Contrasting effects of historical contingency on phenotypic and genomic trajectories during a two-step evolution experiment with bacteria.

BACKGROUND: The impact of historical contingency, i.e. the past evolutionary history of a population, on further adaptation is mostly unknown at both the phenotypic and genomic levels. We addressed this question using a two-step evolution experiment. First, replicate populations of Escherichia coli were propagated in four different environmental conditions for 1000 generations. Then, all replicate populations were transferred and propagated for further 1000 generations to a single new environment. RESULTS: Using this two-step experimental evolution strategy, we investigated, at both the phenotypic and genomic levels, whether and how adaptation in the initial historical environments impacted evolutionary trajectories in a new environment. We showed that both the growth rate and fitness of the evolved populations obtained after the second step of evolution were contingent upon past evolutionary history. In contrast however, the genes that were modified during the second step of evolution were independent from the previous history of the populations. CONCLUSIONS: Our work suggests that historical contingency affects phenotypic adaptation to a new environment. This was however not reflected at the genomic level implying complex relationships between environmental factors and the genotype-to-phenotype map.

Inulin prebiotic reinforces host cancer immunosurveillance via ɣδ T cell activation.

The gut microbiota is now recognized as a key parameter affecting the host's anti-cancer immunosurveillance and ability to respond to immunotherapy. Therefore, optimal modulation for preventive and therapeutic purposes is very appealing. Diet is one of the most potent modulators of microbiota, and thus nutritional intervention could be exploited to improve host anti-cancer immunity. Here, we show that an inulin-enriched diet, a prebiotic known to promote immunostimulatory bacteria, triggers an enhanced Th1-polarized CD4+ and CD8+ αß T cell-mediated anti-tumor response and attenuates tumor growth in three preclinical tumor-bearing mouse models. We highlighted that the inulin-mediated anti-tumor effect relies on the activation of both intestinal and tumor-infiltrating ɣδ T cells that are indispensable for αß T cell activation and subsequent tumor growth control, in a microbiota-dependent manner. Overall, our data identified these cells as a critical immune subset, mandatory for inulin-mediated anti-tumor immunity in vivo, further supporting and rationalizing the use of such prebiotic approaches, as well as the development of immunotherapies targeting ɣδ T cells in cancer prevention and immunotherapy.

Effect of dietary fibre of barley variety 'Rihane' on azoxymethane-induced aberrant crypt foci development and on colonic microbiota diversity in rats.

Many epidemiological and experimental studies have suggested an important role for dietary fibre (DF) of cereals in the prevention of colon cancer. The objective of the present study was to explain the effects of the DF of barley Rihane (BR) on azoxymethane (AOM)-induced aberrant crypt foci (ACF) and colonic bacterial diversity in rats. Following an acclimatisation period, rats were divided into four groups and fed a control (C) diet or experimental diet containing 30 % of BR. DF content in the experimental diet was twice that of the C diet (total DF was 8·69 % in the C diet and 15·24 % in the BR diet). At 7 and 8 weeks of age, rats received two successive subcutaneous injections of AOM at 20 mg/kg body weight. At 12 weeks after the first injection, ten animals from each group were killed. The BR diet decreased colonic pH (P < 0·05) compared with the C diet. The total number of ACF observed decreased considerably in the BR/AOM group compared with the C/AOM group (P < 0·05). Comparison of similarity coefficients showed variability of colonic microbiota species between the different groups. In addition, we showed inter-individual variability within the same group. This similarity was affected by BR and AOM. The present results show that bifidobacteria numbers were lower in rats fed the BR diet compared with those fed the C diet. However, the number of enterobacteria in colonic content was increased (P < 0·05) in the BR group compared with the C group. The results from the present study show that the DF of BR reduced the incidence of AOM-induced ACF and increased microbiota biodiversity.

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 mother's 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.

Faecal bifidobacteria in Indian neonates & the effect of asymptomatic rotavirus infection during the first month of life.

BACKGROUND AND OBJECTIVES: Bifidobacteria colonize the gut after the first week of life and remain an important component of the gut microbiota in infancy. This study was carried out to characterize the diversity and number of bifidobacteria colonizing the gut in Indian neonates and to investigate whether asymptomatic infection with rotavirus in the first month of life affected gut colonization by bifidobacteria. METHODS: DNA was isolated from faeces of 14 term-born neonates who were under surveillance for rotavirus infection. Bacterial and bifidobacterial diversity was evaluated by temporal temperature gradient electrophoresis (TTGE) of 16S rDNA amplified using total bacteria and bifidobacteria-specific primers. Real time PCR, targeting 16S rDNA, was used to quantitate faecal bifidobacteria and enterobacteria. RESULTS: TTGE of conserved bacterial 16S rDNA showed 3 dominant bands of which Escherichia coli (family Enterobacteriaceae) and Bifidobacterium (family Bifidobacteriaceae) were constant. TTGE of Bifidobacterium genus-specific DNA showed a single band in all neonates identified by sequencing as Bifidobacterium longum subsp. infantis. Faecal bifidobacterial counts (log 10 cfu/g faeces) ranged from 6.1 to 9.3 and enterobacterial counts from 6.3 to 9.5. Neonates without and with rotavirus infection in the first week of life did not show significant differences in the median count of bifidobacteria (log 10 count 7.48 vs. 7.41) or enterobacteria (log 10 count 8.79 vs. 7.92). INTERPRETATION AND CONCLUSIONS: B. longum subsp. infantis was the sole bifidobacterial species colonizing the gut of Indian neonates. Asymptomatic rotavirus infection in the first month of life was not associated with alteration in faecal bifidobacteria or enterobacteria.

Differential effects of Bifidobacterium pseudolongum strain Patronus and metronidazole in the rat gut.

In the luminal contents of metronidazole-treated rats, there was a dominant Bifidobacterium species. A strain has been isolated, its 16S rRNA gene has been sequenced, and the strain has been named Bifidobacterium pseudolongum strain Patronus. In this study, using an experimental model of healthy rats, the effects of metronidazole treatment and B. pseudolongum strain Patronus administration on the luminal and mucosa-associated microbiota and on gut oxidation processes were investigated. Metronidazole treatment and the daily gavage of rats with B. pseudolongum strain Patronus increased the numbers of bifidobacteria in cecal contents and in cecal mucosa-associated microbiota compared with those in control rats. Metronidazole reduced the colonic oxidative damage to proteins. This is the first evidence that B. pseudolongum strain Patronus exerts an effect on a biomarker of oxidative damage by reducing the susceptibility to oxidation of proteins in the colon and the small bowel. Antioxidant effects of metronidazole could be linked to the bifidobacterial increase but also to other bacterial modifications.

Time course of bacterial diversity in stool samples of malnourished children with cholera receiving treatment.

BACKGROUND: Recent nutritional interventions have targeted colonic functions in patients with infectious diarrhea during rehydration and during recovery from malnutrition, with the assumption that the effects will be influenced by metabolism of complex carbohydrates by colonic bacteria. However, the diversity of colonic bacteria in patients with cholera is not known. AIM: To study the diversity of colonic bacteria in malnourished children with cholera before and during treatment with oral rehydration salt solutions containing 1 of these 3 substrates: glucose, rice, or amylase-resistant starch. PATIENTS AND METHODS: Serial fecal samples were collected from 30 malnourished children with cholera until completion of rehydration and partial nutritional recovery; 11 malnourished children without diarrhea; and 6 better nourished children. Polymerase chain reaction, using universal primers for 16S rDNA, was performed on chromosomal DNA extracted from the stool samples, and the products were separated by temporal temperature gradient gel electrophoresis. RESULTS: The Vibrio cholerae band was detected in all children at enrollment and disappeared within 2 days. On day 2, a rapid and significant increase in the band numbers was observed, which was followed by a steady increase until day 28. After full recovery from cholera and partial recovery from malnutrition, the number of bands (11.5+/-2.8) was lower than in healthy children (22.2+/-1.3). On day 3, the number of bands was greater with rice or amylase-resistant starch than with glucose (P<.05). CONCLUSIONS: Bacterial diversity was markedly but transiently altered in severely malnourished children with cholera receiving therapy.

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.

Oral administration of viable Bifidobacterium pseudolongum strain Patronus modified colonic microbiota and increased mucus layer thickness in rat.

This study aimed at evaluating the alteration of the colonic microbiota and the changes in the mucus layer thickness induced by oral administration of living bifidobacteria in rats. The study was performed on rats fed with Bifidobacterium pseudolongum strain Patronus (1010 bacteria per day for 7 days). This bacterial administration led to a large increase of mucus thickness (57%, P < 0.05). Both quantitative PCR and high-throughput sequencing of bacterial 16S rRNA gene revealed a significant increase of the amount of the Bifidobacterium genus in the microbiota of rats fed with the strain Patronus, associated with a decrease of Akkermansia muciniphila. The increase in mucus thickness could be due to an increase of the bifidobacteria per se or via the decrease of A. muciniphila, a major mucin-degrading species. As the mucus layer plays an essential role in gut protection, our data enlighten the importance of studying mucus-degrading bacteria for understanding the underlying etiology of diseases such as intestinal bowel diseases and to implement new therapeutic strategies.

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 Crohn's disease. We analyzed and compared the MAB in noninflamed and inflamed ileal mucosa of Crohn's 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 gamma 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 Crohn's disease. This argues against a localized dysbiosis to explain the patchy distribution of mucosal lesions.

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 mother's 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.

Low species diversity and high interindividual variability in faeces of preterm infants as revealed by sequences of 16S rRNA genes and PCR-temporal temperature gradient gel electrophoresis profiles.

Little information regarding the composition of the gut microbiota in preterm infants is available. The purpose of this study was to investigate the bacterial diversity in faeces of preterm infants, using analysis of randomly cloned 16S rRNA genes and PCR-TTGE (temporal temperature gradient gel electrophoresis) profiles, to determine whether noncultivated bacteria represented an important part of the community. The 288 clones obtained from faecal samples of 16 preterm infants were classified into 25 molecular species. All but one molecular species had a cultivated representative in public databases: molecular tools did not reveal any unexplored diversity. The mean number of molecular species per infant was 3.25, ranging from one to eight. There was a high interindividual variability. The main groups encountered were the Enterobacteriaceae family and the genera Enterococcus, Streptococcus and Staphylococcus. Seven preterm infants were colonized by anaerobes and only four by bifidobacteria. TTGE profiles were composed of one to nine bands (mean value: 4.3). Furthermore, 51 of 59 clones (86%) comigrated with a band of the corresponding faecal sample. This study will form a comparative framework for other studies, e.g. on the faecal microbiota of preterm infants with different pathologies or the impact of diet on colonization.

Characterization of human intestinal bifidobacteria using competitive PCR and PCR-TTGE.

In this study, a competitive PCR was developed to estimate the quantity of bifidobacteria in human faecal samples using two 16S rRNA gene Bifidobacterium genus-specific primers, Bif164f and Bif662r. A PCR-temporal temperature gradient gel electrophoresis (TTGE) with the same primers also allowed us to describe the Bifidobacterium species present in these faecal samples. The PCR product obtained from the competitor had 467 bp, and was 47 bp shorter than the PCR products obtained from Bifidobacterium strains. The number of bifidobacterial cells was linear from 10 to 10(8) cells per PCR assay. Taking into account the dilutions of the extracted DNA, the linear range was over 8 x 10(5) bifidobacteria g(-1) of faeces. Reproducibility was assessed from 10 independent DNA extractions from the same stool and the coefficient of variation was 0.5%. When the competitive PCR was compared with the culture method, a similar count of seven out of nine Bifidobacterium pure cultures were obtained, or had a difference inferior or equal to 1 log(10). In faecal samples, the enumeration of Bifidobacterium genus in most cases gave higher results with competitive PCR than with culture on selective Columbia-Beerens agar pH 5 (P < 0.05). In conclusion, this competitive PCR allows a rapid, highly specific and reproducible quantification of Bifidobacterium genus in faecal samples. TTGE fragments co-migrating with B. longum CIP64.63 fragment were found in 10 out of 11 faecal samples. Bifidobacterium adolescentis and B. bifidum were detected in five out of 11 subjects. Thus, cPCR and PCR-TTGE can be associated in order to characterize human faecal bifidobacteria.

A longitudinal study of infant faecal microbiota during weaning.

For infants, the introduction of food other than breast milk is a high risk period due to diarrheal diseases, and may be corroborated with a shift in the faecal microbiota. This longitudinal study was the first undertaken to understand the effect of the supplementation on the infant's faecal microbiota and particularly the bifidobacteria. Eleven infants were enrolled. Their faecal microbiota were analysed using temporal temperature gradient gel electrophoresis (TTGE) with bacterial and bifidobacterial primers. In parallel, bifidobacterial counts were followed using competitive PCR. Three periods were distinguished: exclusive breastfeeding (Bf period), weaning (i.e. formula-milk addition, W period) and postweaning (i.e. breastfeeding cessation, Pw period). The bifidobacterial counts were not modified, reaching 10.5 (Log10 cells g(-1) wet weight). In the TTGE profiles, the main identified bands corresponded to Escherichia coli, Ruminococcus sp. and Bifidobacterium sp., more precisely Bifidobacterium longum, Bifidobacterium infantis and Bifidobacterium breve. For both TTGE profiles, the analysis of the distance suggested a maturation of the faecal microbiota but no correlation could be established with the diet. Despite a high interindividual variability, composition of the faecal microbiota appeared more homogenous after weaning and this point may be correlated with the cessation of breastfeeding.

Modulation of the fecal microbiota by the intake of a Lactobacillus johnsonii La1-containing product in human volunteers.

Lactobacillus johnsonii La1 (La1) is a probiotic strain capable of stimulating the immune system of the host and interfering with gastrointestinal pathogens. This study evaluates how the ingestion of different amounts of La1 influences the main bacterial populations of the fecal microbiota. Eight asymptomatic volunteers participated in the study. After a basal period, they ingested daily 100 mL of a product containing 10(8) CFU mL(-1) of La1 during the first week, 200 mL during the second week and 500 mL during the third week. Fecal samples were obtained at the end of each period and subsequently during 7 weeks. Lactobacilli were determined by culture on MRS agar and La1 colonies were confirmed by ERIC-PCR. The main populations of fecal bacteria were identified by FISH and flow cytometry. At baseline, 37.7% of the total fluorescent bacteria were Eubacterium rectale, 18.3% Fusobacterium prausnitzii, 13.2% Bacteroides, 8.6% Atopobium, 2.30%, Clostridium histolyticum, 2.05% Bifidobacterium and 0.95% Lactobacillus. Fecal excretion of La1 increased during the intake period and decreased during the post-ingestion period, so that no La1 was observed in the stools of the volunteers seven weeks after the intake product has been finished. La1 intake increased the populations of C. histolyticum (p=0.049), Lactobacillus (p=0.056) and Bifidobacterium (p=0.067), and decreased those of F. prausnitzii (p=0.005) while it did not affect Bacteroides, E. rectale and Atopobium populations. These bacterial populations returned to their baseline levels during the post-ingestion period. The regular intake of a La1-containing product beneficially affects the homeostasis of the human fecal microbiota, probably contributing to the health-promoting effects of this probiotic.

Enumeration of Bacteroides species in human faeces by fluorescent in situ hybridisation combined with flow cytometry using 16S rRNA probes.

Bacteroides is a predominant group of the faecal microbiota in healthy adults. To investigate the species composition of Bacteroides by fluorescent in situ hybridisation (FISH) combined with flow cytometry, we developed five species-specific probes targeting the 16S rRNA. Probes were designed to identify cells belonging to Bacteroides distasonis, B. fragilis, B. ovatus, B. vulgatus and B. putredinis. The species-specificity of the probes was assessed against a collection of reference strains from the Cytophaga-Flavobacterium-Bacteroides group. The results of the FISH experiments showed that the probes were specific as they only detected strains of the target species. Determining the fluorescence intensity of each probe relative to that of the EUB 338 probe (domain bacteria) further showed that each species probe easily accessed the targeted site. The probes were applied to quantify the Bacteroides species in faeces collected from 20 healthy adults. All five species were detected in the faecal samples. Cells hybridised with Bfra 998 were the most frequent as they were observed in 90% of individuals (18/20 samples, mean proportion of 3.9 +/- 2.2%). The cells hybridised with Bvulg 1017 were observed in 85% of individuals (17/20 samples) and represented with a mean proportion of 4.2 +/- 6.1%, the most abundant Bacteroides species in human faeces. Cells hybridising with probes for B. ovatus, B. distasonis and B. putredinis were less frequently detected. The large distribution of B. vulgatus and B. fragilis in human faeces is in accordance with previous reports based on culture or molecular studies. This work showed that fluorescent in situ hybridisation is a tool appropriate for a high-resolution analysis of the species composition of complex ecosystems and especially of the Bacteroides group within the faecal microbiota.

Long-term changes in human colonic Bifidobacterium populations induced by a 5-day oral amoxicillin-clavulanic acid treatment.

The objective of this study was to assess the possible modifications due to amoxicillin-clavulanic acid (AMC) treatment on total bacteria and on Bifidobacterium species balance in human colonic microbiota. Eighteen healthy volunteers (19 to 36 years old) were given a 875/125 mg dose of AMC twice a day for 5 days. Fecal samples were obtained before and after antibiotic exposure. After total DNA extraction, total bacteria and bifidobacteria were specifically quantified using real-time PCR. Dominant species were monitored over time using bacterial and bifidobacterial Temporal Temperature Gradient gel Electrophoresis (TTGE). At the end of AMC exposure, total bacterial concentrations as well as bifidobacteria concentrations were significantly reduced compared to before AMC exposure:10.7±0.1 log(10) 16S rRNA gene copies/g vs 11.1±0.1 log(10) (p = 0.003) and 8.1±0.5 log(10) 16S rRNA gene copies/g vs 9.4±0.3 log(10) (p = 0.003), respectively. At the same time, the mean similarity percentages of TTGE bacteria and TTGE bifidobacteria profiles were significantly reduced compared to before AMC exposure: 51.6%±3.5% vs 81.4%±2.1% and 55.8%±7.6% vs 84.5%±4.1%, respectively. Occurrence of B. adolescentis, B. bifidum and B. pseudocatenulatum/B. catenulatum species significantly decreased. Occurrence of B. longum remained stable. Moreover, the number of distinct Bifidobacterium species per sample significantly decreased (1.5±0.3 vs 2.3±0.3; p = 0.01). Two months after AMC exposure, the mean similarity percentage of TTGE profiles was 55.6% for bacteria and 62.3% for bifidobacteria. These results clearly demonstrated that a common antibiotic treatment may qualitatively alter the colonic microbiota. Such modifications may have potential long-term physiological consequences.

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.

Metronidazole effects on microbiota and mucus layer thickness in the rat gut.

Both mucus and mucosa-associated bacteria form a specific environment in the gut; their disruption may play a crucial role in the development of intestinal bowel disease (IBD). Metronidazole, an antibiotic used in the treatment of IBD, alters gut microbiota and reduces basal oxidative stress to proteins in colonic tissue of healthy rats. The aim of this study was to evaluate the impact of the altered microbiota due to the metronidazole on the thickness of the mucus layer. This study was performed in healthy untreated rats (control group) or rats treated by metronidazole (metronidazole-treated rats, 1 mg mL(-1) in drinking water for 7 days). Both PCR-temporal temperature gradient gel electrophoresis and quantitative PCR (qPCR) revealed an altered microbiota with an increase in bifidobacteria and enterobacteria in metronidazole-treated rats compared with control rats. Moreover, a dominant bifidobacterial species, Bifidobacterium pseudolongum, was detected. Using qPCR and FISH, we showed that bifidobacteria were also increased in the microbiota-associated mucosa. At the same time, the mucus layer thickness was increased approximately twofold. These results could explain the benefits of metronidazole treatment and warrant further investigations to define the role of bifidobacteria in the colonic mucosa.

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.