Riina A. Kekkonen

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.

Comparative analysis of fecal DNA extraction methods with phylogenetic microarray: Effective recovery of bacterial and archaeal DNA using mechanical cell lysis

Several different protocols are used for fecal DNA extraction, which is an integral step in all phylogenetic and metagenomic approaches to characterize the highly diverse intestinal ecosystem. We compared four widely used methods, and found their DNA yields to vary up to 35-fold. Bacterial, archaeal and human DNA was quantified by real-time PCR, and a compositional analysis of different extracts was carried out using the Human Intestinal Tract Chip, a 16S rRNA gene-based phylogenetic microarray. The overall microbiota composition was highly similar between the methods in contrast to the profound differences between the subjects (Pearson correlations >0.899 and 0.735, respectively). A detailed comparative analysis of mechanical and enzymatic methods showed that despite their overall similarity, the mechanical cell disruption by repeated bead beating showed the highest bacterial diversity and resulted in significantly improved DNA extraction efficiency of archaea and some bacteria, including Clostridium cluster IV. By applying the mechanical disruption method a high prevalence (67%) of methanogenic archaea was detected in healthy subjects (n=24), exceeding the typical values reported previously. The assessment of performance differences between different methodologies serves as a concrete step towards the comparison and reliable meta-analysis of the results obtained in different laboratories.

Intestinal Microbiota in Healthy Adults: Temporal Analysis Reveals Individual and Common Core and Relation to Intestinal Symptoms

Background While our knowledge of the intestinal microbiota during disease is accumulating, basic information of the microbiota in healthy subjects is still scarce. The aim of this study was to characterize the intestinal microbiota of healthy adults and specifically address its temporal stability, core microbiota and relation with intestinal symptoms. We carried out a longitudinal study by following a set of 15 healthy Finnish subjects for seven weeks and regularly assessed their intestinal bacteria and archaea with the Human Intestinal Tract (HIT)Chip, a phylogenetic microarray, in conjunction with qPCR analyses. The health perception and occurrence of intestinal symptoms was recorded by questionnaire at each sampling point. Principal Findings A high overall temporal stability of the microbiota was observed. Five subjects showed transient microbiota destabilization, which correlated not only with the intake of antibiotics but also with overseas travelling and temporary illness, expanding the hitherto known factors affecting the intestinal microbiota. We identified significant correlations between the microbiota and common intestinal symptoms, including abdominal pain and bloating. The most striking finding was the inverse correlation between Bifidobacteria and abdominal pain: subjects who experienced pain had over five-fold less Bifidobacteria compared to those without pain. Finally, a novel computational approach was used to define the common core microbiota, highlighting the role of the analysis depth in finding the phylogenetic core and estimating its size. The in-depth analysis suggested that we share a substantial number of our intestinal phylotypes but as they represent highly variable proportions of the total community, many of them often remain undetected. Conclusions/Significance A global and high-resolution microbiota analysis was carried out to determine the temporal stability, the associations with intestinal symptoms, and the individual and common core microbiota in healthy adults. The findings provide new approaches to define intestinal health and to further characterize the microbial communities inhabiting the human gut.

Intestinal microbiome is related to lifetime antibiotic use in Finnish pre-school children

Early-life antibiotic use is associated with increased risk for metabolic and immunological diseases, and mouse studies indicate a causal role of the disrupted microbiome. However, little is known about the impacts of antibiotics on the developing microbiome of children. Here we use phylogenetics, metagenomics and individual antibiotic purchase records to show that macrolide use in 2–7 year-old Finnish children (N=142; sampled at two time points) is associated with a long-lasting shift in microbiota composition and metabolism. The shift includes depletion of Actinobacteria, increase in Bacteroidetes and Proteobacteria, decrease in bile-salt hydrolase and increase in macrolide resistance. Furthermore, macrolide use in early life is associated with increased risk of asthma and predisposes to antibiotic-associated weight gain. Overweight and asthmatic children have distinct microbiota compositions. Penicillins leave a weaker mark on the microbiota than macrolides. Our results support the idea that, without compromising clinical practice, the impact on the intestinal microbiota should be considered when prescribing antibiotics.

Associations between the human intestinal microbiota, Lactobacillus rhamnosus GG and serum lipids indicated by integrated analysis of high-throughput profiling data

Accumulating evidence indicates that the intestinal microbiota regulates our physiology and metabolism. Bacteria marketed as probiotics confer health benefits that may arise from their ability to affect the microbiota. Here high-throughput screening of the intestinal microbiota was carried out and integrated with serum lipidomic profiling data to study the impact of probiotic intervention on the intestinal ecosystem, and to explore the associations between the intestinal bacteria and serum lipids. We performed a comprehensive intestinal microbiota analysis using a phylogenetic microarray before and after Lactobacillus rhamnosus GG intervention. While a specific increase in the L. rhamnosus-related bacteria was observed during the intervention, no other changes in the composition or stability of the microbiota were detected. After the intervention, lactobacilli returned to their initial levels. As previously reported, also the serum lipid profiles remained unaltered during the intervention. Based on a high-resolution microbiota analysis, intake of L. rhamnosus GG did not modify the composition of the intestinal ecosystem in healthy adults, indicating that probiotics confer their health effects by other mechanisms. The most prevailing association between the gut microbiota and lipid profiles was a strong positive correlation between uncultured phylotypes of Ruminococcus gnavus-group and polyunsaturated serum triglycerides of dietary origin. Moreover, a positive correlation was detected between serum cholesterol and Collinsella (Coriobacteriaceae). These associations identified with the spectrometric lipidome profiling were corroborated by enzymatically determined cholesterol and triglyceride levels. Actinomycetaceae correlated negatively with triglycerides of highly unsaturated fatty acids while a set of Proteobacteria showed negative correlation with ether phosphatidylcholines. Our results suggest that several members of the Firmicutes, Actinobacteria and Proteobacteria may be involved in the metabolism of dietary and endogenous lipids, and provide a scientific rationale for further human studies to explore the role of intestinal microbes in host lipid metabolism.

Nutritional modulation of exercise-induced immunodepression in athletes: a systematic review and meta-analysis

Background:Heavy exercise induces marked immunodepression that is multifactorial in origin. Nutrition can modulate normal immune function.Objective:To assess the efficacy of nutritional supplements in exercise-induced immunodepression in athletes.Design:Systematic review.Review methods:Randomised and/or controlled trials of athletes undertaking nutritional supplements to minimise the immunodepression after exercise were retrieved. The primary outcome measure was incidence of upper respiratory tract (URT) illness symptoms after exercise, and secondary outcomes included cortisol, cell counts, plasma cytokine concentration, cell proliferative response, oxidative burst, natural killer cell activity and immunoglobulins. When data were available for a pooled estimate of the effect of intervention, meta-analyses were conducted for direct comparisons.Results:Forty-five studies were included (1603 subjects). The studies were heterogeneous in terms of exercise interventions, selection of athletes, settings and outcomes. The overall methodological quality of most of the trials was poor. Twenty studies addressed carbohydrate supplementation, eight glutamine, 13 vitamin C and four others interventions. Three trials assessed the effect of intervention on prevention of URT infections. The pooled rate ratio for URT infections after vitamin C supplementation against placebo was 0.49 (0.34–0.71). Carbohydrate supplementation attenuated the increase in cortisol and neutrophils after exercise; vitamin C attenuated the decrease in lymphocytes after exercise. No other interventions had significant or consistent effect on any of the studied outcomes.Conclusions:Although the prevention of URT infections by vitamin C was supported by two trials, further studies are needed. The available evidence failed to support a role for other nutritional supplements in preventing exercise-induced immune suppression. Larger trials with clinically relevant and uniform end points are necessary to clarify the role of these nutritional interventions.

Milk containing probiotic Lactobacillus rhamnosus GG and respiratory illness in children: a randomized, double-blind, placebo-controlled trial

Background/Objectives:To determine whether long-term daily consumption of milk containing probiotic Lactobacillus rhamnosus GG (GG) decreases respiratory illness in children.Subjects/Methods:A randomized, double-blind, placebo-controlled trial was conducted with 523 children aged 2–6 years attending day care centers in Finland. Subjects received either normal milk or the same milk with GG on three daily meals for 28 weeks. Daily recording of childrens’ symptoms was done by parents. Primary outcome data from 501 subjects were available for analysis, and data from 128 subjects were analyzed as completed cases in terms of recovery of GG in fecal samples.Results:Number of days with at least one respiratory symptom in all subjects was 5.03/month (95% confidence interval (CI): 4.92–5.15) in the GG group and 5.17/month (95% CI: 5.05–5.29) in the placebo group incidence rate ratio (IRR) 0.97; 95% CI: 0.94–1.00; P=0.098). In the completed cases, the figures were 4.71 days/month (95% CI: 4.52–4.90) in the GG group and 5.67 days/month (95% CI: 5.40–5.94) in the placebo group (IRR 0.83; 95% CI: 0.78–0.88; P<0.001).Conclusions:Consumption of GG reduced the occurence of respiratory illness in children attending day care centers in the completed cases subgroup, but not in the total population. Thus, future clinical trials are warranted to clarify the association between fecal recovery of a probiotic and the symptom prevalence.

Probiotic Lactobacillus rhamnosus downregulates FCER1 and HRH4 expression in human mast cells

AIM To investigate the effects of four probiotic bacteria and their combination on human mast cell gene expression using microarray analysis. METHODS Human peripheral-blood-derived mast cells were stimulated with Lactobacillus rhamnosus (L. rhamnosus) GG (LGG(®)), L. rhamnosus Lc705 (Lc705), Propionibacterium freudenreichii ssp. shermanii JS (PJS) and Bifidobacterium animalis ssp. lactis Bb12 (Bb12) and their combination for 3 or 24 h, and were subjected to global microarray analysis using an Affymetrix GeneChip(®) Human Genome U133 Plus 2.0 Array. The gene expression differences between unstimulated and bacteria-stimulated samples were further analyzed with GOrilla Gene Enrichment Analysis and Visualization Tool and MeV Multiexperiment Viewer-tool. RESULTS LGG and Lc705 were observed to suppress genes that encoded allergy-related high-affinity IgE receptor subunits α and γ (FCER1A and FCER1G, respectively) and histamine H4 receptor. LGG, Lc705 and the combination of four probiotics had the strongest effect on the expression of genes involved in mast cell immune system regulation, and on several genes that encoded proteins with a pro-inflammatory impact, such as interleukin (IL)-8 and tumour necrosis factor alpha. Also genes that encoded proteins with anti-inflammatory functions, such as IL-10, were upregulated. CONCLUSION Certain probiotic bacteria might diminish mast cell allergy-related activation by downregulation of the expression of high-affinity IgE and histamine receptor genes, and by inducing a pro-inflammatory response.

Lactobacillus rhamnosus GG and Streptococcus thermophilus induce suppressor of cytokine signalling 3 (SOCS3) gene expression directly and indirectly via interleukin-10 in human primary macrophages

In the present study we have characterized T helper type 2 (Th2) [interleukin (IL)‐10]/Th1 (IL‐12) cytokine expression balance in human primary macrophages stimulated with multiple non‐pathogenic Gram‐positive bacteria used in the food industry and as probiotic substances. Bacteria representing Lactobacillus, Bifidobacterium, Lactococcus, Leuconostoc, Propionibacterium and Streptococcus species induced anti‐inflammatory IL‐10 production, although quantitative differences between the bacteria were observed. S. thermophilus was able to induce IL‐12 production, while the production of IL‐12 induced by other bacteria remained at a low level. The highest anti‐inflammatory potential was seen with bifidobacteria, as evidenced by high IL‐10/IL‐12 induction ratios. All studied non‐pathogenic bacteria were able to stimulate the expression of suppressor of cytokine signalling (SOCS) 3 that controls the expression of proinflammatory cytokine genes. Lactobacillus and Streptococcus species induced SOCS3 mRNA expression directly in the absence of protein synthesis and indirectly via bacteria‐induced IL‐10 production, as demonstrated by experiments with cycloheximide (CHX) and anti‐IL‐10 antibodies, respectively. The mitogen‐activated protein kinase (MAPK) p38 signalling pathway played a key role in bacteria‐induced SOCS3 gene expression. Enhanced IL‐10 production and SOCS3 gene expression induced by live non‐pathogenic Lactobacillus and Streptococcus is also likely to contribute to their immunoregulatory effects in vivo.

Association of Early-Life Antibiotic Use and Protective Effects of Breastfeeding: Role of the Intestinal Microbiota

IMPORTANCE Long duration of breastfeeding is known to reduce the frequency of infections and the risk of overweight, both of which are prevalent health problems among children, but the mechanisms are unclear. OBJECTIVES To test whether early-life antibiotic use in children prevents the beneficial long-term effects of breastfeeding on weight development and lifetime antibiotic use, and to investigate whether the duration of breastfeeding is associated with long-term microbiota development. DESIGN, SETTING, AND PARTICIPANTS Retrospective cohort study, conducted from June 2015 to December 2015, of the association between the duration of breastfeeding and lifetime antibiotic use by children as well as body mass index (BMI; calculated as weight in kilograms divided by height in meters squared) z score in a cohort of 226 healthy children aged 2 to 6 years attending day care at the study area in northern Finland and participating in a probiotic trial from October 1, 2009, through April 30, 2010. Fecal microbiota composition analysis was performed in a subcohort of 42 of these children. EXPOSURES Duration of breastfeeding and the number of different antibiotic courses purchased for the child. MAIN OUTCOMES AND MEASURES The BMI z score, lifetime antibiotic use after weaning, and fecal microbiota composition. RESULTS A total of 226 children (mean [SD] age, 55 [1.4] months; 54% male) were included in the study. Among the 113 children with no antibiotics before weaning, each month of breastfeeding decreased the mean number of postweaning antibiotic courses by 5% (95% CI, 2% to 8%; P = .001) and mean BMI z scores by 0.08 unit (95% CI, 0.04 to 0.11; P < .001). Among the 113 early-life antibiotic users, the effect of breastfeeding on postweaning antibiotic use was borderline significant (estimated 4% decrease per month; 95% CI, 0% to 7%; P = .04) and the effect on BMI z score disappeared (estimated 1% increase; 95% CI, -3% to 5%; P = .50). In the subcohort of 42 children with fecal microbiota composition analysis, the children with short breastfeeding duration (0-6 months) and no early-life antibiotic use or with long breastfeeding duration (8-16 months) and early-life use of antibiotics had a significantly lower abundance of Bifidobacterium (by 55%; 95% CI, 43% to 87%; P = .006; and 39%, 95% CI, 30% to 68%; P < .001, respectively) and Akkermansia (by 71%; 95% CI, 28% to 87%; P = .008; and 69%; 95% CI, 22% to 90%; P = .02, respectively) compared with those with long duration of breastfeeding and no early-life antibiotics. CONCLUSIONS AND RELEVANCE Antibiotic use in a child during breastfeeding may weaken the beneficial effects of long breastfeeding duration. The results suggest that particularly the long-term metabolic benefits of breastfeeding are conveyed by the intestinal microbiota.