Novel Developmental Analyses Identify Longitudinal Patterns of Early Gut Microbiota that Affect Infant Growth

It is acknowledged that some obesity trajectories are set early in life, and that rapid weight gain in infancy is a risk factor for later development of obesity. Identifying modifiable factors associated with early rapid weight gain is a prerequisite for curtailing the growing worldwide obesity epidemic. Recently, much attention has been given to findings indicating that gut microbiota may play a role in obesity development. We aim at identifying how the development of early gut microbiota is associated with expected infant growth. We developed a novel procedure that allows for the identification of longitudinal gut microbiota patterns (corresponding to the gut ecosystem developing), which are associated with an outcome of interest, while appropriately controlling for the false discovery rate. Our method identified developmental pathways of Staphylococcus species and Escherichia coli that were associated with expected growth, and traditional methods indicated that the detection of Bacteroides species at day 30 was associated with growth. Our method should have wide future applicability for studying gut microbiota, and is particularly important for translational considerations, as it is critical to understand the timing of microbiome transitions prior to attempting to manipulate gut microbiota in early life

Cohort profile: Finnish Health and Early Life Microbiota (HELMi) longitudinal birth cohort

Peer reviewe

Dichotomous development of the gut microbiome in preterm infants.

BackgroundPreterm infants are at risk of developing intestinal dysbiosis with an increased proportion of Gammaproteobacteria. In this study, we sought the clinical determinants of the relative abundance of feces-associated Gammaproteobacteria in very low birth weight (VLBW) infants. Fecal microbiome was characterized at ≤ 2 weeks and during the 3rd and 4th weeks after birth, by 16S rRNA amplicon sequencing. Maternal and infant clinical characteristics were extracted from electronic medical records. Data were analyzed by linear mixed modeling and linear regression.ResultsClinical data and fecal microbiome profiles of 45 VLBW infants (gestational age 27.9 ± 2.2 weeks; birth weight 1126 ± 208 g) were studied. Three stool samples were analyzed for each infant at mean postnatal ages of 9.9 ± 3, 20.7 ± 4.1, and 29.4 ± 4.9 days. The average relative abundance of Gammaproteobacteria was 42.5% (0-90%) at ≤ 2 weeks, 69.7% (29.9-86.9%) in the 3rd, and 75.5% (54.5-86%) in the 4th week (p < 0.001). Hierarchical and K-means clustering identified two distinct subgroups: cluster 1 started with comparatively low abundance that increased with time, whereas cluster 2 began with a greater abundance at ≤ 2 weeks (p < 0.001) that decreased over time. Both groups resembled each other by the 3rd week. Single variants of Klebsiella and Staphylococcus described variance in community structure between clusters and were shared between all infants, suggesting a common, hospital-derived source. Fecal Gammaproteobacteria was positively associated with vaginal delivery and antenatal steroids.ConclusionsWe detected a dichotomy in gut microbiome assembly in preterm infants: some preterm infants started with low relative gammaproteobacterial abundance in stool that increased as a function of postnatal age, whereas others began with and maintained high abundance. Vaginal birth and antenatal steroids were identified as predictors of Gammaproteobacteria abundance in the early (≤ 2 weeks) and later (3rd and 4th weeks) stool samples, respectively. These findings are important in understanding the development of the gut microbiome in premature infants

Towards large-cohort comparative studies to define the factors influencing the gut microbial community structure of ASD patients.

Differences in the gut microbiota have been reported between individuals with autism spectrum disorders (ASD) and neurotypical controls, although direct evidence that changes in the microbiome contribute to causing ASD has been scarce to date. Here we summarize some considerations of experimental design that can help untangle causality in this complex system. In particular, large cross-sectional studies that can factor out important variables such as diet, prospective longitudinal studies that remove some of the influence of interpersonal variation in the microbiome (which is generally high, especially in children), and studies transferring microbial communities into germ-free mice may be especially useful. Controlling for the effects of technical variables, which have complicated efforts to combine existing studies, is critical when biological effect sizes are small. Large citizen-science studies with thousands of participants such as the American Gut Project have been effective at uncovering subtle microbiome effects in self-collected samples and with self-reported diet and behavior data, and may provide a useful complement to other types of traditionally funded and conducted studies in the case of ASD, especially in the hypothesis generation phase

The influence of maternal and infant nutrition on cardiometabolic traits: novel findings and future research directions from four Canadian birth cohort studies

A mother's nutritional choices while pregnant may have a great influence on her baby's development in the womb and during infancy. There is evidence that what a mother eats during pregnancy interacts with her genes to affect her child's susceptibility to poor health outcomes including childhood obesity, pre-diabetes, allergy and asthma. Furthermore, after what an infant eats can change his or her intestinal bacteria, which can further influence the development of these poor outcomes. In the present paper, we review the importance of birth cohorts, the formation and early findings from a multi-ethnic birth cohort alliance in Canada and summarise our future research directions for this birth cohort alliance. We summarise a method for harmonising collection and analysis of self-reported dietary data across multiple cohorts and provide examples of how this birth cohort alliance has contributed to our understanding of gestational diabetes risk; ethnic and diet-influences differences in the healthy infant microbiome; and the interplay between diet, ethnicity and birth weight. Ongoing work in this birth cohort alliance will focus on the use of metabolomic profiling to measure dietary intake, discovery of unique diet–gene and diet–epigenome interactions, and qualitative interviews with families of children at risk of metabolic syndrome. Our findings to-date and future areas of research will advance the evidence base that informs dietary guidelines in pregnancy, infancy and childhood, and will be relevant to diverse and high-risk populations of Canada and other high-income countries

Discordant transmission of bacteria and viruses from mothers to babies at birth

BACKGROUND: The earliest microbial colonizers of the human gut can have life-long consequences for their hosts. Precisely how the neonatal gut bacterial microbiome and virome are initially populated is not well understood. To better understand how the maternal gut microbiome influences acquisition of the infant gut microbiome, we studied the early life bacterial microbiomes and viromes of 28 infant twin pairs and their mothers. RESULTS: Infant bacterial and viral communities more closely resemble those of their related co-twin than unrelated infants. We found that 63% of an infant\u27s bacterial microbiome can be traced to their mother\u27s gut microbiota. In contrast, only 15% of their viral communities are acquired from their mother. Delivery route did not determine how much of the bacterial microbiome or virome was shared from mother to infant. However, bacteria-bacteriophage interactions were altered by delivery route. CONCLUSIONS: The maternal gut microbiome significantly influences infant gut microbiome acquisition. Vertical transmission of the bacterial microbiome is substantially higher compared to vertical transmission of the virome. However, the degree of similarity between the maternal and infant gut bacterial microbiome and virome did not vary by delivery route. The greater similarity of the bacterial microbiome and virome between twin pairs than unrelated twins may reflect a shared environmental exposure. Thus, differences of the inter-generation transmissibility at birth between the major kingdoms of microbes indicate that the foundation of these microbial communities are shaped by different rules. Video Abstract

Maternal prenatal stress, infant microbiota, brain, and behavioral development : The FinnBrain Birth Cohort Study

The gut microbiota and its manipulation have been shown to affect behavior and neurodevelopment in rodents. Likewise, maternal prenatal stress is known to influence offspring health and development as well as gut microbiota composition in rodents and non-human primates. However, how infant fecal microbiota is associated with infant behavioral and brain developmental phenotypes or with exposure to prenatal stress remains largely unknown. The first aim of this dissertation was to explore how infant fecal microbiota associates with temperament, emotional attention, and amygdala volume, all of which may relate to later socioemotional and behavioral development. The second aim was to investigate if maternal prenatal chronic psychological distress and chronic cortisol levels, which are measures of prenatal stress in this study, associate with infant fecal microbiota composition and diversity. The studies were conducted in the prospective, general population-based FinnBrain Birth Cohort Study. First, the results showed that early fecal microbiota composition was associated with temperament traits and attention bias towards fearful faces. Specifically, Bifidobacterium, Stroptococcus, and Atopobium were positively associated with positive emotionality, whereas Bifidobacterium was negatively and Clostridium was positively associated with greater attention bias towards fearful faces. Both temperament and attention bias towards faces showed an interaction by sex regarding fecal microbiota composition. The left amygdala volume as well as negative emotionality and fear reactivity were negatively associated with fecal microbiota diversity. Second, maternal prenatal stress associated with fecal microbiota composition, including increases in abundances of genera within the Proteobacteria phylum and decreases in Lactobacillus abundance. This dissertation argues that infant fecal microbiota associates with later brain and behavioral phenotypes and encourages future longitudinal and mechanistic studies. Likewise, we corroborate some earlier findings regarding maternal prenatal stress and infant fecal microbiota.Äidin raskaudenaikainen stressi, lapsen mikrobisto, käyttäytymisen ja aivojen kehitys Eläintöiden perusteella on ehdotettu, että suolistomikrobisto vaikuttaa aivojen toimintaan ja käyttäytymiseen. Lisäksi jyrsijöillä ja kädellisillä on osoitettu, että äidin raskaudenaikainen stressi vaikuttaa jälkeläisten kasvuun ja terveyteen sekä suolistomikrobiston koostumukseen. Vielä ei kuitenkaan täysin ymmärretä, että liittyykö äidin raskaudenaikainen stressi lapsen suolistomikrobiston koostumukseen tai liittyykö lapsen suolistomirkobiston koostumus varhaiseen käyttäytymiseen tai aivojen kehittymiseen ihmisillä. Tässä väitöskirjassa kartoitettiin FinnBrain-syntymäkohorttitutkimuksessa lapsen varhaisen mikrobiston yhteyksiä temperamenttiin, kasvoihin ja kasvojen ilmeisiin kohdistuvaan kognitiiviseen tarkkaavuuteen sekä mantelitumakkeen kokoon. Lisäksi väitöskirjassa tutkittiin äidin raskauden aikaisen pitkäaikaisten psyykkisten oireiden ja kortisolipitoisuuksien – joita käytettiin raskaudenaikaisen stressin mittareina tässä tutkimuksessa – yhteyksiä lapsen suolistomikrobiston koostumukseen ja monimuotoisuuteen. Lapsen suolistomikrobiston koostumus oli yhteydessä temperamenttiin ja varhaiseen pelokkaisiin kasvoihin kohdistuvaan tarkkaavaisuuteen. Bifidobacterium, Streptococcus, Atopobium bakteerisuvut olivat yhteydessä positiiviseen emotionaalisuuteen, ja toisaalta Clostridium ja Bifidobacterium suvut olivat yhteydessä pelokkaisiin kasvoisiin kohdistuvaan tarkkaavaisuuteen. Sukupuoli vaikutti suolistomikrobiston ja temperamentin sekä lapsen kasvoihin kohdistuvan tarkkaavaisuuden välisiin yhteyksiin. Suolistomikrobiston vähäisempi monimuotoisuus oli yhteydessä suurempaan vasemman mantelitumakkeen kokoon ja voimakkaampaan negatiiviseen emotionaalisuuteen ja pelkoreagoivuuteen. Äidin raskaudenaikaisten stressi oli yhteydessä Proteobakteereihin kuuluvien sukujen ja maitohappobakteerien pitoisuuksiin. Väitöskirjan löydökset tukevat väitettä, että suolistomikrobisto on yhteydessä aivojen kehitykseen ja käyttäytymiseen, mutta löydökset eivät vielä kerro taustalla olevista syy-seuraussuhteesta. Äidin raskaudenaikaisen stressin yhteydet lapsen suolistomikrobistoon vahvistivat jo aiemmin raportoituja löydöksiä

Mechanisms Affecting the Gut of Preterm Infants in Enteral Feeding Trials

Large randomized controlled trials (RCTs) in preterm infants offer unique opportunities for mechanistic evaluation of the risk factors leading to serious diseases, as well as the actions of interventions designed to prevent them. Necrotizing enterocolitis (NEC) a serious inflammatory gut condition and late-onset sepsis (LOS) are common feeding and nutrition-related problems that may cause death or serious long-term morbidity and are key outcomes in two current UK National Institutes for Health Research (NIHR) trials. Speed of increasing milk feeds trial (SIFT) randomized preterm infants to different rates of increases in milk feeds with a primary outcome of survival without disability at 2 years corrected age. Enteral lactoferrin in neonates (ELFIN) randomizes infants to supplemental enteral lactoferrin or placebo with a primary outcome of LOS. This is a protocol for the mechanisms affecting the gut of preterm infants in enteral feeding trials (MAGPIE) study and is funded by the UK NIHR Efficacy and Mechanistic Evaluation programme. MAGPIE will recruit ~480 preterm infants who were enrolled in SIFT or ELFIN. Participation in MAGPIE does not change the main trial protocols and uses non-invasive sampling of stool and urine, along with any residual resected gut tissue if infants required surgery. Trial interventions may involve effects on gut microbes, metabolites (e.g., short-chain fatty acids), and aspects of host immune function. Current hypotheses suggest that NEC and/or LOS are due to a dysregulated immune system in the context of gut dysbiosis, but mechanisms have not been systematically studied within large RCTs. Microbiomic analysis will use next-generation sequencing, and metabolites will be assessed by mass spectrometry to detect volatile organic and other compounds produced by microbes or the host. We will explore differences between disease cases and controls, as well as exploring the actions of trial interventions. Impacts of this research are multiple: translation of knowledge of mechanisms promoting gut health may explain outcomes or suggest alternate strategies to improve health. Results may identify new non-invasive diagnostic or monitoring techniques, preventative or treatment strategies for NEC or LOS, or provide data useful for risk stratification in future studies. Mechanistic evaluation might be especially informative where there are not clear effects on the primary outcome (ISRCTN 12554594)

Strain-resolved analysis of the human intestinal microbiota

The gut microbiota is ascribed a crucial role in human health, particularly in regulating immune and inflammatory responses, which is why it is being associated with a wide range of diseases, including obesity, diabetes, and cancer. Nonetheless, fundamental ecological questions of microbiome establishment, stability and resilience, as well as its transmission across hosts and generations remain incompletely understood, partly due to the lack of methods for high-resolution microbiome profiling. New insights in this field can therefore directly contribute to the development of bacterial and microbiota-based therapies. This work introduces SameStr, a novel bioinformatic program for strain-resolved metagenomics that allows for the specific tracking of microbes across samples, enabling the detection and quantification of microbial transmission and persistence, as well as the observation of direct strain competition. Deployed across cohorts to process over 4200 metagenomes, SameStr enabled analysis of the microbiome with unprecedented phylogenetic resolution. The data included both publicly available metagenomes and sequence data generated in collaboration with our research partners, and was examined using multivariate statistics and machine learning frameworks. First, the establishment and development of the neonatal microbiota was studied, revealing a birth mode-dependent vertical transmission of the maternal microbiota. The microbiota of neonates born by cesarean section was characterized by increased relative abundance of oxygen-tolerant and atypical organisms and showed signs of a delayed establishment of a strictly anaerobic gut environment in these children. Such birth mode-dependent differences diminished over time, yet were measurable within the first two years of life. Furthermore, strain analysis verified the transmission and colonization of parental microbes, which indicated a possible lifelong colonization by microbes from selected species. The temporal persistence of microbes was also characterized in healthy adults, revealing similar taxonomy-dependent patterns of stability. For some species, persistence has been demonstrated both in children and in adults over a period of at least two years. These species are known for their capability to metabolize host-derived glycans found both in breastmilk and intestinal mucus, pointing to a potential strategy for effective cross-generational microbiota transmission, and warranting additional research to assess the implications of their disturbed transfer for long-term health. Since their specificity allows assignment to individual hosts, fingerprints of individual microbial strains offer the potential to be used in forensics and data quality control applications. Finally, to gain new insights into the microbiota dynamics during Fecal Microbiota Transplantation (FMT), microbial strain transmission was analyzed in the context of a diverse set of patient, microbiome, and clinical conditions. In the analyzed studies, FMT was used for the experimental treatment of a variety of diseases, including colonization with drug-resistant and pathogenic microbes, metabolic and inflammatory bowel diseases, and as an adjunct to the immunotherapeutic treatment of cancer. Analyses uncovered what appear to be the universal drivers of post-FMT microbiota assembly, including clinical and ecological factors that are important for successful transplantation of donor strains. In particular, the relevance of the microbiota dysbiosis of the recipient was emphasized, which was inducible by pre-treating the patient with antibiotics or laxatives. Presumably, this can open up ecological niches in the patients intestines, which favors colonization with donor strains. Colonization rates did not play a role for the treatment success of recurrent C. difficile infections and inflammatory bowel disease, but indicated a trend associated with an improved immune response in cancer patients. Concerningly, the transfer of an atypical and potentially pro-inflammatory microbial community from one donor was also observed, calling for further investigations into the immediate and long-term clinical consequences of FMT. These analyses demonstrate the advantages of a strain-based microbiome analysis. Due to the achieved methodological accuracy, strain-resolved microbial dynamics could be precisely disentangled when comparing longitudinal samples from healthy adults as well as parent-child and patient-donor pairs. This revealed taxonomic, clinical, and ecological factors that are critical to microbiome assembly, including microbial transmission, persistence, and competition. Together, these findings lay the groundwork for future developments of precision personalized microbiota modulation therapies.Der Darmmikrobiota wird eine entscheidende Rolle für die menschliche Gesundheit zugeschrieben, was insbesondere die Regulation von Immun- und Entzündungsreaktionen betrifft, weshalb sie mit einer Vielzahl von Krankheiten wie etwa Fettleibigkeit, Diabetes oder Krebs in Verbindung gebracht wird. Nichtsdestotrotz sind grundlegende ökologische Fragen der Etablierung, Stabilität und Resilienz von Mikrobiomen sowie ihrer Übertragung über Wirte und Generationen hinweg noch immer unvollständig untersucht, was teilweise auf das Fehlen von Methoden zur hochauflösenden Mikrobiom-Profilierung zurückzuführen ist. Neue Erkenntnisse auf diesem Gebiet können daher unmittelbar zur Entwicklung von Bakterien- und Mikrobiota-basierten Therapien beitragen. Diese Arbeit stellt SameStr vor, ein neues bioinformatisches Programm für stammaufgelöste Metagenomik, das die spezifische probenübergreifende Untersuchung von Mikroorganismen ermöglicht. Hiermit können der Nachweis und die Quantifizierung der Übertragung und Persistenz, sowie die Beobachtung der direkten Konkurrenz mikrobieller Stämme erfolgen. SameStr wurde kohortenübergreifend für die Analyse von über 4200 Metagenomen eingesetzt und ermöglichte die Profilierung des Mikrobioms mit einer beispiellosen phylogenetischen Auflösung. Die Metagenome, welche sowohl öffentlich verfügbare als auch in Zusammenarbeit mit unseren Forschungspartnern generierte Daten beinhalteten, konnten mittels multivariater Statistik und maschinellen Lernens beleuchtet werden. Zunächst wurde die Etablierung und Entwicklung der neonatalen Mikrobiota analysiert, was eine vom Geburtsmodus abhängige vertikale Übertragung der mütterlichen Mikrobiota aufzeigte. Die Mikrobiota von Neugeborenen die durch einen Kaiserschnitt zur Welt gekommen waren, war vermehrt von Sauerstoff-toleranten und Darm-untypischen Organismen besiedelt und deutete darauf hin, dass sich ein strikt anaerobes Darmmilieu bei diesen Kindern mit einer gewissen Verzögerung einstellte. Derartige geburtsabhängige Veränderungen schwächten sich mit der Zeit ab, waren jedoch bis zum zweiten Lebensjahr messbar. Weiterhin konnte die Übertragung und Kolonisierung elterlicher Organismen mittels Stamm-Analyse nachgewiesen werden, was außerdem auf eine mögliche lebenslange Besiedlung durch Mikroben ausgewählter Spezies hindeutete. Die zeitliche Persistenz von Mikroorganismen wurde darüber hinaus auch bei gesunden Erwachsenen charakterisiert, was ebenfalls Taxonomie-abhängige Stabilitätsmuster zum Vorschein brachte. Bei einigen Spezies, die bekannt dafür sind vom menschlichen Wirt stammende Glykane zu metabolisieren, wurde die Persistenz sowohl bei Kindern als auch bei Erwachsenen über einen Zeitraum von mindestens zwei Jahren nachgewiesen. Diese Glykane kommen sowohl in der Muttermilch als auch im Darmschleim vor, was auf eine potenzielle Strategie für eine effektive generationsübergreifende Übertragung der Mikrobiota hinweist. Um die langfristigen Auswirkungen einer gestörten Mikrobiota-Übertragung auf die Gesundheit bewerten zu können, wird jedoch weitere Forschung benötigt. Da ihre Spezifität die Zuordnung zu individuellen Wirten ermöglicht, bieten mikrobielle Stämme zudem das Potenzial in der Forensik und bei Datenqualitätstests Anwendung zu finden. Um schließlich neue Erkenntnisse zur Mikrobiota-Dynamik während der fäkalen Mikrobiota-Transplantation (FMT) zu gewinnen, wurde die Stammübertragung im Kontext einer Vielzahl von Patienten-, Mikrobiom- und klinischen Parametern analysiert. FMT wurde in den vorliegenden Studien zur experimentellen Behandlung verschiedenster Erkrankungen eingesetzt, darunter Kolonisierung mit resistenten und pathogenen Keimen, metabolische Erkrankungen, entzündliche Erkrankungen des Darms, sowie begleitend zur immuntherapeutischen Behandlung von Krebs. Die Analysen zeigten scheinbar universelle klinische und ökologische Faktoren auf, welche für eine erfolgreiche Integration von Spenderstämmen von Bedeutung sind. Insbesondere wurde die Relevanz der Mikrobiota-Dysbiose des Empfängers hervorgehoben, welche zudem durch Vorbehandlung der Patienten mittels Gabe von Antibiotika oder Laxativa induziert werden kann. Vermutlich können hierdurch ökologische Nischen im Darm der Patienten eröffnet werden, was eine Kolonisierung mit Spenderstämmen begünstigt. Kolonisierungsraten spielten für den Behandlungserfolg wiederkehrender Clostridien-Infektionen und entzündlicher Darmerkrankungen keine Rolle, deuteten jedoch auf einen Trend hin, der mit einer verbesserten Immunantwort bei Krebspatienten einhergeht. Beunruhigenderweise wurde auch die Übertragung einer atypischen und potenziell entzündungsfördernden Mikrobiota eines Donoren beobachtet, was weitere Untersuchungen zu unmittelbaren und langfristigen klinischen Folgen der FMT erforderlich macht. Die Ergebnisse dieser Arbeit zeigen die Vorteile einer Stamm-basierten Mikrobiom-Analyse auf. Durch die erreichte methodische Genauigkeit konnten bei Vergleichen von Zeitverlaufsproben gesunder Erwachsener sowie Eltern-Kind- und Patienten-Spender-Paaren, die Dynamiken mikrobieller Stämme präzise entschlüsselt werden. Dabei kamen taxonomische, klinische und ökologische Faktoren zum Vorschein, welche für die Zusammensetzung der Mikrobiota, einschließlich der mikrobiellen Übertragung, Persistenz und Kompetition, maßgebend sind. Diese neuen Erkenntnisse bilden die Grundlage für künftige Entwicklungen von Therapien zur präzisen, personalisierten Modulation der Mikrobiota