Culture dependent and independent analyses suggest a low level of sharing of endospore-forming species between mothers and their children.

Spore forming bacteria comprise a large part of the human gut microbiota. However, study of the endospores in gut microbiota is limited due to difficulties of culturing and numerous unknown germination factors. In this study we propose a new method for culture-independent characterization of endospores in stool samples. We have enriched DNA of spore-forming bacterial species from stool samples of 40 mother-child pairs from a previously described mother-child cohort. The samples were exposed to a two-step purification process comprising ethanol and ethidium monoazide (EMA) treatment to first kill vegetative cells and to subsequently eliminate their DNA from the samples. The composition of the ethanol-EMA resistant DNA was characterized by 16S rRNA marker gene sequencing. Operational taxonomic units (OTUs) belonging to the Clostridia class (OTU1: Romboutsia, OTU5: Peptostreptococcaceae and OTU14: Clostridium senso stricto) and one belonging to the Bacillus class (OTU20: Turicibacter) were significantly more abundant in the samples from mothers and children after ethanol-EMA treatment than in those treated with ethanol only. No correlation was observed between ethanol-EMA resistant OTUs detected in children and in their mothers, which indicates that a low level of spore-forming species are shared between mothers and their children. Anaerobic ethanol-resistant bacteria were isolated from all mothers and all children over 1 year of age. Generally, in 70% of the ethanol-treated samples used for anaerobic culturing, 16S rRNA gene sequences of bacterial isolates corresponded to OTUs detected in these samples after EMA treatment. We report a new DNA-based method for the characterization of endospores in gut microbiota. Our method has high degree of correspondence to the culture-based method, although it requires further optimization. Our results also indicate a high turnover of endospores in the gut during the first two years of life, perhaps with a high environmental impact.

Fish Consumption at One Year of Age Reduces the Risk of Eczema, Asthma and Wheeze at Six Years of Age.

BACKGROUND: The role of dietary fish and n-3 polyunsaturated fatty acids (n-PUFAs) in the primary prevention of allergic diseases remains uncertain. The aim of this study was to investigate associations between the consumption of fish and cod liver oil (rich in n-PUFAs) from pregnancy to the first two years of life, and parental reported allergic diseases at six years of age. METHODS: We used data from the Prevention of Allergy among Children in Trondheim study and included mother-infant pairs who had submitted questionnaires detailing both maternal or infant diet and allergic disease at six years of age. RESULTS: Eating fish at least once a week at one year of age was associated with a 28-34% reduction in the odds of current eczema, asthma, and wheeze at six years of age. Cod liver oil consumption at least four times per week at one year of age tended to be associated with a lower risk of allergy-related outcomes at six years. We found no consistent associations between allergy-related outcomes and fish or cod liver oil consumption by mothers. CONCLUSION: The preventive effect of fish consumption is best achieved by increasing dietary fish in the first year of life.

Liquid array diagnostics: a novel method for rapid detection of microbial communities in single-tube multiplex reactions.

We present a novel liquid array diagnostics (LAD) method, which enables rapid and inexpensive detection of microbial markers in a single-tube multiplex reaction. We evaluated LAD both on pure cultures, and on infant gut microbiota for a 15-plex reaction. LAD showed more than 80% accuracy of classification and a detection limit lower than 2% of the Illumina reads per sample. The results on the clinical dataset showed that there was a rapid decrease of staphylococci from 10-day- to 4-month-old children, a peak of bifidobacteria at 4 months, and a peak of Bacteroides in 2-year-old children, which is in accordance with findings described in the literature. Being able to detect up to 50 biomarkers, LAD is a suitable method for assays where high throughput is essential.

Low Maternal Microbiota Sharing across Gut, Breast Milk and Vagina, as Revealed by 16S rRNA Gene and Reduced Metagenomic Sequencing.

The maternal microbiota plays an important role in infant gut colonization. In this work we have investigated which bacterial species are shared across the breast milk, vaginal and stool microbiotas of 109 women shortly before and after giving birth using 16S rRNA gene sequencing and a novel reduced metagenomic sequencing (RMS) approach in a subgroup of 16 women. All the species predicted by the 16S rRNA gene sequencing were also detected by RMS analysis and there was good correspondence between their relative abundances estimated by both approaches. Both approaches also demonstrate a low level of maternal microbiota sharing across the population and RMS analysis identified only two species common to most women and in all sample types (Bifidobacterium longum and Enterococcus faecalis). Breast milk was the only sample type that had significantly higher intra- than inter- individual similarity towards both vaginal and stool samples. We also searched our RMS dataset against an in silico generated reference database derived from bacterial isolates in the Human Microbiome Project. The use of this reference-based search enabled further separation of Bifidobacterium longum into Bifidobacterium longum ssp. longum and Bifidobacterium longum ssp. infantis. We also detected the Lactobacillus rhamnosus GG strain, which was used as a probiotic supplement by some women, demonstrating the potential of RMS approach for deeper taxonomic delineation and estimation.

Breastfeeding-associated microbiota in human milk following supplementation with Lactobacillus rhamnosus GG, Lactobacillus acidophilus La-5, and Bifidobacterium animalis ssp. lactis Bb-12.

Breastfeeding is one of the major factors affecting the early development of the infant gut microbiota, and weaning is associated with a shift in the gut microbiota toward a more adult composition. Through breastfeeding, infants receive bioactive components that shape their microbiota while also being exposed to the breast milk and breast surface microbial communities. Recent studies have suggested the possibility of an entero-mammary route of microbial transfer, opening the possibility of infant gut microbiota modulation through maternal probiotic supplementation. In this study, we have analyzed breast milk samples collected at 10 d and 3 mo postpartum from women participating in the Probiotics in the Prevention of Allergy among Children in Trondheim placebo controlled trial. Women who were randomized to the probiotic arm of the Probiotics in the Prevention of Allergy among Children in Trondheim trial received a fermented milk supplemented with Lactobacillus rhamnosus GG, Lactobacillus acidophilus La-5, and Bifidobacterium animalis ssp. lactis Bb-12, consuming this daily from 4 wk before their expected due date until 3 mo after birth. In total, 472 breast milk samples were assessed for the administered bacteria using quantitative real-time PCR and the microbiota transferred during breastfeeding was analyzed using 16S ribosomal RNA gene sequencing of 142 samples. We found that breastfeeding is unlikely to be a significant source of L. rhamnosus GG, L. acidophilus La-5, and B. animalis ssp. lactis Bb-12 for infants in the probiotic arm of the trial. Furthermore, maternal supplementation did not significantly affect the overall composition of the breast milk microbiota transferred during breastfeeding. We also present a descriptive analysis of this microbiota, which was largely dominated by Streptococcus and Staphylococcus genera at both 10 d and 3 mo postpartum. Samples collected at 3 mo postpartum had a statistically significant lower presence and relative abundance of the Staphylococcus genus. These samples also had a greater number of observed species and diversity, including more operational taxonomic units from the Rothia, Veillonella, Granulicatella, and Methylbacterium genera.

Diversity of vaginal microbiota increases by the time of labor onset.

Vaginal microbiota is an important early source of bacterial colonization for newborns. However, only a few small studies have investigated the composition of vaginal microbiota during labor. In this work, we analyzed vaginal swabs collected at 36 weeks gestation and at the onset of labor from 256 women participating in a randomized placebo-controlled study of probiotic supplementation for the prevention of atopic dermatitis in offspring. Although individuals' vaginal microbiota was stable over time, several bacterial families, which are characteristic of mixed community state type (CST) IV, were overrepresented in vaginal swabs sampled at labor. Alpha-diversity also tended to increase by between 36 weeks gestation and the onset of birth. In the majority of women, CST remained the same throughout the study. Among the women who switched their vaginal microbiota from one CST to another, approximately half shifted towards CST IV. Although CST IV is often associated with bacterial vaginosis, which in turn may lead to preterm birth, in our cohort this shift was not associated with self-reported vaginosis, preterm delivery or birthweight. Probiotic consumption did not alter vaginal microbiota.

Atopic dermatitis prevention in children following maternal probiotic supplementation does not appear to be mediated by breast milk TSLP or TGF-ß.

BACKGROUND: The Probiotics in Prevention of Allergy among Children in Trondheim (ProPACT) study, a randomised, placebo controlled trial, demonstrated that maternal supplementation with probiotic milk reduced the incidence of atopic dermatitis (AD) in infancy. The mechanisms behind this effect are incompletely understood and breast milk cytokines have been postulated as possible mediating factors. In this study we aimed to assess whether breast milk TLSP and TGF-ß are affected by a maternal probiotic supplementation regime, and their contribution to the preventive effect of this regime on AD in the offspring. METHODS: TSLP and TGF-ß isoforms (TGF-ß1, TGF-ß2 and TGF-ß3) were measured using ELISA and multiplex assays, respectively, in breast milk samples collected at 10 days and 3 months postpartum from women participating in the ProPACT trial (n = 259). The natural indirect and direct effects of maternal probiotics on AD, due to changes in breast milk cytokines, were estimated using causal mediation techniques. RESULTS: Probiotic supplementation tend to lead to high levels of breast milk TSLP at 10 days postpartum (p = 0.062), but this change did not contribute to the prevention of AD according to the mediation analysis. Probiotics had no apparent effect on TSLP at 3 months or TGF-ßs at either time points. Thus, these are unlikely to be mediators of the effect of maternal probiotics on AD in offspring. CONCLUSIONS: Whilst maternal probiotic supplementation resulted in higher breast milk concentrations of TLSP at 10 days postpartum, this does not appear to be a mechanism for prevention of AD by maternal probiotics. Trial registration The original trial protocol is registered in ClinicalTrials.gov (identifier NCT00159523).

Human Breast Milk miRNA, Maternal Probiotic Supplementation and Atopic Dermatitis in Offspring.

BACKGROUND: Perinatal probiotic ingestion has been shown to prevent atopic dermatitis (AD) in infancy in a number of randomised trials. The Probiotics in the Prevention of Allergy among Children in Trondheim (ProPACT) trial involved a probiotic supplementation regime given solely to mothers in the perinatal period and demonstrated a ~40% relative risk reduction in the cumulative incidence of AD at 2 years of age. However, the mechanisms behind this effect are incompletely understood. Micro-RNAs (miRNA) are abundant in mammalian milk and may influence the developing gastrointestinal and immune systems of newborn infants. The objectives of this study were to describe the miRNA profile of human breast milk, and to investigate breast milk miRNAs as possible mediators of the observed preventative effect of probiotics. METHODS: Small RNA sequencing was conducted on samples collected 3 months postpartum from 54 women participating in the ProPACT trial. Differential expression of miRNA was assessed for the probiotic vs placebo and AD vs non-AD groups. The results were further analysed using functional prediction techniques. RESULTS: Human breast milk samples contain a relatively stable core group of highly expressed miRNAs, including miR-148a-3p, miR-22-3p, miR-30d-5p, let-7b-5p and miR-200a-3p. Functional analysis of these miRNAs revealed enrichment in a broad range of biological processes and molecular functions. Although several miRNAs were found to be differentially expressed on comparison of the probiotic vs placebo and AD vs non-AD groups, none had an acceptable false discovery rate and their biological significance in the development of AD is not immediately apparent from their predicted functional consequences. CONCLUSION: Whilst breast milk miRNAs have the potential to be active in a diverse range of tissues and biological process, individual miRNAs in breast milk 3 months postpartum are unlikely to play a major role in the prevention of atopic dermatitis in infancy by probiotics ingestion in the perinatal period. TRIAL REGISTRATION: ClinicalTrials.gov NCT00159523.

Potential association of vacuum cleaning frequency with an altered gut microbiota in pregnant women and their 2-year-old children.

BACKGROUND: Westernized lifestyle and hygienic behavior have contributed to dramatic changes in the human-associated microbiota. This particularly relates to indoor activities such as house cleaning. We therefore investigated the associations between washing and vacuum cleaning frequency and the gut microbiota composition in a large longitudinal cohort of mothers and their children. The gut microbiota composition was determined using 16S ribosomal RNA (rRNA) gene Illumina deep sequencing. RESULTS: We found that high vacuum cleaning frequency about twice or more a week was associated with an altered gut microbiota composition both during pregnancy and for 2-year-old children, while there were no associations with house washing frequency. In total, six Operational Taxonomic Units (OTUs) showed significant False Discovery Rate (FDR) corrected associations with vacuum cleaning frequency for mothers (two positive and four negative) and five for 2-year-old children (four positive and one negative). For mothers and the 2-year-old children, OTUs among the dominant microbiota (average >5 %) showed correlation to vacuum cleaning frequency, with an increase in Faecalibacterium prausnitzii for mothers (p = 0.013, FDR corrected), and Blautia sp. for 2-year children (p = 0.012, FDR corrected). CONCLUSIONS: Bacteria showing significant associations are among the dominant gut microbiota, which may indicate indirect immunomodulation of the gut microbiota possibly through increased allergen (dust mites) exposure as a potential mechanism. However, further exploration is needed to unveil mechanistic details.

The commensal infant gut meta-mobilome as a potential reservoir for persistent multidrug resistance integrons.

Despite the accumulating knowledge on the development and establishment of the gut microbiota, its role as a reservoir for multidrug resistance is not well understood. This study investigated the prevalence and persistence patterns of an integrase gene (int1), used as a proxy for integrons (which often carry multiple antimicrobial resistance genes), in the fecal microbiota of 147 mothers and their children sampled longitudinally from birth to 2 years. The study showed the int1 gene was detected in 15% of the study population, and apparently more persistent than the microbial community structure itself. We found int1 to be persistent throughout the first two years of life, as well as between mothers and their 2-year-old children. Metagenome sequencing revealed integrons in the gut meta-mobilome that were associated with plasmids and multidrug resistance. In conclusion, the persistent nature of integrons in the infant gut microbiota makes it a potential reservoir of mobile multidrug resistance.

Perinatal probiotic supplementation in the prevention of allergy related disease: 6 year follow up of a randomised controlled trial.

BACKGROUND: Perinatal probiotics supplementation has been shown to be effective in the primary prevention of atopic dermatitis (AD) in early childhood, although the long term effects of probiotics on AD and other allergic diseases is less certain. We have previously reported a significant reduction in the cumulative incidence of AD at 2 years after maternal probiotic supplementation. In this study we present the effects of perinatal probiotics given to women from a general population on allergy related diseases in their offspring at 6 years. METHODS: Four hundred and fifteen pregnant women were randomised to receive probiotic or placebo milk in a double-blinded trial from 36 week gestation until 3 months postpartum. Probiotic milk contained Lactobacillus rhamnosos GG, L. acidophilus La-5 and Bifidobacterium animalis subsp. lactis Bb-12. At 6 years, children were re-assessed for AD, atopic sensitisation, asthma and allergic rhinoconjunctivitis (ARC). RESULTS: At 6 years, 81 and 82 children were assessed for AD in the probiotic and placebo groups, respectively. In a multiple imputation analysis, there was as trend towards a lower cumulative incidence of AD in the probiotic group compared to the placebo group (OR 0.64, 95 % CI 0.39-1.07, p = 0.086; NNT = 10). This finding was statistically significantly in the complete case analysis (OR 0.48, 95 % CI 0.25-0.92, p = 0.027, NNT = 6). The prevalence of asthma and atopic sensitisation, and the cumulative incidence of ARC were not significantly affected by the probiotic regime at 6 years of age. CONCLUSIONS: Maternal probiotic ingestion alone may be sufficient for long term reduction in the cumulative incidence of AD, but not other allergy related diseases. TRIAL REGISTRATION: ClinicalTrials.gov identifier: NCT00159523.

High-Resolution Analyses of Overlap in the Microbiota Between Mothers and Their Children.

Understanding the transmission of the human microbiota from mother to child is of major importance. Although we are gaining knowledge using 16S rRNA gene analyses, the resolution of this gene is not sufficient to determine transmission patterns. We therefore developed an Illumina deep sequencing approach targeting the 16-23S rRNA Internal Transcribed Spacer (ITS) for high-resolution microbiota analyses. Using this approach, we analyzed the composition and potential mother to child transmission patterns of the microbiota (milk and stool) in a longitudinal cohort of 20 mother/child pairs. Our results show overlap in the infant stool microbiota with both mother's milk and stool, and that the overlap with stool increases with age. We found an Operational Taxonomic Unit resembling Streptococcus gordonii as the most widespread colonizer of both mothers and their children. In conclusion, the increased resolution of 16-23S rRNA ITS deep sequencing revealed new knowledge about potential transmission patterns of human-associated bacteria.

Does Maternal Perinatal Probiotic Supplementation Alter the Intestinal Microbiota of Mother and Child?

OBJECTIVES: Maternal probiotic supplementation has been shown to prevent the development of atopic dermatitis in the offspring. We aimed to investigate whether probiotics in pregnant and breast-feeding mothers altered the colonization pattern and the diversity of the mothers' and children's intestinal microbiota. METHODS: In a randomized, double-blind trial, women received probiotic milk or placebo from 36 weeks of gestation up to 3 months postnatally while breast-feeding. The probiotic milk contained Lactobacillus rhamnosus GG, L acidophilus La-5, and Bifidobacterium animalis subsp. lactis Bb-12. Stool samples were collected from the mothers at 30 to 36 weeks of gestation and 3 months after birth, and from the child at age 10 days, 3 months, 1 year, and 2 years, and bacteria were analyzed by quantitative polymerase chain reaction. Additionally, stool samples from 3-month-old and 2-year-old children were characterized using 16S ribosomal RNA gene deep sequencing to estimate the bacterial classes and genera, and the α- and ß-diversity. RESULTS: Three months after birth, both the prevalence and the relative abundance of the administered probiotic bacteria were significantly increased among the mothers in the probiotic group compared with among those in the placebo group. Only the Lactobacillus rhamnosus GG bacteria colonized the children at 10 days and at 3 months of age. There were no significant differences in the abundance of the administered probiotic bacteria between the groups at 1 and 2 years of age. For the bacterial classes and genera, and α- and ß-diversity, there were no significant differences between the groups. CONCLUSIONS: Different probiotic bacteria seem to have different ability to transfer from the mother to the child. We found no evidence that the probiotics altered the microbial composition or α- and ß-diversity of the children.

The predictive value of allergen skin prick tests and IgE tests at pre-school age: the PACT study.

BACKGROUND: Sensitization toward allergens, as determined by skin prick test (SPT) or specific IgE (sIgE), is a predictor for the later presence of allergy-related disease (atopic eczema, allergic rhinoconjuctivitis and asthma). However, it is not known whether SPT or sIgE should be the preferred test. The aim of this study was to compare the predictive ability of SPT and sIgE when performed in a general population of 2-yr-old children. METHODS: In a prospective, longitudinal population-based study of children aged 2-6 yr, SPT and sIgE for nine common allergens were performed at 2 yr. Allergy-related disease was evaluated by clinical examination and questionnaire at 2 and 6 yr of age (n = 199). RESULTS: Skin prick test or sIgE was positive in 10.6% and 21.1% in the 2-yr-old children, respectively. The prevalence of allergy-related disease was 25.6% at 2 yr and 25.1% at 6 yr. Half of the cases at 2 yr were transient. Both SPT and sIgE were statistically significant predictors for later allergy-related disease, OR = 6.5 (95% CI 2.3-18.6) and OR = 4.1 (95% CI 1.9-9.0), respectively. Receiver operating characteristic analysis showed that SPT and sIgE had comparable predictive ability for atopic eczema, asthma or any allergy-related disease, but sIgE had better ability to predict later allergic rhinoconjunctivitis. CONCLUSION: Sensitization at 2 yr may be useful predictors of allergy-related disease later in childhood. The predictive ability of SPT and sIgE were mainly comparable; however, it may be that sIgE is the preferred choice in young children when the aim is to predict allergic rhinoconjunctivitis.

Major faecal microbiota shifts in composition and diversity with age in a geographically restricted cohort of mothers and their children.

Despite the importance, the diversity of the human infant gut microbiota still remains poorly characterized at the regional scale. Here, we investigated the faecal microbiota diversity in a large 16S rRNA gene data set from a healthy cohort of 86 mothers and their children from the Trondheim region in Norway. Samples were collected from mothers during early and late pregnancy, as well as from their children at 3 days, 10 days, 4 months, 1 year and 2 years of age. Using a combination of Sanger sequencing of amplicon mixtures (without cloning), real-time quantitative PCR and deep pyrosequencing, we observed a clear age-related colonization pattern in children that was surprisingly evident between 3- and 10-day samples. In contrast, we did not observe any shifts in microbial composition during pregnancy. We found that alpha-diversity was highest at 2 years and lowest at 4 months, whereas beta-diversity estimates indicated highest interindividual variation in newborns. Variation significantly decreased by the age of 10 days and was observed to be convergent over time; however, there were still major differences between 2 years and adults whom exhibited the lowest interindividual diversity. Taken together, the major age-affiliated population shift within gut microbiota suggests that there are important mechanisms for transmission and persistence of gut bacteria that remain unknown.

Diversity of intestinal microbiota in infancy and the risk of allergic disease in childhood.

PURPOSE OF REVIEW: Numerous studies have attempted to describe specific microbiota deviations that may precede atopic sensitization and atopic disease in childhood. This has given rise to a hypothesis suggesting that a reduced intestinal microbial diversity in infancy increases the risk of allergic manifestations. This review intends to sum up the main findings and discuss relevant exposures that regulate intestinal microbial diversity. RECENT FINDINGS: Taken together the three studies in this review lend support to the diversity hypothesis, but reported differences related to atopic sensitization and clinical expression are discussed. A summary on analytic methods and functional aspect of the microbiota in allergic disease is presented to ameliorate a presentation of recent articles on environmental and host-factors regulating microbiota composition and diversity. SUMMARY: The current evidence indicates that intestinal microbiota diversity can be associated with allergic diseases, but the exact mechanisms and interactions contributing to this effect are far from understood and need further investigation.

The impact of pre- and postnatal exposures on allergy related diseases in childhood: a controlled multicentre intervention study in primary health care.

BACKGROUND: Environmental factors such as tobacco exposure, indoor climate and diet are known to be involved in the development of allergy related diseases. The aim was to determine the impact of altered exposure to these factors during pregnancy and infancy on the incidence of allergy related diseases at 2 years of age. METHODS: Children from a non-selected population of mothers were recruited to a controlled, multicenter intervention study in primary health care. The interventions were an increased maternal and infant intake of n-3 PUFAs and oily fish, reduced parental smoking, and reduced indoor dampness during pregnancy and the children's first 2 years of life. Questionnaires on baseline data and exposures, and health were collected at 2 years of age. RESULTS: The prevalence of smoking amongst the mothers and fathers was approximately halved at 2 years of age in the intervention cohort compared to the control cohort. The intake of n-3 PUFA supplement and oily fish among the children in the intervention cohort was increased. There was no significant change for indoor dampness. The odds ratio for the incidence of asthma was 0.72 (95% CI, 0.55-0.93; NNTb 53), and 0.75 for the use of asthma medication (95% CI, 0.58-0.96). The odds ratio for asthma among girls was 0.41 (95% CI 0.24-0.70; NNTb 32), and for boys 0.93 (95% CI 0.68-1.26). There were no significant change for wheeze and atopic dermatitis. CONCLUSION: Reduced tobacco exposure and increased intake of oily fish during pregnancy and early childhood may be effective in reducing the incidence of asthma at 2 years of age. The differential impact in boys and girls indicates that the pathophysiology of asthma may depend on the sex of the children. TRIAL REGISTRATION: Current Controlled Trials ISRCTN28090297.

Early eczema and the risk of childhood asthma: a prospective, population-based study.

BACKGROUND: Severe eczema in young children is associated with an increased risk of developing asthma and rhino-conjunctivitis. In the general population, however, most cases of eczema are mild to moderate. In an unselected cohort, we studied the risk of current asthma and the co-existence of allergy-related diseases at 6 years of age among children with and without eczema at 2 years of age. METHODS: Questionnaires assessing various environmental exposures and health variables were administered at 2 years of age. An identical health questionnaire was completed at 6 years of age. The clinical investigation of a random subsample ascertained eczema diagnoses, and missing data were handled by multiple imputation analyses. RESULTS: The estimate for the association between eczema at 2 years and current asthma at 6 years was OR=1.80 (95% CI 1.10-2.96). Four of ten children with eczema at 6 years had the onset of eczema after the age of 2 years, but the co-existence of different allergy-related diseases at 6 years was higher among those with the onset of eczema before 2 years of age. CONCLUSIONS: Although most cases of eczema in the general population were mild to moderate, early eczema was associated with an increased risk of developing childhood asthma. These findings support the hypothesis of an atopic march in the general population. TRIAL REGISTRATION: The Prevention of Allergy among Children in Trondheim study has been identified as ISRCTN28090297 in the international Current Controlled Trials database.