A synbiotic mixture of selected oligosaccharides and bifidobacteria assists murine gut microbiota restoration following antibiotic challenge.

BACKGROUND: Typically, animal models studying gastrointestinal microbiotas compromised in early life have employed either germ-free animals or mice treated with a cocktail of antibiotics. Such studies intend to mimic scenarios of infants born by caesarean section and/or subjected to antibiotic treatment. However, the antibiotics used in these studies are rarely prescribed to infants. Therefore, an early life model was developed in which the murine gastrointestinal microbiota was severely disrupted by clindamycin treatment. RESULTS: In this mouse model, we investigated the extent supplementation with a synbiotic mixture of prebiotics, being scGOS/lcFOS with the human milk oligosaccharide 2'-Fucosyllactose (2'-FL), in combination with or without single strain or mix of "infant type" bifidobacteria, can rescue an antibiotic-compromised microbiota. Shotgun metagenomic sequencing showed that the microbiota was severely disrupted by the clindamycin challenge. No recovery was observed 3 weeks post-challenge in the scGOS/lcFOS/2'FL group, while the group that received the synbiotic treatment of scGOS/lcFOS/2'-FL with Bifidobacterium breve NRBB01 showed partial recovery. Strikingly in the scGOS/lcFOS/2'-FL group receiving the mixture of bifidobacteria resulted in a recovery of the microbiota disruption. Histological analyses showed that the clindamycin-treated animals at the end of the experiment still suffered from mild oedema and villi/colonic crypt irregularities which was ameliorated by the synbiotic intervention. CONCLUSION: Our study demonstrates that supplementation of synbiotic mixture of scGOS/lcFOS/2'-FL in combination with a specific mix of infant-type bifidobacterial strains is able to partially revive an antibiotic-perturbed gastrointestinal microbiota. Video Abstract.

Quantitative Physiology and Proteome Adaptations of Bifidobacterium breve NRBB57 at Near-Zero Growth Rates.

In natural environments, nutrients are usually scarce, causing microorganisms to grow slowly while staying metabolically active. These natural conditions can be simulated using retentostat cultivations. The present study describes the physiological and proteome adaptations of the probiotic Bifidobacterium breve NRBB57 from high (0.4 h-1) to near-zero growth rates. Lactose-limited retentostat cultivations were carried out for 21 days in which the bacterial growth rate progressively reduced to 0.00092 h-1, leading to a 3.4-fold reduction of the maintenance energy requirement. Lactose was mainly converted into acetate, formate, and ethanol at high growth rates, while in the retentostat, lactate production increased. Interestingly, the consumption of several amino acids (serine, aspartic acid, and glutamine/arginine) and glycerol increased over time in the retentostat. Morphological changes and viable but nonculturable cells were also observed in the retentostat. Proteomes were compared for all growth rates, revealing a downregulation of ribosomal proteins at near-zero growth rates and an upregulation of proteins involved in the catabolism of alternative energy sources. Finally, we observed induction of the stringent response and stress defense systems. Retentostat cultivations were proven useful to study the physiology of B. breve, mimicking the nutrient scarcity of its complex habitat, the human gut. IMPORTANCE In natural environments, nutrients are usually scarce, causing microorganisms to grow slowly while staying metabolically active. In this study we used retentostat cultivation to investigate how the probiotic Bifidobacterium breve adapts its physiology and proteome under severe nutrient limitation resulting in near-zero growth rates (<0.001 h-1). We showed that the nutrient limitation induced a multifaceted response including stress defense and stringent response, metabolic shifts, and the activation of novel alternative energy-producing pathways.

Ligilactobacillus salivarius PS2 Supplementation during Pregnancy and Lactation Prevents Mastitis: A Randomised Controlled Trial.

Mastitis is considered one of the main reasons for unwanted breastfeeding cessation. This study aimed to investigate the preventive effect of the probiotic strain Ligilactobacillus salivarius PS2 on the occurrence of mastitis in lactating women. In this multicountry, multicenter, randomized, double-blind, placebo-controlled trial, 328 women were assigned to the probiotic or the placebo group. The intervention started from the 35th week of pregnancy until week 12 post-partum. The primary outcome was the incidence (hazard) rate of mastitis, defined as the presence of at least two of the following symptoms: breast pain, breast erythema, breast engorgement not relieved by breastfeeding, and temperature > 38 °C. The probability of being free of mastitis during the study was higher in the probiotic than in the placebo group (p = 0.022, Kaplan-Meier log rank test) with 9 mastitis cases (6%) vs. 20 mastitis cases (14%), respectively. The hazard ratio of the incidence of mastitis between both study groups was 0.41 (0.190-0.915; p = 0.029), indicating that women in the probiotic group were 58% less likely to experience mastitis. In conclusion, supplementation of L. salivarius PS2 during late pregnancy and early lactation was safe and effective in preventing mastitis, which is one of the main barriers for continuing breastfeeding.

Broad Purpose Vector for Site-Directed Insertional Mutagenesis in Bifidobacterium breve.

Members of the genus Bifidobacterium are notoriously recalcitrant to genetic manipulation due to their extensive and variable repertoire of Restriction-Modification (R-M) systems. Non-replicating plasmids are currently employed to achieve insertional mutagenesis in Bifidobacterium. One of the limitations of using such insertion vectors is the presence within their sequence of various restriction sites, making them sensitive to the activity of endogenous restriction endonucleases encoded by the target strain. For this reason, vectors have been developed with the aim of methylating and protecting the vector using a methylase-positive Escherichia coli strain, in some cases containing a cloned bifidobacterial methylase. Here, we present a mutagenesis approach based on a modified and synthetically produced version of the suicide vector pORI28 (named pFREM28), where all known restriction sites targeted by Bifidobacterium breve R-M systems were removed by base substitution (thus preserving the codon usage). After validating the integrity of the erythromycin marker, the vector was successfully employed to target an α-galactosidase gene responsible for raffinose metabolism, an alcohol dehydrogenase gene responsible for mannitol utilization and a gene encoding a priming glycosyltransferase responsible for exopolysaccharides (EPS) production in B. breve. The advantage of using this modified approach is the reduction of the amount of time, effort and resources required to generate site-directed mutants in B. breve and a similar approach may be employed to target other (bifido)bacterial species.

Strain-Specific Probiotic Properties of Bifidobacteria and Lactobacilli for the Prevention of Diarrhea Caused by Rotavirus in a Preclinical Model.

Probiotic supplementation with different lactobacilli and bifidobacterial strains has demonstrated beneficial effects in infectious diarrhea caused by rotavirus (RV) in young children. Preclinical models of RV infection might be a good strategy to screen for the efficacy of new probiotic strains or to test their comparative efficacy. Neonatal Lewis rats were supplemented with Bifidobacterium breve M-16V, Lactobacillus acidophilus NCFM, Lactobacillus helveticus R0052, or Lactobacillus salivarius PS2 from days 2-14 of life. On day five, animals received RV SA-11 orally. Fecal samples were collected daily, weighed, and scored for the calculation of severity and incidence of diarrhea. In addition, fecal pH and fecal viral shedding were measured. Animals were sacrificed at the end of the study and their blood was obtained for the quantification of RV-specific immunoglobulins. RV infection was induced in ~90% of the animals. All probiotics caused a reduction of several clinical variables of severity and incidence of diarrhea, except L. salivarius PS2. L. acidophilus NCFM, B. breve M-16V, and L. helveticus R0052 seemed to be very effective probiotic strains. In addition, all Lactobacillus strains reduced the viral elimination one day post-inoculation. No differences were detected in the specific anti-RV humoral response. The present study highlights the strain-specific effects of probiotics and identifies promising probiotics for use in ameliorating and preventing RV-induced diarrhea in children, for example by including them in infant formulas.

Effect of Synbiotic on the Gut Microbiota of Cesarean Delivered Infants: A Randomized, Double-blind, Multicenter Study.

We determined the effect of short-chain galacto-oligosaccharides (scGOS), long-chain fructo-oligosaccharides (lcFOS) and Bifidobacterium breve M-16V on the gut microbiota of cesarean-born infants. Infants were randomized to receive a standard formula (control), the same with scGOS/lcFOS and B. breve M-16V (synbiotic), or with scGOS/lcFOS (prebiotic) from birth until week 16, 30 subjects born vaginally were included as a reference group. Synbiotic supplementation resulted in a higher bifidobacteria proportion from day 3/5 (P < 0.0001) until week 8 (P = 0.041), a reduction of Enterobacteriaceae from day 3/5 (P = 0.002) till week 12 (P = 0.016) compared to controls. This was accompanied with a lower fecal pH and higher acetate. In the synbiotic group, B. breve M-16V was detected 6 weeks postintervention in 38.7% of the infants. This synbiotic concept supported the early modulation of Bifidobacterium in C-section born infants that was associated with the emulation of the gut physiological environment observed in vaginally delivered infants.

Early-Life Events, Including Mode of Delivery and Type of Feeding, Siblings and Gender, Shape the Developing Gut Microbiota.

Colonization of the infant gut is believed to be critically important for a healthy growth as it influences gut maturation, metabolic, immune and brain development in early life. Understanding factors that influence this process is important, since an altered colonization has been associated with a higher risk of diseases later in life. Fecal samples were collected from 108 healthy neonates in the first half year of life. The composition and functionality of the microbiota was characterized by measuring 33 different bacterial taxa by qPCR/RT qPCR, and 8 bacterial metabolites. Information regarding gender, place and mode of birth, presence of siblings or pets; feeding pattern and antibiotic use was collected by using questionnaires. Regression analysis techniques were used to study associations between microbiota parameters and confounding factors over time. Bacterial DNA was detected in most meconium samples, suggesting bacterial exposure occurs in utero. After birth, colonization by species of Bifidobacterium, Lactobacillus and Bacteroides was influenced by mode of delivery, type of feeding and presence of siblings, with differences found at species level and over time. Interestingly, infant-type bifidobacterial species such as B. breve or B. longum subsp infantis were confirmed as early colonizers apparently independent of the factors studied here, while B. animalis subsp. lactis presence was found to be dependent solely on the type of feeding, indicating that it might not be a common infant gut inhabitant. One interesting and rather unexpected confounding factor was gender. This study contributes to our understanding of the composition of the microbiota in early life and the succession process and the evolution of the microbial community as a function of time and events occurring during the first 6 months of life. Our results provide new insights that could be taken into consideration when selecting nutritional supplementation strategies to support the developing infant gut microbiome.

Mother-to-infant transmission of intestinal bifidobacterial strains has an impact on the early development of vaginally delivered infant's microbiota.

OBJECTIVES: Bifidobacterium species are one of the major components of the infant's intestine microbiota. Colonization with bifidobacteria in early infancy is suggested to be important for health in later life. However, information remains limited regarding the source of these microbes. Here, we investigated whether specific strains of bifidobacteria in the maternal intestinal flora are transmitted to their infant's intestine. MATERIALS AND METHODS: Fecal samples were collected from healthy 17 mother and infant pairs (Vaginal delivery: 12; Cesarean section delivery: 5). Mother's feces were collected twice before delivery. Infant's feces were collected at 0 (meconium), 3, 7, 30, 90 days after birth. Bifidobacteria isolated from feces were genotyped by multilocus sequencing typing, and the transitions of bifidobacteria counts in infant's feces were analyzed by quantitative real-time PCR. RESULTS: Stains belonging to Bifidobacterium adolescentis, Bifidobacterium bifidum, Bifidobacterium catenulatum, Bifidobacterium longum subsp. longum, and Bifidobacterium pseudocatenulatum, were identified to be monophyletic between mother's and infant's intestine. Eleven out of 12 vaginal delivered infants carried at least one monophyletic strain. The bifidobacterial counts of the species to which the monophyletic strains belong, increased predominantly in the infant's intestine within 3 days after birth. Among infants delivered by C-section, monophyletic strains were not observed. Moreover, the bifidobacterial counts were significantly lower than the vaginal delivered infants until 7 days of age. CONCLUSIONS: Among infants born vaginally, several Bifidobacterium strains transmit from the mother and colonize the infant's intestine shortly after birth. Our data suggest that the mother's intestine is an important source for the vaginal delivered infant's intestinal microbiota.

Relevance of pre- and postnatal nutrition to development and interplay between the microbiota and metabolic and immune systems.

Early-life programming is becoming an established concept that states that the environment during early development affects health and disease in adulthood, probably via epigenetic mechanisms such as DNA methylation, histone modifications, RNA silencing, or a combination. Accumulating evidence suggests that nutrition during pregnancy and early postnatal life is one of the most important environmental cues that programs microbiological, metabolic, and immunologic development. The neonatal period is crucial for the early microbial colonization of the almost sterile gastrointestinal tract of the newborn infant. These first colonizers play an important role in host health because they are involved in nutritional, immunologic, and physiologic functions. Evidence from animal and human studies indicates that the composition of the gut microbiota has an effect on body composition, digestion, and metabolic homeostasis. Furthermore, the functionality of the metabolism develops after birth when the newborn is first exposed to nutrition via the gastrointestinal tract. Exposure to environmental microbial components is also suggested to have a key role in the maturation process of the immune system, and in turn the immune system shapes the composition of the microbiota. Therefore, the use of nutritional strategies to program the microbiota composition to favor a more beneficial bacterial population and to support the development of the metabolic and immune systems may provide a good opportunity to prevent later health problems such as obesity, diabetes, and allergy.

Intestinal microbiology in early life: specific prebiotics can have similar functionalities as human-milk oligosaccharides.

Human milk is generally accepted as the best nutrition for newborns and has been shown to support the optimal growth and development of infants. On the basis of scientific insights from human-milk research, a specific mixture of nondigestible oligosaccharides has been developed, with the aim to improve the intestinal microbiota in early life. The mixture has been extensively studied and has been shown to be safe and to have potential health benefits that are similar to those of human milk. The specific mixture of short-chain galacto-oligosaccharides and long-chain fructo-oligosaccharides has been found to affect the development of early microbiota and to increase the Bifidobacterium amounts as observed in human-milk-fed infants. The resulting gut ecophysiology is characterized by high concentrations of lactate, a slightly acidic pH, and specific short-chain fatty acid profiles, which are high in acetate and low in butyrate and propionate. Here, we have summarized the main findings of dietary interventions with these specific oligosaccharides on the gut microbiota in early life. The gut ecophysiology in early life may have consequences for the metabolic, immunologic, and even neurologic development of the child because reports increasingly substantiate the important function of gut microbes in human health. This review highlights major findings in the field of early gut colonization and the potential impact of early nutrition in healthy growth and development.

The fecal bifidobacterial transcriptome of adults: a microarray approach.

Bifidobacteria are a predominant group present among adult human intestinal microbiota and are considered to be beneficial to host health. Both the dynamics and functional activity of bifidobacteria from the intestinal tract of four adults, following ingestion of a mix consisting of short chain galactooligosaccharides, long chain fructooligosaccharides and acidic oligosaccharides from pectin hydrolysate (GFP), was investigated. The percentage of total bifidobacteria, monitored by quantitative real time PCR, was not significantly altered but marked species-specific changes occurred in all individuals over time, indicating a dynamic bifidobacterial community. Insight into the functional activity of the bifidobacteria was acquired using a clone library-based microarray comprising the genomes of various bifidobacteria to reveal the bifidobacterial transcriptome within the fecal community. Total RNA from the fecal microbial community was hybridized to the microarray and 310 clones were selected for sequencing which revealed genes belonging to a wide range of functional groups demonstrating substantial metabolic activity. While the intake of GFP did not have a significant effect on the overall change in gene expression, 82 genes showed a significant change. Most of the predicted genes were involved in metabolism of carbohydrates of plant origin, house keeping functions such as DNA replication and transcription, followed by membrane transport of a wide variety of substrates including sugars and metals and amino acid metabolism. Other genes were involved in transport, nucleotide metabolism, amino acid metabolism, environmental information processing and cellular processes and signalling. A smaller number of genes were involved in general metabolism, glycan metabolism, energy metabolism, lipid metabolism and cell surface. These results support the notion that bifidobacteria utilize mainly indigestible polysaccharides as their main source of energy and biosynthesis of cellular components.

Transmission of intestinal Bifidobacterium longum subsp. longum strains from mother to infant, determined by multilocus sequencing typing and amplified fragment length polymorphism.

The gastrointestinal tracts of neonates are colonized by bacteria immediately after birth. It has been discussed that the intestinal microbiota of neonates includes strains transferred from the mothers. Although some studies have indicated possible bacterial transfer from the mother to the newborn, this is the first report confirming the transfer of bifidobacteria at the strain level. Here, we investigated the mother-to-infant transmission of Bifidobacterium longum subsp. longum by genotyping bacterial isolates from the feces of mothers before delivery and of their infants after delivery. Two hundred seven isolates from 8 pairs of mothers and infants were discriminated by multilocus sequencing typing (MLST) and amplified fragment length polymorphism (AFLP) analysis. By both methods, 11 strains of B. longum subsp. longum were found to be monophyletic for the feces of the mother and her infant. This finding confirms that these strains were transferred from the intestine of the mother to that of the infant. These strains were found in the first feces (meconium) of the infant and in the feces at days 3, 7, 30, and 90 after birth, indicating that they stably colonize the infant's intestine immediately after birth. The strains isolated from each family did not belong to clusters derived from any of the other families, suggesting that each mother-infant pair might have unique family-specific strains.

Bifidobacterium population analysis in the infant gut by direct mapping of genomic hybridization patterns: potential for monitoring temporal development and effects of dietary regimens.

A bifidobacterial mixed-species microarray platform was used in a proof-of-principle study to address the composition and development of bifidobacteria in DNA extracted from faecal samples. These were collected in a time-course of 2 years since birth and derived from human infants that were breastfed, standard formula-fed or received a prebiotic formula during their weaning period. A set of over 50 samples was analysed, testifying for the throughput of the designed platform for multiple genome hybridizations. The generated data revealed that faecal samples of breastfed infants contained a high abundance of genomic DNA homologous to Bifidobacterium breve. In contrast, faecal samples from standard formula-fed infants lacked detectable amounts of this B. breve DNA but contained genes with high similarity to B. longum. Remarkably, infants that received breastmilk and later a prebiotic formula consisting of a standard formula milk containing a mixture of specific galacto- and fructo-oligosaccharides, continued to harbour a B. breve-dominant faecal population. One infant that received standard formula in combination with the additional B. lactis Bb12 culture, contained significant amounts of faecal DNA belonging to Bb12 but only during the period of ingestion. The microarray platform showed sufficient sensitivity to analyse the B. breve group at the strain level. Overall, the B. breve populations observed in the faecal samples of the studied infants showed a stable composition over time and were unique per infant. In conclusion, our results show the applicability of comparative genome hybridization to study bifidobacterial populations in infant faecal samples without the use of any amplification step.

Molecular analysis of faecal and duodenal samples reveals significantly higher prevalence and numbers of Pseudomonas aeruginosa in irritable bowel syndrome.

Intestinal microbiota may play a role in the pathophysiology of irritable bowel syndrome (IBS). In this case-control study, mucosa-associated small intestinal and faecal microbiota of IBS patients and healthy subjects were analysed using molecular-based methods. Duodenal mucosal brush and faecal samples were collected from 37 IBS patients and 20 healthy subjects. The bacterial 16S rRNA gene was amplified and analysed using PCR denaturing gradient gel electrophoresis (DGGE). Pooled average DGGE profiles of all IBS patients and all healthy subjects from both sampling sites were generated and fingerprints of both groups were compared. The DGGE band fragments which were confined to one group were further characterized by sequence analysis. Quantitative real-time PCR (q-PCR) was used to quantify the disease-associated microbiota. Averaged DGGE profiles of both groups were identical for 78.2 % in the small intestinal samples and for 86.25 % in the faecal samples. Cloning and sequencing of the specific bands isolated from small intestinal and faecal DGGE patterns of IBS patients showed that 45.8 % of the clones belonged to the genus Pseudomonas, of which Pseudomonas aeruginosa was the predominant species. q-PCR analysis revealed higher levels (P<0.001) of P. aeruginosa in the small intestine of IBS patients (8.3 %±0.950) than in the small intestine of healthy subjects (0.1 %±0.069). P. aeruginosa was also significantly (P<0.001) more abundant (2.34 %±0.31) in faeces of IBS patients than in faeces of healthy subjects (0.003 %±0.0027). This study shows that P. aeruginosa is detected more frequently and at higher levels in IBS patients than in healthy subjects, suggesting its potential role in the pathophysiology of IBS.

Effect of multifibre mixture with prebiotic components on bifidobacteria and stool pH in tube-fed children.

The objective of the present study was to evaluate the effect of a paediatric tube feed supplemented with a multifibre mixture on the gut microbiota and nutritional and micronutrient status of children on long-term enteral nutrition (EN). A randomised, controlled, double-blind, cross-over trial (2 × 3 months) with a washout period of 1 month was carried out. Twenty-seven children (80% neurologically impaired) aged 11.9 (SD 3.9) years, on long-term EN (4.8 (SD 3.9) years) were recruited. The analyses of the children's faecal pH, microbiota along with anthropometric measures, bowel movements and markers of blood micronutrient status were made. Twenty children completed the study. A significant increase in the proportion of stool bifidobacteria (+16.6%, P < 0.05) was observed during the multifibre period than during the fibre-free period, together with a significant reduction in stool pH (P < 0.001). Stool frequency and consistency as well as growth did not differ between the two periods. There was a significant increase (P < 0.05) in plasma ferritin at the end of the fibre-free period, but plasma ferritin levels remained within normal ranges during both periods. No diet effects on other blood parameters were observed. In conclusion, addition of a multifibre mixture with prebiotic components to paediatric EN is well tolerated, promotes bifidobacteria and reduces stool pH, indicating an improved gut health.

Lower Bifidobacteria counts in both duodenal mucosa-associated and fecal microbiota in irritable bowel syndrome patients.

AIM: To determine the composition of both fecal and duodenal mucosa-associated microbiota in irritable bowel syndrome (IBS) patients and healthy subjects using molecular-based techniques. METHODS: Fecal and duodenal mucosa brush samples were obtained from 41 IBS patients and 26 healthy subjects. Fecal samples were analyzed for the composition of the total microbiota using fluorescent in situ hybridization (FISH) and both fecal and duodenal brush samples were analyzed for the composition of bifidobacteria using real-time polymerase chain reaction. RESULTS: The FISH analysis of fecal samples revealed a 2-fold decrease in the level of bifidobacteria (4.2 +/- 1.3 vs 8.3 +/- 1.9, P < 0.01) in IBS patients compared to healthy subjects, whereas no major differences in other bacterial groups were observed. At the species level, Bifidobacterium catenulatum levels were significantly lower (6 +/- 0.6 vs 19 +/- 2.5, P < 0.001) in the IBS patients in both fecal and duodenal brush samples than in healthy subjects. CONCLUSION: Decreased bifidobacteria levels in both fecal and duodenal brush samples of IBS patients compared to healthy subjects indicate a role for microbiotic composition in IBS pathophysiology.

The Mucin degrader Akkermansia muciniphila is an abundant resident of the human intestinal tract.

A 16S rRNA-targeted probe, MUC-1437, was designed and validated in order to determine the presence and numbers of cells of Akkermansia muciniphila, a mucin degrader, in the human intestinal tract. As determined by fluorescent in situ hybridization, A. muciniphila accounted more than 1% of the total fecal cells and was shown to be a common bacterial component of the human intestinal tract.

Early impairment of gut function and gut flora supporting a role for alteration of gastrointestinal mucosa in human immunodeficiency virus pathogenesis.

Our results show that impairment of the gastrointestinal tracts in human immunodeficiency virus (HIV)-positive patients is present in the early phases of HIV disease. This impairment is associated with alterations in gut microbiota and intestinal inflammatory parameters. These findings support the hypothesis that alterations at the gastrointestinal-tract level are a key factor in HIV pathogenesis.

Advanced molecular tools for the identification of lactic acid bacteria.

Recent years have seen an explosion in the development and application of molecular tools for identifying microbes and analyzing their activity. These tools are increasingly applied to strains of lactic acid bacteria (LAB), including those used in fermentation and as well as those marketed as probiotics, for identification and analysis of their activity. Many of these tools are based on 16S ribosomal DNA sequences and exploit either hybridization or PCR techniques. Furthermore, complete or partial genomes of various LAB and bifidobacteria have been determined and offer omics-based approaches to analyze the activity of the bacteria provided that the mechanisms of their action are known. Finally, fluorescent probes coupled to flow cytometry are used to monitor the physiological capacity of bacterial cells in situ. All these approaches can be used for the screening and selection of LAB, assessing their role in fermentation and flavor development in fermented products. Additional aspects of probiotic LAB include their viability and vitality during processing and analysis of their presence, persistence, and performance in the gastrointestinal tract. An overview of these approaches is provided, and specific examples of their application to lactic cultures are presented. Because of their abundant use in tracing and tracking of LAB, a complete listing of 16S ribosomal RNA probes for lactobacilli and bifidobacteria is provided.

Genetic diversity of viable, injured, and dead fecal bacteria assessed by fluorescence-activated cell sorting and 16S rRNA gene analysis.

A novel approach combining a flow cytometric in situ viability assay with 16S rRNA gene analysis was used to study the relationship between diversity and activity of the fecal microbiota. Simultaneous staining with propidium iodide (PI) and SYTO BC provided clear discrimination between intact cells (49%), injured or damaged cells (19%), and dead cells (32%). The three subpopulations were sorted and characterized by denaturing gradient gel electrophoresis (DGGE) of 16S rRNA gene amplicons obtained from the total and bifidobacterial communities. This analysis revealed that not only the total community but also the distinct subpopulations are characteristic for each individual. Cloning and sequencing of the dominant bands of the DGGE patterns showed that most of clones retrieved from the live, injured, and dead fractions belonged to Clostridium coccoides, Clostridium leptum, and Bacteroides. We found that some of the butyrate-producing related bacteria, such as Eubacterium rectale and Eubacterium hallii, were obviously viable at the time of sampling. However, amplicons affiliated with Bacteroides and Ruminococcus obeum- and Eubacterium biforme-like bacteria, as well as Butyrivibrio crossotus, were obtained especially from the dead population. Furthermore, some bacterial clones were recovered from all sorted fractions, and this was especially noticeable for the Clostridium leptum cluster. The bifidobacterial phylotypes identified in total samples and sorted fractions were assigned to Bifidobacterium adolescentis, Bifidobacterium longum, Bifidobacterium infantis, Bifidobacterium pseudocatenulatum, and Bifidobacterium bifidum. Phylogenetic analysis of the live, dead, and injured cells revealed a remarkable physiological heterogeneity within these bacterial populations; B. longum and B. infantis were retrieved from all sorted fractions, while B. adolescentis was recovered mostly from the sorted dead fraction.