Antonia Suau

Quantification of Bacterial Groups within Human Fecal Flora by Oligonucleotide Probe Hybridization

ABSTRACT To investigate the population structure of the predominant phylogenetic groups within the human adult fecal microbiota, a new oligonucleotide probe designated S-G-Clept-1240-a-A-18 was designed, validated, and used with a set of five 16S rRNA-targeted oligonucleotide probes. Application of the six probes to fecal samples from 27 human adults showed additivity of 70% of the total 16S rRNA detected by the bacterial domain probe. The Bacteroidesgroup-specific probe accounted for 37% ± 16% of the total rRNA, while the enteric group probe accounted for less than 1%.Clostridium leptum subgroup and Clostridium coccoides group-specific probes accounted for 16% ± 7% and 14% ± 6%, respectively, while Bifidobacterium andLactobacillus groups made up less than 2%.

Gut flora and inflammatory bowel disease

The pathogenesis of inflammatory bowel disease involves interactions between the host susceptibility, mucosal immunity and intestinal microflora. There is therefore great interest in the changes in the endogenous flora in inflammatory bowel disease patients and in the establishment of potential genetic variations in host responses to endogenous bacteria. In this review, we summarize the modifications in the various regional ecosystems in the gastrointestinal tract during inflammatory bowel disease (luminal bacteria in faeces or inside the gastrointestinal tract, bacteria in mucus and bacteria directly attached to the mucosa). Results were obtained following a ‘candidate microorganism strategy’ and, as is occurring increasingly frequently, following a ‘full description strategy’, which has progressed largely due to the development of culture‐independent techniques. The possibility of modifying the ecosystem using prebiotics or probiotics offers hope for new treatment developments, particularly in the prevention of relapse.

Conditions of bifidobacterial colonization in preterm infants : A prospective analysis

Background: Premature birth results in a delayed and abnormal qualitative pattern of gut colonization. This abnormal pattern is thought to affect intestinal development and contribute to a higher risk of gastrointestinal infectious diseases such as neonatal necrotizing enterocolitis (NEC). In particular, bifidobacteria are thought to play a major role. We therefore studied bifidobacterial colonization in preterm infants during the first month of life. Patients and Methods: Fecal samples were prospectively analyzed in 52 infants born at a gestational age ranging from 30 to 35 weeks fed with a preterm formula alone and, in 18, with their mothers milk. Fecal samples were collected twice per week during the hospital stay. Bifidobacterial colonization was analyzed with culture and a molecular method. Results: Bifidobacterial colonization occurred in 18 infants at a median age of 11 days, always greater than the corrected mean gestational age of 35.4 weeks (SD, 0.9) and greater than 34 weeks for 16 of 18. Colonization by bifidobacteria was affected by neither birthweight nor mode of delivery nor antibiotics given to the mother or infant. In contrast, birth gestational age had a significant impact on colonization by bifidobacteria (P < 0.05), which always occurred in children born at a birth gestational age greater than 32.9 weeks (P < 0.05). Conclusions: Birth gestational age seems to act as a major determinant of bifidobacterial colonization in the premature infant, suggesting the role of gut maturation, a finding that should probably be taken into account in manipulations of the gut flora aimed at reducing NEC.

Differences in rDNA libraries of faecal bacteria derived from 10- and 25-cycle PCRs

In spite of its shortcoming, analysis of PCR-derived rDNA libraries is being employed increasingly to investigate microbial diversity within many ecosystems. In the present investigation, the effects of the number of PCR cycles (10 vs 25 cycles) on the inferred structure of a 16S rDNA library have been examined. Seventy-five 25-cycle sequences were retrieved and analysed in comparison with 284 10-cycle sequences already described in a previous study. The 359 clones obtained were classified into 94 molecular species (at least 98% sequence similarity). At the level of large phylogenetic groups, the two cloned rDNA libraries were not different. A mathematical model was developed in order to estimate the number of molecular species expected if further sequencing was performed. Coverage-based computing, projections and statistical analysis demonstrated that the structures of the two PCR-derived rDNA libraries were different and that the 25-cycle rDNA library displayed reduced diversity. It is suggested that the number of PCR cycles used for amplification of 16S rDNA genes for phylogenetic diversity studies must therefore be kept as small as possible.

Molecular analysis of intestinal microbiota of rainbow trout (Oncorhynchus mykiss).

The aim of this study was to evaluate different molecular tools based on the 16S rRNA gene, internal transcribed spacer, and the rpoB gene to examine the bacterial populations present in juvenile rainbow trout intestines. DNA was extracted from both pooled intestinal samples and bacterial strains. Genes were PCR-amplified and analysed using both temporal temperature gradient gel electrophoresis (TTGE) and restriction fragment length polymorphism methods. Because of the high cultivability of the samples, representative bacterial strains were retrieved and we compared the profiles obtained from isolated bacteria with the profile of total bacteria from intestinal contents. Direct analysis based on rpoB-TTGE revealed a simple bacterial composition with two to four bands per sample, while the 16S rRNA gene-TTGE showed multiple bands and comigration for a few species. Sequencing of the 16S rRNA gene- and rpoB-TTGE bands revealed that the intestinal microbiota was dominated by Lactococcus lactis, Citrobacter gillenii, Kluyvera intermedia, Obesumbacterium proteus, and Shewanella marinus. In contrast to 16S rRNA gene-TTGE, rpoB-TTGE profiles derived from bacterial strains produced one band per species. Because the single-copy state of rpoB leads to a single band in TTGE, the rpoB gene is a promising molecular marker for investigating the bacterial community of the rainbow trout intestinal microbiota.

Fecal microbial community in preterm infants

The gastrointestinal tract of a healthy fetus is sterile. In full term infants, the bacterial colonization of the gastrointestinal tract has been extensively studied if not entirely understood. During the birth process and rapidly thereafter, microbes from the mother and the surrounding environment colonize the gastrointestinal tract until a dense and complex bacterial community is established. In vaginally delivered neonates, bacteria appear in the stools during the first day of life, with usually Escherichia coli and Enterococcus spp., among the first, followed within the first 5 days by Bifidobacterium spp. Because, at this stage, the composition of the gut bacterial community is strongly influenced by the diet, a shift in the bacterial composition can be observed. By 10 days of age, most healthy full term neonates are colonized with a heterogeneous bacterial flora, with bifidobacteria dominant in breast-fed infants and a more diversified flora in formula-fed infants (1). A dynamic balance exists between the bacterial community, the host physiology, and the diet: all of them influence initial acquisition, subsequent development and eventual stability of the gut ecosystem (2). In contrast, in preterm infants, especially extremely low birth weight preterm infants (weighting less than 1,000 g at birth), bacterial colonization and its consequences on health have not been extensively studied. Many factors influence the biodiversity of the intestinal flora and may increase the risk of gastrointestinal disease such as necrotizing enterocolitis (NEC): immaturity of the main vital functions, the characteristics of the medical environment, from delivery to hospital discharge, the developmental stage of gastrointestinal and immune functions, the mode and environment of delivery, the feeding regimen, the kind of drug therapy (such as antibiotics, corticoids, etc.), or of other therapy (such as oxygenation). In adults, the human cecal flora differs quantitatively and qualitatively from the fecal flora. Facultative anaerobes represented 25% of total bacteria in the cecum versus 1% in the feces (3). So, the biodiversity of the bacterial community should be studied at these different levels of the gastrointestinal tract. For obvious reasons, most of the studies were performed on stool samples. In recent years, the use of ribosomal RNA, in particular, sequences of the 16S rRNA genes, has greatly facilitated the study of gastrointestinal tract ecology because it allows a culture-independent analysis of the fecal microbial community. In adult fecal samples, molecular tools have indicated that 60% to 80% of the total human microflora has not been cultivated (4). Techniques such as polymerase chain reaction, gene sequencing, in situ hybridization, and denaturing gradient gel electrophoresis are routinely used to study gut microbial ecology and have been recently reviewed by Suau (5). So far, only three studies inspected the microbial composition of premature infants using molecular methods (6–8). Thus, the aim of the present review is to describe the composition of fecal microbial community in preterm infants from the colonization of sterile gut, to present the different factors that contribute to its alterations, and to link the gut microflora to diseases such as NEC. This review is essentially based on data collected using traditional bacterial culture from fecal samples.

Effects on faecal microbiota of dietary and acidic oligosaccharides in children during partial formula feeding.

Objective: To test the safety and effect on faecal microbiota of a formula with prebiotic oligosaccharides alone or in combination with acidic oligosaccharides in infants at the age of partial formula feeding. Patients and Methods: The study was a double-blind, placebo-controlled, randomised intervention trial in which 82 healthy, full-term, partially breast-fed children, from 1 week to 3 months old, were given 1 of the following formulae: whey-based formula (control group), whey-based formula with galacto- and long-chain fructo-oligosaccharides (scGOS/lcFOS group), or whey-based formula with galacto- and long-chain fructo-oligosaccharides added with pectin-derived acidic oligosaccharides (scGOS/lcFOS/pAOS group). Children were studied for the duration of the partial formula feeding period and every 2 weeks for 2 months after breast-feeding cessation. The total bacteria count and the proportion of 7 bacterial families were determined using in situ hybridisation coupled to flow cytometry. Results: The total bacterial count did not alter with time or type of feeding (9.9 ± 0.1 log10 cells per gram wet weight). Compared with the control group, there was an increase of the Bifidobacterium genus (P = 0.0001), and a decrease of proportions for the Bacteroides group (P = 0.02) and the Clostridium coccoides group (P = 0.01) in both oligosaccharide groups. The proportion of bifidobacteria was significantly higher in the scGOS/lcFOS/pAOS compared with the scGOS/lcFOS group (P < 0.01). Conclusions: Infant formulae appear to be clinically safe and effective on infant microbiota. They minimize the alteration of faecal microbiota after cessation of breast-feeding and promote bifidobacteria proportions, with a stronger effect when acidic oligosaccharides are present.

Patchy distribution of mucosal lesions in ileal Crohn's disease is not linked to differences in the dominant mucosa‐associated bacteria: A study using fluorescence in situ hybridization and temporal temperature gradient gel electrophoresis

Background The mucosa‐associated bacteria (MAB) are suspected of being involved in the pathogenesis of Crohns disease. We analyzed and compared the MAB in noninflamed and inflamed ileal mucosa of Crohns disease patients (n = 22). Methods Tissue samples from the inflamed ileal mucosa and from the adjacent noninflamed ileal mucosa were taken from surgical resection specimens. The MAB were investigated using fluorescence in situ hybridization with 7 group‐specific probes and temporal temperature gradient gel electrophoresis (TTGE). Results Samples from both noninflamed and inflamed mucosa were obtained from 15 patients. The distribution of the bacterial populations was not different between noninflamed and inflamed mucosa. The Bacteroidetes phylum was dominant and accounted for 29% of MAB (0%–74%) in noninflamed tissues and 32% (0%–70%) in inflamed areas. The &ggr; Proteobacteria represented 12% (0%–70%) of MAB both in noninflamed and inflamed areas. The Clostridium coccoides group (Firmicutes phylum) represented 15% of MAB in noninflamed tissues versus 7% in inflamed areas. For most of the patients the similarity index between TTGE paired profiles was very high. Conclusion The dominant MAB do not differ between noninflamed and inflamed ileal mucosa in Crohns disease. This argues against a localized dysbiosis to explain the patchy distribution of mucosal lesions. (Inflamm Bowel Dis 2007)

A fermented formula in pre-term infants: clinical tolerance, gut microbiota, down-regulation of faecal calprotectin and up-regulation of faecal secretory IgA

Intestinal bacterial colonisation in pre-term infants is delayed compared with full-term infants, leading to an increased risk of gastrointestinal disease. Modulation of colonisation through dietary supplementation with probiotics or prebiotics could decrease such a risk. The present study evaluated clinical tolerance, the effects on gut microbiota, and inflammatory and immunological mucosal responses to an infant formula adapted for pre-term infants that included in its manufacturing process a fermentation step with two probiotic strains, Bifidobacterium breve C50 and Streptococcus thermophilus 065, inactivated by heat at the end of the process. A total of fifty-eight infants (gestational age: 30-35 weeks), fed either the fermented pre-term formula or a standard pre-term formula, were followed up during their hospital stay. Clinical tolerance, faecal microbiota using a culture and a culture-independent method (temporal temperature gel electrophoresis), faecal calprotectin and secretory IgA were analysed weekly. No difference was observed regarding anthropometric data and digestive tolerance, except for abdominal distension, the incidence of which was lower in infants fed the fermented formula for 2 weeks. Bacterial colonisation was not modified by the type of feeding, particularly for bifidobacteria. Faecal calprotectin was significantly lower in infants fed the fermented formula for 2 weeks, and secretory IgA increased with both mothers milk and the fermented formula. The fermented formula was well tolerated and did not significantly modulate the bacterial colonisation but had benefits on inflammatory and immune markers, which might be related to some features of gastrointestinal tolerance.

Amoxicillin treatment modifies the composition of Bifidobacterium species in infant intestinal microbiota.

OBJECTIVES Amoxicillin is a beta-lactam antibiotic largely used in childhood. However only few studies described its impact on composition of children gut microbiota, in particular on Bifidobacterium populations considered as beneficial microorganisms. In this study, the impact on faecal Bifidobacterium species of a seven-day amoxicillin treatment was quantitatively and qualitatively assessed in infants during an episode of acute respiratory infection. METHODS Faecal samples from 31 infants were obtained on day 0 (just before amoxicillin therapy) and on day 7 (the end of therapy). Total DNA was extracted and bifidobacteria were quantified using real-time PCR. Predominant Bifidobacterium species were then identified using specific PCR-TTGE. RESULTS Bifidobacteria concentrations were not significantly altered by amoxicillin compared to the healthy group. However, amoxicillin treatment induced a complete disappearance of Bifidobacterium adolescentis species (occurrence rate of 0% versus 36.4% in healthy group, P < 0.001), a significant decrease in the occurrence rate of Bifidobacterium bifidum (23% versus 54.5% in healthy group, P < 0.05), but did not affect Bifidobacterium longum (93.5% versus 100% in healthy group) and Bifidobacterium pseudocatenulatum/B. catenulatum (about 55% in both groups). The number of Bifidobacterium species per microbiota significantly decreased from 2.5 +/- 1 for healthy group to 1.8 +/- 0.9 for treated infants (P < 0.05). CONCLUSIONS This study showed that a 7 day amoxicillin treatment did not alter the counts of Bifidobacterium. However amoxicillin can have an impact by changing the microbiota at the species level and decreased the diversity of this population.