The developing gut microbiota and its consequences for health.

The developmental origin of health and disease highlights the importance of the period of the first 1000 days (from the conception to the 2 years of life). The process of the gut microbiota establishment is included in this time window. Various perinatal determinants, such as cesarean section delivery, type of feeding, antibiotics treatment, gestational age or environment, can affect the pattern of bacterial colonization and result in dysbiosis. The alteration of the early bacterial gut pattern can persist over several months and may have long-lasting functional effects with an impact on disease risk later in life. As for example, early gut dysbiosis has been involved in allergic diseases and obesity occurrence. Besides, while it was thought that the fetus developed under sterile conditions, recent data suggested the presence of a microbiota in utero, particularly in the placenta. Even if the origin of this microbiota and its eventual transfer to the infant are nowadays unknown, this placental microbiota could trigger immune responses in the fetus and would program the infant's immune development during fetal life, earlier than previously considered. Moreover, several studies demonstrated a link between the composition of placental microbiota and some pathological conditions of the pregnancy. All these data show the evidence of relationships between the neonatal gut establishment and future health outcomes. Hence, the use of pre- and/or probiotics to prevent or repair any early dysbiosis is increasingly attractive to avoid long-term health consequences.

Comparative phenotypic analysis of "Clostridium neonatale" and Clostridium butyricum isolates from neonates.

"Clostridium neonatale" was recently described as a new species within the Cluster I of the Clostridium genus sensu stricto. In this study, we characterized "C. neonatale" isolates (n = 42) and compared their phenotypic properties with those of Clostridium butyricum (n = 26), a close related species. Strains isolated from fecal samples of healthy neonates were tested for different phenotypic characteristics. Compared to C. butyricum, "C. neonatale" showed a significant higher surface hydrophobicity (p = 0.0047), exopolysaccharide production (p = 0.0069), aero-tolerance (p = 0.0222) and viability at 30 °C (p = 0.0006). A lower swimming ability (p = 0.0146) and tolerance against bile (0.3%) (p = 0.0494), acid (pH 4.5) (p < 0.0001), osmolarity (NaCl 5%, p = 0.0188) and temperature at 50 °C (p = 0.0013) characterized "C. neonatale" strains. Our results showed that "C. neonatale" behaves very differently from C. butyricum and suggests specific responses to environmental changes. Besides it is the first study on clinical isolates for these two anaerobic members of the newborns' gut microbiota and broadens our knowledge about their phenotypic traits.

[Fecal microbiota transplantation: review]. / Le transfert de flore digestive : une revue de la littérature.

Fecal microbiota transplantation (FMT) has gained an increasing medical interest, since the recognition of the role of disturbed microbiota in the development of various diseases. To date, FMT is an established treatment modality for multiple recurrent Clostridium difficile infection (RCDI), despite lack of standardization of the procedure. Persisting normalization of the disturbed colonic microbiota associated with RCDI seems to be responsible for the therapeutic effect of FMT. For other diseases, FMT should be considered strictly experimental, only offered to patients in an investigational clinical setting. Although the concept of FMT is appealing, current expectations should be damped until future evidence arises.

[Why could gut microbiota become a medication?]. / Pourquoi la flore intestinale a-t-elle vocation à devenir médicament ?

The gut microbiota (or gut flora) is a set of bacteria living in symbiosis with the host. Strictly associated with the intestinal tract and interacting with it, the gut microbiota is not a tissue nor an organ, but a supra-organism. A disruption of dialogue between bacteria and human cells is a risk factor or a possible cause of various diseases. The restoration of this dialogue, thanks to the transfer of the gut microbiota of a healthy individual to a patient whose balance of gut flora has been broken, is a new therapeutic approach. If its exact effect still eludes scientific understanding, its clinical benefit is well established for an indication, and is recently being tested for many others. The proven contribution of gut microbiota in the human physiological balance calls for intensifying research throughout the world about the state of knowledge and technologies, as well as on the legal and ethical dimension of fecal microbiota transfer. This didactic paper updates the questions in relation with this therapeutic act.

Probiotics, gut microbiota and health.

The human gut is a huge complex ecosystem where microbiota, nutrients, and host cells interact extensively, a process crucial for the gut homeostasis and host development with a real partnership. The various bacterial communities that make up the gut microbiota have many functions including metabolic, barrier effect, and trophic functions. Hence, any dysbiosis could have negative consequences in terms of health and many diseases have been associated to impairment of the gut microbiota. These close relationships between gut microbiota, health, and disease, have led to great interest in using probiotics (i.e. live micro-organisms), or prebiotics (i.e. non-digestible substrates) to positively modulate the gut microbiota to prevent or treat some diseases. This review focuses on probiotics, their mechanisms of action, safety, and major health benefits. Health benefits remain to be proven in some indications, and further studies on the best strain(s), dose, and algorithm of administration to be used are needed. Nevertheless, probiotic administration seems to have a great potential in terms of health that justifies more research.

Longitudinal analysis and genotyping of infant dominant bifidobacterial populations.

Bifidobacterial population dynamics were investigated by the longitudinal analysis of the dominant population isolated from the feces of young infants. After molecular identification and fingerprinting comparison, clone identity of the consecutive strains belonging to the same species for one individual was performed by pulsed-field gel electrophoresis. The results, obtained from 15 individuals sampled four times over a five-week period suggested a turnover of the dominant bifidobacteria in the population not only at the species but also at its species representative levels. This study provides new insights of the in vivo dynamics of commensal bifidobacteria. It highlights the need to take into consideration the fluctuation of bifidobacterial populations that may occur in one individual in order to investigate reliably the impact of dietary components, such as probiotics or prebiotics, on the intestinal ecosystem.

Diversity of gut Bifidobacterium species is not altered between allergic and non-allergic French infants.

Some clinical studies have suggested a relationship between allergic diseases and gut microbiota. We aimed to study bifidobacterial colonization at species and strain levels in ten allergic French infants included at their first clinical consultation and 20 controls matching for age at sampling, mode of delivery, per partum antibiotics, type of feeding and antibiotics in the first weeks of life. The faecal microbiota was analyzed by culture methods and TTGE. Bifidobacterial species and strains were identified using multiplex PCR and Box-PCR fingerprinting. No differences were observed between groups in the number of colonized infants or in the levels of colonization by the main aerobic and anaerobic genera. All infants were colonized with high levels of Bifidobacterium except for one in each group. One to 5 Bifidobacterium species and 1 to 7 strains were observed per subject independently of allergic status and age at sampling. Our study showed the infants to be colonized by several species and strains, including several strains from the same species. This diversity in Bifidobacterium colonization was not related with the allergic status and showed that the link between Bifidobacterium colonization and allergic diseases is complex and cannot be restricted to the role attributed to Bifidobacterium species.

Fecal expression of human ß-defensin-2 following birth.

BACKGROUND: Newborns display high intestinal permeability and a naive adaptive immune system, but infections are rare, indicating strong innate defense mechanisms. OBJECTIVE: To measure the kinetics of fecal ß-defensin-2 (HBD2), an inducible endogenous antimicrobial peptide produced by intestinal epithelial cells, in full-term and preterm infants. METHODS: As a first step of this bicentric study, we enrolled 30 healthy full-term infants and 20 healthy preterm infants, with fecal samples collected at days 3, 7, 12 and 30 in full-term infants and at days 15, 30 and 60 in preterm infants. As a second step, we enrolled 10 preterm infants with intestinal distress, either necrotizing enterocolitis (NEC) Bell's stage III (n = 3) or isolated rectal bleeding (n = 7) and 20 controls, cross-matched for gestational age and age at sampling. RESULTS: HBD2 decreased significantly from day 3 to day 7 (227 ng/g; 14-440 vs. 117 ng/g; 30-470, p = 0.01) then moderately until day 30 (84 ng/g; 10-500) in healthy full-term infants. Healthy preterm infants showed similar high levels between days 15 and 60 (82 ng/g; 30-154 and 85 ng/g; 26-390, respectively). No significant variation of fecal HBD2 levels was observed between infants with clinical features of intestinal distress (77 ng/g, 2-1,271) and cross-matched controls (56 ng/g, 31-164). However, 2/3 infants with NEC and 1/7 infants with isolated rectal bleeding had HBD2 levels above the maximal level observed in controls. CONCLUSIONS: The kinetics of fecal HBD2 in the neonatal period indicate that this inducible defensin can be detected at high level in the feces of full-term and preterm infants, independently of gestational age or mode of feeding. The potential role of fecal HBD2 in detecting NEC is suggested.

Short-chain fatty acids and polyamines in the pathogenesis of necrotizing enterocolitis: Kinetics aspects in gnotobiotic quails.

Despite years of investigation, pathogenesis of necrotizing enterocolitis (NEC) remains elusive. Bacterial metabolites were implicated by several authors but their roles remain controversial. The aim of our study was to investigate the role of SCFAs and polyamines through a kinetic study of histological and macroscopical digestive lesions in monobiotic quails. Germ-free quails, inoculated with a Clostridium butyricum strain involved in a NEC case, were fed or not with a diet including lactose (7%). Quails were sacrificed at various times between D7 and D24 after bacterial inoculation. NEC-like lesions, i.e. thickening, pneumatosis, and hemorrhages, occurred only in lactose-fed quails and increased with time. The main histological characteristics were infiltrates of mononuclear cells, then heterophilic cells, then gas cyst and necrosis. The first event observed, before histological and macroscopical lesions, is a high production of butyric acid, which precedes an increase of iNOS gene expression. No difference in polyamines contents depending on the diet was observed. These results show the major role of butyric acid produced by commensal bacteria in the onset of the digestive lesions.

Tetracycline resistance mediated by tet(W), tet(M), and tet(O) genes of Bifidobacterium isolates from humans.

MICs of tetracyclines were determined for 86 human Bifidobacterium isolates and three environmental strains. The tet(O) gene was found to be absent in these isolates. tet(W) and tet(M) were found in 26 and 7%, respectively, of the Bifidobacterium isolates, and one isolate contained both genes. Chromosomal DNA hybridization showed that there was one chromosomal copy of tet(W) and/or tet(M).

Inhibitory impact of bifidobacteria on the transfer of beta-lactam resistance among Enterobacteriaceae in the gnotobiotic mouse digestive tract.

While looking for new means to limit the dissemination of antibiotic resistance, we evaluated the role of potentially probiotic bifidobacteria on the transfer of resistance genes between enterobacteria. Transfers of bla genes encoding extended-spectrum beta-lactamases (SHV-5 and CTX-M-15) were studied in the absence or presence of bifidobacteria. In vitro, transfer frequencies of these bla genes decreased significantly in the presence of three of five tested strains, i.e., Bifidobacterium longum CUETM-89-215, Bifidobacterium bifidum CIP-56.7T, and Bifidobacterium pseudocatenulatum CIP-104168T. Four transfer experiments were conducted in the digestive tract of gnotobiotic mice, the first three observing the effect of B. longum CUETM-89-215, B. bifidum CIP-56.7T, and B. pseudocatenulatum CIP-104168T on blaSHV-5 transfer and the fourth experiment studying the effect of B. bifidum CIP-56.7T on blaCTX-M-15 transfer. These experiments revealed significant decreases in the transconjugant levels (up to 3 logs) in mice having received B. bifidum CIP-56.7T or B. pseudocatenulatum CIP-104168T compared to control mice. Bifidobacteria appear to have an inhibitory impact on the transfer of antibiotic resistance genes. The inhibitory effect is associated to specific bifidobacterial strains and may be related to the production of thermostable metabolites by these strains.

[Linezolid: antibacterial activity, clinical efficacy and resistance]. / Le linézolide: activité antibactérienne, intérêts cliniques et résistance.

Linezolid is a synthetic antibiotic, the first available agent in a new class of antibiotic called the oxazolidinones, whose particular mechanism of action consists in inhibiting the initiation of protein synthesis. Its spectrum of in vitro and in vivo activity includes staphylococci, streptococci, enterococci, corynebacteria and some anaerobic bacteria (Peptostreptococcus, Clostridium, and Fusobacterium). The first therapeutic results were very encouraging, leading to the marketing of the product in France in 2002. Linezolid is indicated in the treatment of pneumonia and the complicated infections of the skin. Pharmacocinetics studies have shown that linezolid has an excellent bioavailability allowing a fast relay per os. However, failures of treatment under linezolid were reported and resistant strains of staphylococci and enterococci were obtained in vitro and in vivo after therapeutic use of this antibiotic. Changes in the domain V of 23S rRNA were found in the site of fixation, the most frequent was (G out of U) in position 2576 (numbering E. coli). In a context where resistance to traditional treatments in enterococci, pneumococci and S. aureus do not cease to increase, linezolid can be regarded as a therapeutic alternative to treat the infections with Gram-positive cocci.

Antimicrobial susceptibility of bifidobacteria.

OBJECTIVES: The aim of our study was to analyse the antibiotic susceptibility of various strains of Bifidobacterium spp. to a wide range of antimicrobial agents. METHODS: Fifty strains belonging to eight species of bifidobacteria, isolated from humans, animals or probiotic products, were tested for susceptibility to 30 antibiotics by disc diffusion on Brucella agar supplemented with 5% laked sheep blood and vitamin K1 (1 mg/L). MICs of nine anti-anaerobe agents, including three new molecules (telithromycin, linezolid and gatifloxacin), were determined using the reference agar-dilution method. RESULTS: All strains of bifidobacteria, whatever the species, were sensitive to penicillins: penicillin G, amoxicillin (MIC(50) 0.06 mg/L), piperacillin, ticarcillin, imipenem and usually anti-Gram-positive antibiotics (macrolides, clindamycin, pristinamycin, vancomycin and teicoplanin). Susceptibility to cefalothin and cefotetan was variable. Most isolates (70%) were resistant to fusidic acid. As expected, high resistance rates were observed for aminoglycosides. Metronidazole, an agent known for its anti-anaerobe activity, was ineffective against 38% of the strains. The newly commercialized molecules, telithromycin, linezolid and gatifloxacin, were active with MIC(50)S of 1 mg/L. The only variation in susceptibility observed among the different species concerned Bifidobacterium breve, which appeared to be generally more resistant. Potentially acquired resistance was only observed against tetracycline and minocycline, in 14% of the strains. CONCLUSIONS: With regard to a general concern about the safety of probiotics, such as potential transferability of resistance determinants, bifidobacteria, with their low natural and acquired resistance to 30 antibiotics, appear risk-free.

High faecal calprotectin concentrations in newborn infants.

BACKGROUND: Calprotectin, a major component of soluble cytosolic proteins in human neutrophil granulocytes, is excreted in excess in stools during inflammatory bowel disease in adults and children. Faecal calprotectin concentrations are also higher during the first year of life than in adults. OBJECTIVES: To measure faecal calprotectin concentrations in the neonatal period and define reference values according to the mode of feeding: standard infant formula, prebiotic infant formula (Calisma, Blédina SA, France), or breast feeding. PATIENTS AND METHODS: A prospective study was carried out over three months in 69 full term, healthy newborns with a median gestational age of 39.8 weeks (range 37-41.5) and a birth weight of 3300 g (range 2600-4460). Three groups were formed depending on the mode of feeding: group 1 (n = 18) received a standard infant formula, group 2 (n = 19) the prebiotic infant formula, and group 3 (n = 32) was breast fed. One stool sample was taken from each newborn on day 4 (3-7), and faecal calprotectin analysed using a commercial enzyme linked immunoassay (Calprest, Eurospital, Italy). RESULTS: Faecal calprotectin concentrations (median 167 micro g/g) were higher than reference values in healthy adults. The concentration was below the upper reference limit for adults (50 micro g/g) for three infants only, one fed the standard formula and two fed the prebiotic formula. Concentrations did not differ significantly according to method of feeding. CONCLUSIONS: Compared with healthy adults, newborns have high calprotectin concentrations in the first days of life. There is no obvious influence of the mode of feeding.

[Risk factors and screening strategy for group B streptococcal colonization in pregnant women: results of a prospective study]. / Facteurs de risque et stratégie de dépistage de la colonisation par le streptocoque du groupe B chez la femme enceinte: résultats d'une étude prospective.

OBJECTIVES: Group B Streptococcus (GBS) continues to be the most important bacterial cause of sepsis, meningitidis in newborns. American consensus guidelines have been published. They recommended the use of either risk-based strategy or screening-based approach for GBS colonization in pregnant women to identify candidates for intrapartum prophylaxis. Screening consists of obtaining vaginal and anorectal specimens for culture at 35 to 37 weeks' gestation. The aim of this prospective study was to assess the usefulness of systematic and concomitant GBS screening by rectal and vaginal swab in pregnant women. A questionnaire designed to determine the risk factors for colonization by GBS was completed. MATERIALS AND METHODS: We have screened 370 pregnant women with rectal and vaginal swab. RESULTS: Fifty seven (15.4%) women had positive GBS cultures. Of those women, the rectum and the vagina were the only site of colonization in 16 (4.3%) and 8 (2.2%) women respectively. None of the factors studied was significantly associated with GBS colonization. CONCLUSION: Detection of GBS is enhanced by 40% by using vaginal and anorectal swabs compared to a vaginal swab alone. No studied factor appeared to predict GBS colonization, which incited us to screen all pregnant women.

Oligofructose and experimental model of neonatal necrotising enterocolitis.

The gut of preterm neonates is colonised with a paucity of bacterial species originating more from the environment than from the mother. Furthermore, a delayed colonisation by bifidobacteria promotes colonisation by potentially pathogenic bacteria. This may contribute towards the development of neonatal necrotising enterocolitis (NEC). The physiopathology of NEC is still unclear but immaturity of the gut, enteral feeding and bacterial colonisation are all thought to be involved. None of the current preventive treatments are considered satisfactory. Modulating the autochthonous microflora by probiotics or prebiotics could be a more reliable approach to prevention. Using gnotobiotic quails as an experimental model of NEC we have shown that onset of intestinal lesions requires a combination of low endogenous lactase activity, lactose in diet, and colonisation by lactose-fermenting bacteria such as the clostridia. The protective role of bifidobacteria was demonstrated in this model through a decrease in clostridial populations and in butyric acid. Oligofructose dietary supplementation was shown to enhance this effect with an increase in the bifidobacterial level and consequently a greater decrease in clostridia. However, oligofructose was unable to promote a bifidobacterial acquisition when the microflora was initially deprived of this group. Nevertheless, oligofructose can act as an anti-infective agent and decrease the occurrence or severity of the lesions depending on the bacteria involved. According to these results and to the fact that oligosaccharides are a major component of breast milk, the addition of oligofructose in formula milks may be a nutritional approach to favouring colonisation by a beneficial flora.

Diet, length of gestation, and fecal short chain fatty acids in healthy premature neonates.

BACKGROUND: Excretion of fecal short-chain fatty acids (SCFAs) may indicate changes in colonic or colonocyte metabolism. The aim of this study was to detect the influence of gestational age and feeding practices on SCFA concentrations and profiles in healthy preterm infants. METHODS: A total of 198 fecal samples (28 infants) were collected from 8 to 21 days of age from 3 groups of preterm infants born at 33 to 37 weeks of gestation and fed either breast milk (group I) or Nutramigen, a lactose-free formula (group II), and extremely preterm infants born before 33 weeks of gestation and fed breast milk (group III). Total SCFA concentrations and SCFA profiles were analyzed using a gas chromographic (GC) procedure. RESULTS: Total fecal SCFA excretion did not differ significantly between group I (mean, 24.0 micromol/g; range, 1.3 to 118.8 micromol/g) and group II (mean, 23.0 micromol/g; range, 3.0 to 73.3 micromol/g). Conversely, differences occurred between SCFA profiles and became significant after day 17. The main differences were a significant increase in the butyric acid concentration (12% versus 30%) with group II. Compared with group I, fecal SCFA concentrations were 3.2-fold lower (7.4 micromol/g; range, 0.3 to 37.4 micromol/g) in group III with no significant changes in the profiles. CONCLUSIONS: Fecal SCFA excretion may vary in absence of any digestive disease. During this study, in terms of gestational age, total SCFA concentrations were significantly lower in extremely premature infants compared with infants born less premature, despite their known higher deficiency in intestinal lactase activity. In terms of diet, the absence of lactose did not lead to a decrease in colonic fermentation and induced changes in SCFA patterns. These new baseline data may offer clues to further development of milk formulas.