Hans G.H.J. Heilig

Molecular Diversity of Lactobacillus spp. and Other Lactic Acid Bacteria in the Human Intestine as Determined by Specific Amplification of 16S Ribosomal DNA

ABSTRACT A Lactobacillus group-specific PCR primer, S-G-Lab-0677-a-A-17, was developed to selectively amplify 16S ribosomal DNA (rDNA) from lactobacilli and related lactic acid bacteria, including members of the genera Leuconostoc, Pediococcus, and Weissella. Amplicons generated by PCR from a variety of gastrointestinal (GI) tract samples, including those originating from feces and cecum, resulted predominantly in Lactobacillus-like sequences, of which ca. 28% were most similar to the 16S rDNA of Lactobacillus ruminis. Moreover, four sequences of Leuconostoc species were retrieved that, so far, have only been detected in environments other than the GI tract, such as fermented food products. The validity of the primer was further demonstrated by using Lactobacillus-specific PCR and denaturing gradient gel electrophoresis (DGGE) of the 16S rDNA amplicons of fecal and cecal origin from different age groups. The stability of the GI-tract bacterial community in different age groups over various time periods was studied. The Lactobacillus community in three adults over a 2-year period showed variation in composition and stability depending on the individual, while successional change of the Lactobacillus community was observed during the first 5 months of an infant’s life. Furthermore, the specific PCR and DGGE approach was tested to study the retention in fecal samples of a Lactobacillus strain administered during a clinical trial. In conclusion, the combination of specific PCR and DGGE analysis of 16S rDNA amplicons allows the diversity of important groups of bacteria that are present in low numbers in specific ecosystems to be characterized, such as the lactobacilli in the human GI tract.

Global and Deep Molecular Analysis of Microbiota Signatures in Fecal Samples From Patients With Irritable Bowel Syndrome

BACKGROUND & AIMS Irritable bowel syndrome (IBS) has been associated with disruptions to the intestinal microbiota, but studies have had limited power, coverage, and depth of analysis. We aimed to define microbial populations that can be used discriminate the fecal microbiota of patients with IBS from that of healthy subjects and correlate these with IBS intestinal symptom scores. METHODS The microbiota composition was assessed by global and deep molecular analysis of fecal samples from 62 patients with IBS patients and 46 healthy individuals (controls). We used a comprehensive and highly reproducible phylogenetic microarray in combination with quantitative polymerase chain reaction. RESULTS The intestinal microbiota of IBS patients differed significantly (P = .0005) from that of controls. The microbiota of patients, compared with controls, had a 2-fold increased ratio of the Firmicutes to Bacteroidetes (P = .0002). This resulted from an approximately 1.5-fold increase in numbers of Dorea, Ruminococcus, and Clostridium spp (P < .005); a 2-fold decrease in the number of Bacteroidetes (P < .0001); a 1.5-fold decrease in numbers of Bifidobacterium and Faecalibacterium spp (P < .05); and, when present, a 4-fold lower average number of methanogens (3.50 × 10(7) vs 8.74 × 10(6) cells/g feces; P = .003). Correlation analysis of the microbial groups and IBS symptom scores indicated the involvement of several groups of Firmicutes and Proteobacteria in the pathogenesis of IBS. CONCLUSIONS Global and deep molecular analysis of fecal samples indicates that patients with IBS have a different composition of microbiota. This information might be used to develop better diagnostics and ultimately treatments for IBS.

Development and application of the human intestinal tract chip, a phylogenetic microarray: analysis of universally conserved phylotypes in the abundant microbiota of young and elderly adults

In this paper we present the in silico assessment of the diversity of variable regions of the small subunit ribosomal RNA (SSU rRNA) gene based on an ecosystem-specific curated database, describe a probe design procedure based on two hypervariable regions with minimal redundancy and test the potential of such probe design strategy for the design of a flexible microarray platform. This resulted in the development and application of a phylogenetic microarray for studying the human gastrointestinal microbiota – referred as the human intestinal tract chip (HITChip). Over 4800 dedicated tiling oligonucleotide probes were designed based on two hypervariable regions of the SSU rRNA gene of 1140 unique microbial phylotypes (< 98% identity) following analysis of over 16 000 human intestinal SSU rRNA sequences. These HITChip probes were hybridized to a diverse set of human intestinal samples and SSU rRNA clones to validate its fingerprinting and quantification potential. Excellent reproducibility (median Pearsons correlation of 0.99) was obtained following hybridization with T7 polymerase transcripts generated in vitro from SSU rRNA gene amplicons. A linear dose–response was observed with artificial mixtures of 40 different representative amplicons with relative abundances as low as 0.1% of total microbiota. Analysis of three consecutively collected faecal samples from ten individuals (five young and five elderly adults) revealed temporal dynamics and confirmed that the adult intestinal microbiota is an individual-specific and relatively stable ecosystem. Further analysis of the stable part allowed for the identification of a universal microbiota core at the approximate genus level (90% sequence similarity). This core consists of members of Actinobacteria, Bacteroidetes and Firmicutes. Used as a phylogenetic fingerprinting tool with the possibility for relative quantification, the HITChip has the potential to bridge the gaps in our knowledge in the quantitative and qualitative description of the human gastrointestinal microbiota composition.

Isolation of Bifidobacteria from Breast Milk and Assessment of the Bifidobacterial Population by PCR-Denaturing Gradient Gel Electrophoresis and Quantitative Real-Time PCR

ABSTRACT The objective of this work was to elucidate if breast milk contains bifidobacteria and whether they can be transmitted to the infant gut through breastfeeding. Twenty-three women and their respective infants provided samples of breast milk and feces, respectively, at days 4 to 7 after birth. Gram-positive and catalase-negative isolates from specific media with typical bifidobacterial shapes were identified to the genus level by F6PPK (fructose-6-phosphate phosphoketolase) assays and to the species level by 16S rRNA gene sequencing. Bifidobacterial communities in breast milk were assessed by PCR-denaturing gradient gel electrophoresis (PCR-DGGE), and their levels were estimated by quantitative real-time PCR (qRTi-PCR). Bifidobacteria were present in 8 milk samples and 21 fecal samples. Bifidobacterium breve, B. adolescentis, and B. bifidum were isolated from milk samples, while infant feces also contained B. longum and B. pseudocatenulatum. PCR-DGGE revealed the presence of one to four dominant bifidobacterial bands in 22 milk samples. Sequences with similarities above 98% were identified as Bifidobacterium breve, B. adolescentis, B. longum, B. bifidum, and B. dentium. Bifidobacterial DNA was detected by qRTi-PCR in the same 22 milk samples at a range between 40 and 10,000 16S rRNA gene copies per ml. In conclusion, human milk seems to be a source of living bifidobacteria for the infant gut.

Bacterial Diversity in Meconium of Preterm Neonates and Evolution of Their Fecal Microbiota during the First Month of Life

The establishment and succession of bacterial communities in infants may have a profound impact in their health, but information about the composition of meconium microbiota and its evolution in hospitalized preterm infants is scarce. In this context, the objective of this work was to characterize the microbiota of meconium and fecal samples obtained during the first 3 weeks of life from 14 donors using culture and molecular techniques, including DGGE and the Human Intestinal Tract Chip (HITChip) analysis of 16S rRNA amplicons. Culture techniques offer a quantification of cultivable bacteria and allow further study of the isolate, while molecular techniques provide deeper information on bacterial diversity. Culture and HITChip results were very similar but the former showed lower sensitivity. Inter-individual differences were detected in the microbiota profiles although the meconium microbiota was peculiar and distinct from that of fecal samples. Bacilli and other Firmicutes were the main bacteria groups detected in meconium while Proteobacteria dominated in the fecal samples. Culture technique showed that Staphylococcus predominated in meconium and that Enterococcus, together with Gram-negative bacteria such as Escherichia coli, Escherichia fergusonii, Klebsiella pneumoniae and Serratia marcescens, was more abundant in fecal samples. In addition, HITChip results showed the prevalence of bacteria related to Lactobacillus plantarum and Streptococcus mitis in meconium samples whereas those related to Enterococcus, Escherichia coli, Klebsiella pneumoniae and Yersinia predominated in the 3rd week feces. This study highlights that spontaneously-released meconium of preterm neonates contains a specific microbiota that differs from that of feces obtained after the first week of life. Our findings indicate that the presence of Serratia was strongly associated with a higher degree of immaturity and other hospital-related parameters, including antibiotherapy and mechanical ventilation.

High temporal and inter‐individual variation detected in the human ileal microbiota

The diversity and temporal stability of the predominant bacteria in the human ileum was studied with the use of ileal effluent samples of seven individuals with Brooke ileostomies. The total number of bacteria within the ileal effluent was in the range of 10⁷ -10⁸ bacteria per gram (wet weight). The diversity of the bacteria in the ileal effluent showed marked differences compared with that in faecal samples from age-matched healthy adults. The ileal effluent had a higher relative abundance of species within the orders Lactobacillales and Clostridiales, mainly Streptococcus bovis-related species, and the Veillonella group, and a lower proportion of species related to Ruminococcus gnavus, R. obeum and Bacteroides plebeius. In addition, inter-individual differences were found, indicative of a highly personal ileal microbiota profile. Furthermore, temporal profiles showed large fluctuations per individual over a period of 9-28 days (average similarity over a period of 9 days was as low as 44%), and differences between morning and afternoon profiles were observed. Parallel cloning and sequencing efforts revealed several phylotypes that were not identified in previous studies (12 out of 65 phylotypes showed less than 97% sequence similarity with previously reported sequences). Achaea were found to be below detection limit by quantitative PCR. Overall, the results indicate that the microbiota of the human ileum is relatively unstable, less complex and consisting of different dominating phylotypes when compared with the colonic microbiota.

Isolation of DNA from bacterial samples of the human gastrointestinal tract.

The human gastrointestinal (GI) tract contains a complex microbial community that develops in time and space. The most widely used approaches to study microbial diversity and activity are all based on the analysis of nucleic acids, DNA, rRNA and mRNA. Here, we present a DNA isolation protocol that is suitable for a wide variety of GI tract samples, including biopsies with minute amounts of material. The protocol is set up in such a way that sampling can be performed outside the laboratory, which offers possibilities for implementation in large intervention studies. The DNA isolation is based on mechanical disruption, followed by isolation of nucleic acids using phenol:chloroform:isoamylalcohol extraction. In addition, it includes an alternative DNA isolation protocol that is based on a commercial kit. These protocols have all been successfully used in our laboratory, resulting in isolation of DNA of sufficient quality for microbial diversity studies. Depending on the number of samples and sample type, the whole procedure will take approximately 2.5–4 hours.

Genetic Diversity of Viable, Injured, and Dead Fecal Bacteria Assessed by Fluorescence-Activated Cell Sorting and 16S rRNA Gene Analysis

ABSTRACT 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.

Long‐term monitoring of the human intestinal microbiota composition

The microbiota that colonizes the human intestinal tract is complex and its structure is specific for each of us. In this study we expand the knowledge about the stability of the subject-specific microbiota and show that this ecosystem is stable in short-term intervals (< 1 year) but also during long periods of time (> 10 years). The faecal microbiota composition of five unrelated and healthy subjects was analysed using a comprehensive and highly reproducible phylogenetic microarray, the HITChip. The results show that the use of antibiotics, application of specific dietary regimes and distant travelling have limited impact on the microbiota composition. Several anaerobic genera, including Bifidobacterium and a number of genera within the Bacteroidetes and the Firmicutes phylum, exhibit significantly higher similarity than the total microbiota. Although the gut microbiota contains subject-specific species, the presence of which is preserved throughout the years, their relative abundance changes considerably. Consequently, the recently proposed enterotype status appears to be a varying characteristic of the microbiota. Our data show that the intestinal microbiota contains a core community of permanent colonizers, and that environmentally introduced changes of the microbiota throughout adulthood are primarily affecting the abundance but not the presence of specific microbial species.

The microbial eukaryote Blastocystis is a prevalent and diverse member of the healthy human gut microbiota

To date, the majority of research into the human gut microbiota has focused on the bacterial fraction of the community. Inevitably, this has resulted in a poor understanding of the diversity and functionality of other intestinal microorganisms in the human gut. One such nonbacterial member is the microbial eukaryote Blastocystis, which has been implicated in the aetiology of a range of different intestinal and extra-intestinal diseases. However, prevalence data from different studies are conflicting, and crucially, there is limited information on its incidence and diversity in healthy individuals. Here, we survey the prevalence, genetic diversity and temporal stability of Blastocystis in a group of healthy adults (n = 105) using a sensitive PCR assay. Blastocystis was present in 56% of our sample set, which is much higher than previously reported from an industrialised county (Ireland). Moreover, a diversity of different subtypes (species) were detected, and Blastocystis was present in a subset of individuals sampled over a period of time between 6 and 10 years, indicating that it is capable of long-term host colonisation. These results show that Blastocystis is a common and diverse member of the healthy gut microbiota, thereby extending our knowledge of the microbial ecology of the healthy human intestine.