Paul W. O'Toole

Composition, variability, and temporal stability of the intestinal microbiota of the elderly

Alterations in the human intestinal microbiota are linked to conditions including inflammatory bowel disease, irritable bowel syndrome, and obesity. The microbiota also undergoes substantial changes at the extremes of life, in infants and older people, the ramifications of which are still being explored. We applied pyrosequencing of over 40,000 16S rRNA gene V4 region amplicons per subject to characterize the fecal microbiota in 161 subjects aged 65 y and older and 9 younger control subjects. The microbiota of each individual subject constituted a unique profile that was separable from all others. In 68% of the individuals, the microbiota was dominated by phylum Bacteroides, with an average proportion of 57% across all 161 baseline samples. Phylum Firmicutes had an average proportion of 40%. The proportions of some phyla and genera associated with disease or health also varied dramatically, including Proteobacteria, Actinobacteria, and Faecalibacteria. The core microbiota of elderly subjects was distinct from that previously established for younger adults, with a greater proportion of Bacteroides spp. and distinct abundance patterns of Clostridium groups. Analyses of 26 fecal microbiota datasets from 3-month follow-up samples indicated that in 85% of the subjects, the microbiota composition was more like the corresponding time-0 sample than any other dataset. We conclude that the fecal microbiota of the elderly shows temporal stability over limited time in the majority of subjects but is characterized by unusual phylum proportions and extreme variability.

Bacteriocin production as a mechanism for the antiinfective activity of Lactobacillus salivarius UCC118.

The mechanisms by which probiotic strains enhance the health of the host remain largely uncharacterized. Here we demonstrate that Lactobacillus salivarius UCC118, a recently sequenced and genetically tractable probiotic strain of human origin, produces a bacteriocin in vivo that can significantly protect mice against infection with the invasive foodborne pathogen Listeria monocytogenes. A stable mutant of Lb. salivarius UCC118 that is unable to produce the Abp118 bacteriocin also failed to protect mice against infection with two strains of L. monocytogenes, EGDe and LO28, confirming that bacteriocin production is the primary mediator of protection against this organism. Furthermore, Lb. salivarius UCC118 did not offer any protection when mice were infected with a strain of L. monocytogenes expressing the cognate Abp118 immunity protein AbpIM, confirming that the antimicrobial effect is a result of direct antagonism between Lb. salivarius and the pathogen, mediated by the bacteriocin Abp118.

Comparative analysis of pyrosequencing and a phylogenetic microarray for exploring microbial community structures in the human distal intestine

Background Variations in the composition of the human intestinal microbiota are linked to diverse health conditions. High-throughput molecular technologies have recently elucidated microbial community structure at much higher resolution than was previously possible. Here we compare two such methods, pyrosequencing and a phylogenetic array, and evaluate classifications based on two variable 16S rRNA gene regions. Methods and Findings Over 1.75 million amplicon sequences were generated from the V4 and V6 regions of 16S rRNA genes in bacterial DNA extracted from four fecal samples of elderly individuals. The phylotype richness, for individual samples, was 1,400–1,800 for V4 reads and 12,500 for V6 reads, and 5,200 unique phylotypes when combining V4 reads from all samples. The RDP-classifier was more efficient for the V4 than for the far less conserved and shorter V6 region, but differences in community structure also affected efficiency. Even when analyzing only 20% of the reads, the majority of the microbial diversity was captured in two samples tested. DNA from the four samples was hybridized against the Human Intestinal Tract (HIT) Chip, a phylogenetic microarray for community profiling. Comparison of clustering of genus counts from pyrosequencing and HITChip data revealed highly similar profiles. Furthermore, correlations of sequence abundance and hybridization signal intensities were very high for lower-order ranks, but lower at family-level, which was probably due to ambiguous taxonomic groupings. Conclusions The RDP-classifier consistently assigned most V4 sequences from human intestinal samples down to genus-level with good accuracy and speed. This is the deepest sequencing of single gastrointestinal samples reported to date, but microbial richness levels have still not leveled out. A majority of these diversities can also be captured with five times lower sampling-depth. HITChip hybridizations and resulting community profiles correlate well with pyrosequencing-based compositions, especially for lower-order ranks, indicating high robustness of both approaches. However, incompatible grouping schemes make exact comparison difficult.

Comparison of two next-generation sequencing technologies for resolving highly complex microbiota composition using tandem variable 16S rRNA gene regions

High-throughput molecular technologies can profile microbial communities at high resolution even in complex environments like the intestinal microbiota. Recent improvements in next-generation sequencing technologies allow for even finer resolution. We compared phylogenetic profiling of both longer (454 Titanium) sequence reads with shorter, but more numerous, paired-end reads (Illumina). For both approaches, we targeted six tandem combinations of 16S rRNA gene variable regions, in microbial DNA extracted from a human faecal sample, in order to investigate their limitations and potentials. In silico evaluations predicted that the V3/V4 and V4/V5 regions would provide the highest classification accuracies for both technologies. However, experimental sequencing of the V3/V4 region revealed significant amplification bias compared to the other regions, emphasising the necessity for experimental validation of primer pairs. The latest developments of 454 and Illumina technologies offered higher resolution compared to their previous versions, and showed relative consistency with each other. However, the majority of the Illumina reads could not be classified down to genus level due to their shorter length and higher error rates beyond 60 nt. Nonetheless, with improved quality and longer reads, the far greater coverage of Illumina promises unparalleled insights into highly diverse and complex environments such as the human gut.

Composition and energy harvesting capacity of the gut microbiota: relationship to diet, obesity and time in mouse models

Background and Aims Increased efficiency of energy harvest, due to alterations in the gut microbiota (increased Firmicutes and decreased Bacteroidetes), has been implicated in obesity in mice and humans. However, a causal relationship is unproven and contributory variables include diet, genetics and age. Therefore, we explored the effect of a high-fat (HF) diet and genetically determined obesity (ob/ob) for changes in microbiota and energy harvesting capacity over time. Methods Seven-week-old male ob/ob mice were fed a low-fat diet and wild-type mice were fed either a low-fat diet or a HF-diet for 8 weeks (n=8/group). They were assessed at 7, 11 and 15 weeks of age for: fat and lean body mass (by NMR); faecal and caecal short-chain fatty acids (SCFA, by gas chromatography); faecal energy content (by bomb calorimetry) and microbial composition (by metagenomic pyrosequencing). Results A progressive increase in Firmicutes was confirmed in both HF-fed and ob/ob mice reaching statistical significance in the former, but this phylum was unchanged over time in the lean controls. Reductions in Bacteroidetes were also found in ob/ob mice. However, changes in the microbiota were dissociated from markers of energy harvest. Thus, although the faecal energy in the ob/ob mice was significantly decreased at 7 weeks, and caecal SCFA increased, these did not persist and faecal acetate diminished over time in both ob/ob and HF-fed mice, but not in lean controls. Furthermore, the proportion of the major phyla did not correlate with energy harvest markers. Conclusion The relationship between the microbial composition and energy harvesting capacity is more complex than previously considered. While compositional changes in the faecal microbiota were confirmed, this was primarily a feature of high-fat feeding rather than genetically induced obesity. In addition, changes in the proportions of the major phyla were unrelated to markers of energy harvest which changed over time. The possibility of microbial adaptation to diet and time should be considered in future studies.

An irritable bowel syndrome subtype defined by species-specific alterations in faecal microbiota

Background and aims Irritable bowel syndrome (IBS) is a common functional gastrointestinal disorder that may be triggered by enteric pathogens and has also been linked to alterations in the microbiota and the host immune response. The authors performed a detailed analysis of the faecal microbiota in IBS and control subjects and correlated the findings with key clinical and physiological parameters. Design The authors used pyrosequencing to determine faecal microbiota composition in 37 IBS patients (mean age 37 years; 26 female subjects; 15 diarrhoea-predominant IBS, 10 constipation-predominant IBS and 12 alternating-type IBS) and 20 age- and gender-matched controls. Gastrointestinal and psychological symptom severity and quality of life were evaluated with validated questionnaires and colonic transit time and rectal sensitivity were measured. Results Associations detected between microbiota composition and clinical or physiological phenotypes included microbial signatures associated with colonic transit and levels of clinically significant depression in the disease. Clustering by microbiota composition revealed subgroups of IBS patients, one of which (n=15) showed normal-like microbiota composition compared with healthy controls. The other IBS samples (n=22) were defined by large microbiota-wide changes characterised by an increase of Firmicutes-associated taxa and a depletion of Bacteroidetes-related taxa. Conclusions Detailed microbiota analysis of a well-characterised cohort of IBS patients identified several clear associations with clinical data and a distinct subset of IBS patients with alterations in their microbiota that did not correspond to IBS subtypes, as defined by the Rome II criteria.

Genome-scale analyses of health-promoting bacteria: probiogenomics

The human body is colonized by an enormous population of bacteria (microbiota) that provides the host with coding capacity and metabolic activities. Among the human gut microbiota are health-promoting indigenous species (probiotic bacteria) that are commonly consumed as live dietary supplements. Recent genomics-based studies (probiogenomics) are starting to provide insights into how probiotic bacteria sense and adapt to the gastrointestinal tract environment. In this Review, we discuss the application of probiogenomics in the elucidation of the molecular basis of probiosis using the well-recognized model probiotic bacteria genera Bifidobacterium and Lactobacillus as examples.

γ‐Aminobutyric acid production by culturable bacteria from the human intestine

To assess the ability of human intestinally derived strains of Lactobacillus and Bifidobacterium to produce γ‐aminobutyric acid (GABA).

Exercise and associated dietary extremes impact on gut microbial diversity

Objective The commensal microbiota, host immunity and metabolism participate in a signalling network, with diet influencing each component of this triad. In addition to diet, many elements of a modern lifestyle influence the gut microbiota but the degree to which exercise affects this population is unclear. Therefore, we explored exercise and diet for their impact on the gut microbiota. Design Since extremes of exercise often accompany extremes of diet, we addressed the issue by studying professional athletes from an international rugby union squad. Two groups were included to control for physical size, age and gender. Compositional analysis of the microbiota was explored by 16S rRNA amplicon sequencing. Each participant completed a detailed food frequency questionnaire. Results As expected, athletes and controls differed significantly with respect to plasma creatine kinase (a marker of extreme exercise), and inflammatory and metabolic markers. More importantly, athletes had a higher diversity of gut micro-organisms, representing 22 distinct phyla, which in turn positively correlated with protein consumption and creatine kinase. Conclusions The results provide evidence for a beneficial impact of exercise on gut microbiota diversity but also indicate that the relationship is complex and is related to accompanying dietary extremes.

Functional genome analysis of Bifidobacterium breve UCC2003 reveals type IVb tight adherence (Tad) pili as an essential and conserved host-colonization factor

Development of the human gut microbiota commences at birth, with bifidobacteria being among the first colonizers of the sterile newborn gastrointestinal tract. To date, the genetic basis of Bifidobacterium colonization and persistence remains poorly understood. Transcriptome analysis of the Bifidobacterium breve UCC2003 2.42-Mb genome in a murine colonization model revealed differential expression of a type IVb tight adherence (Tad) pilus-encoding gene cluster designated “tad2003.” Mutational analysis demonstrated that the tad2003 gene cluster is essential for efficient in vivo murine gut colonization, and immunogold transmission electron microscopy confirmed the presence of Tad pili at the poles of B. breve UCC2003 cells. Conservation of the Tad pilus-encoding locus among other B. breve strains and among sequenced Bifidobacterium genomes supports the notion of a ubiquitous pili-mediated host colonization and persistence mechanism for bifidobacteria.