populations. The treatment also led to an increase in acetic acid, CH4 and CO2 production, suggesting overgrowth of some anaerobic bacteria. Ammonium, generally considered as undesirable, declined. CONCLUSIONS: The whey retentate clearly altered the microbial community in the SHIME. SIGNIFICANCE AND IMPACT OF THE STUDY: Whey retentate appears to exert a beneficial effect on the in vitro gastrointestinal system; these findings warrant confirmation by in vivo studies.

Isolation and characterization of a porin-like protein of 45 kilodaltons from Bacteroides fragilis.

Resistance to the combination of amoxicillin and clavulanic acid in some Bacteroides fragilis strains may be associated with a lack of porin proteins. Comparison of outer membrane protein profiles from one resistant strain (B. fragilis CFPL 358) and two susceptible strains of B. fragilis (ATCC 25285 and CFPL 92125) showed that a few proteins were missing in the resistant strain, especially a 45-kDa protein. To determine whether this protein was a porin-like protein, we attempted to isolate it from the two susceptible strains by using gel filtration (Sephacryl S-200, Superose 6) and ion exchange chromatographies (DEAE Trisacryl, DEAE Sepharose Fast Flow). Elution from DEAE resins was poor compared to the 60-67-kDa region, which suggested that the 45-kDa protein exhibited stronger cationic forms. The use of sodium dodecyl sulfate during elution improved the recovery of the 45-kDa protein, showing that detergent modified its conformation and its ionic bounds with the chromatographic matrices but it was not sufficient for good purification. Superose 6 gel filtration also failed to separate this protein from the 60-67-kDa region. The only method resulting in the positive recovery of a purified 45-kDa band from both susceptible B. fragilis strains was electroelution from SDS-PAGE. The swelling assay showed that the 45-kDa protein was a porin-like protein. From this study, we concluded that the 45-kDa protein from B. fragilis was a porin-like protein which might be involved in the antibiotic resistance of a strain in which this protein was missing.

Cell-free whey from milk fermented with Bifidobacterium breve C50 used to modify the colonic microflora of healthy subjects.

The ingestion of viable bacteria is thought to be required to modify intestinal microflora. In the present study, the effects on fecal flora of consumption of cell-free concentrated whey from milk that had been fermented with Bifidobacterium breve C50 was tested using 10 healthy human volunteers. Results were compared with effects of a commercial milk formula that had been fermented with Streptococcus thermophilus and B. breve C50 and given to 10 control subjects. Nitroreductase and beta-glucuronidase activities were assessed as risk indexes for colon carcinogenesis, and beta-galactosidase was measured as an indicator of the fermentation capacity of the colonic flora. Fecal excretion of Bacteroides fragilis, Clostridium perfringens, and clostridial spores decreased after 7 d of consumption of either preparation; however, counts of bifidobacteria only increased after intake of B. breve whey. Fecal pH was reduced from 7.1 +/- 0.2 to 6.6 +/- 0.3 after intake of whey that had been fermented with Bif. breve. Fecal nitroreductase and beta-glucuronidase significantly decreased, and beta-galactosidase activity increased, after consumption of either preparation. The results indicate that ingestion of viable bifidobacteria was not required to modify intestinal flora of humans. Repression of B. fragilis and clostridia seems to be independent of colonic bifidobacterial overgrowth in humans.

Cell-free wheys from bifidobacteria fermented milks exert a regulatory effect on the intestinal microflora of mice and humans.

Bacteroides fragilis and clostridia are normally present in the human colon but they may exert pathogenic effects when the homeostasis is upset following various forms of stress. One approach to preventing gastrointestinal disorders is to use bifidobacteria fermented milk. It has been suggested that the efficacy of such a product is related to abiotic compounds produced during milk fermentation. Experiments reported in this paper attempt to check this theory. Six whey retentates were prepared by fermenting cow's milk with six human strains of Bifidobacterium breve and acetic and lactic acids were eliminated by ultrafiltration. Their ability to reduce intestinal clostridial carriage was assessed in C3H mice. Only one whey retentate led to a decrease in clostridia, bacilli, B. fragilis and fecal pH and to an increase in bifidobacteria. Assays in ten human volunteers resulted in similar changes in fecal flora and fecal pH within 7 days of whey retentate intake (30 mL/day). No antibiotic-like effect was demonstrated in vitro. Compounds involved in microflora regulation were located in two ultrafiltrated fractions (30-100 and 100-300 kDa). Both fractions contained mainly low molecular weight glycoproteins (20-40 kDa) and two high molecular weight glycoproteins (121, 211 kDa) that were almost undetectable in the inactive 10-30 kDa fraction.

Bifidobacteria and human health: regulatory effect of indigenous bifidobacteria on Escherichia coli intestinal colonization.

Bifidobacteria are assumed to exert colonization resistance to enteric pathogens. We associated C3H germfree mice with either Bifidobacterium longum or Escherichia coli or both strains and studied how they settled in the gut and the lymphoid organs as well as their effect on mucus composition. Within 24 hE. coli colonized the gut of germfree or B. longum ex-germfree mice. In contrast,B. longum was established in the intestine of E. coli ex-germfree mice only 1 month after inoculation whereas it colonized the germfree gut within 24 h. Although B. longum did not exert colonization resistance to E. coli, the establishing of bifidobacteria in the gut partly prevented changes in the E. coli cell wall. After colonization of the germfree or B. longum mono-associated mice, E. coli lipopolysaccharide exhibited a higher concentration of Kdo and the O-antigen side chain disappeared. A reduction in Kdo content was observed within 1 month in E. coli-B. longum diassociated mice whereas it remained at a high level in E. coli mono-associated mice. Association in a second step with B. longum led to Kdo reduction. Changes in E. coli LPS might be related to mucus modification. Inoculation of either bacterium led to a slow increase in mucus protein content which was however twice as high after E. coli implantation. Inoculation of B. longum in a second step led to a reduction in protein content before B. longum colonized the intestine at a high level suggesting that the protein concentration in the mucus was controlled by the host itself. A new glycoprotein of 200-230 kDa detected during the period preceeding colonization seemed to be broken down by B. longum. The resulting end product might participate in the restoration of E. coli LPS. Finally,B. longum inoculation led to the disappearance of E. coli from kidneys, liver, spleen and lung. The organs were cleared of E. coli before B. longum highly colonized the intestine suggesting that high intestinal colonization by B. longum was not required. Regulation of E. coli invasion seemed to depend on the ability of B. longum to stimulate the immune system.

Bacterial translocation, intestinal morphology, and enzyme activities after ileal ischemia in newborn piglets.

Intestinal ischemia was created after a limited laparotomy by ligation of the terminal mesenteric vessels in the last 10 cm of distal ileum in 2-day-old piglets. Five groups (each n = 15) were studied: 1 (unoperated control group, killed on day 4), 2 (sham control with laparotomy, killed on day 4), 3 (ischemia, killed on day 4), 4 (ischemia, killed on day 9), and 5 (unoperated control on day 9, not killed). All animals in groups 1, 2 and 5 survived. Two animals in group 3 and 1 in group 4 died (peritonitis and distal ileal perforation). In animals killed on day 9, less weight gain was observed in group 4 compared to the unoperated controls. Macroscopically, no alteration was found at laparotomy in the animals in group 1, whereas in group 2, 1 animal showed beginning peritonitis and another some degree of peritoneal adhesions in group 3, 1 piglet had an intestinal perforation and 4 had intestinal distention above the ischemic loop. In group 4, 7 animals had dilatation of the upper loops, 4 a complete stricture, and 3 peritonitis with complete necrosis of the distal ileum. Microscopic examination revealed severe lesions of the ischemic area in groups 3 and 4 and mild lesions of the upper loop. The kidney was contaminated by translocation of gram-positive cocci in 36% of cases in group 2. Germ carriage for staphylococci was estimated at 80% in the terminal ileum of animals in group 3 versus 8.3% in group 2. In groups 3 and 4, the translocation rate was 30% in the kidney and 40% in the liver. Low disaccharidase activities were found in ischemic areas in groups 3 and 4, with no difference in activity in the upper loops.

Regulation of the bacterial intestinal implantation in infant born by caesarean section.

Studies were undertaken to determine the regulation of the bacterial intestinal implantation in 19 newborns delivered by caesarean section. Correlation was made with the infant feeding mode. The effect of human milk seemed to be the result of B. bifidum proliferation, in contrast to artificial alimentation that seemed to favour C. perfringens implantation. The question was raised by us as to whether this opposition was only related to alimentation. In fact, B. bifidum itself also had an effect as demonstrated by the lower mean counts of C. perfringens in bottle-fed infants carrying the bifido-bacterial flora (P = 0.05). None of the other faecal bacteria investigated in this study led to the same decrease.

[Host-Bifidobacterium interactions in the axenic mouse: partial characterization of bifidogenic factors in the intestinal contents]. / Etude des interactions hôte--Bifidobacterium chez la souris axénique: caractérisation partielle des facteurs bifidigènes du contenu intestinal.

Growth factors for Bifidobacterium bifidum were detected in faeces of axenic mice strain C3H. Most of these factors were found in the nondialyzable fraction obtained after aqueous extraction and dialysis. SDS-PAGE and filtration chromatography on Sepharose 4B revealed that many glycosylated components harbored a bifidigenic activity. Intestinal colinization of mice by B. bifidum involved the utilization and eventually the disappearance of the intestinal bifidigenic factors. There was no change in the protein concentration in fecal extracts, but the total hexose concentration was lower. Comparison of electrophoretic PAGE profiles after periodic acid Schiff coloration showed that bacteria used up the glycosylated fractions of many glycopeptides, particularly those of mucins and four glycoproteins. There was no correlation between the hexose concentration detected in every active fraction and the degree of in vivo degradation of bifidigenic factors. The attack on active glycopeptides having a molecular mass greater than 670 kDa thus revealed hexose sites that were not detectable previously by the phenol - sulfuric acid method. However, the amount of bifidigenic factors detected in vitro allowed us to measure the importance of the degradation of a component by B. bifidum in vivo.

Modulation of Clostridium perfringens intestinal colonization in infants delivered by caesarean section.

The colonization by Clostridium perfringens was investigated in 19 infants delivered by caesarean section during the two first weeks of life. The pattern of C. perfringens colonization depended upon the feeding. Breast feeding led to the repression of C. perfringens, whereas bottle feeding allowed its maintenance. On the contrary, Bifidobacterium bifidum growth was favoured by breast feeding. However, in one breast-fed infant, B. bifidum was never isolated and C. perfringens decreased. Breast feeding was able to directly modulate C. perfringens numbers. In fact, B. bifidum also had an effect, as demonstrated by the lower mean counts of C. perfringens, in bottle-fed infants carrying the bifidobacteria flora (p = 0.05). None of the bifidobacteria investigated in this study led to the same decrease.