The Bern Birth Cohort (BeBiCo) to study the development of the infant intestinal microbiota in a high-resource setting in Switzerland: rationale, design, and methods.

BACKGROUND: Microbiota composition is fundamental to human health with the intestinal microbiota undergoing critical changes within the first two years of life. The developing intestinal microbiota is shaped by maternal seeding, breast milk and its complex constituents, other nutrients, and the environment. Understanding microbiota-dependent pathologies requires a profound understanding of the early development of the healthy infant microbiota. METHODS: Two hundred and fifty healthy pregnant women (≥20 weeks of gestation) from the greater Bern area will be enrolled at Bern University hospital's maternity department. Participants will be followed as mother-baby pairs at delivery, week(s) 1, 2, 6, 10, 14, 24, 36, 48, 96, and at years 5 and 10 after birth. Clinical parameters describing infant growth and development, morbidity, and allergic conditions as well as socio-economic, nutritional, and epidemiological data will be documented. Neuro-developmental outcomes and behavior will be assessed by child behavior checklists at and beyond 2 years of age. Maternal stool, milk, skin and vaginal swabs, infant stool, and skin swabs will be collected at enrolment and at follow-up visits. For the primary outcome, the trajectory of the infant intestinal microbiota will be characterized by 16S and metagenomic sequencing regarding composition, metabolic potential, and stability during the first 2 years of life. Secondary outcomes will assess the cellular and chemical composition of maternal milk, the impact of nutrition and environment on microbiota development, the maternal microbiome transfer at vaginal or caesarean birth and thereafter on the infant, and correlate parameters of microbiota and maternal milk on infant growth, development, health, and mental well-being. DISCUSSION: The Bern birth cohort study will provide a detailed description and normal ranges of the trajectory of microbiota maturation in a high-resource setting. These data will be compared to data from low-resource settings such as from the Zimbabwe-College of Health-Sciences-Birth-Cohort study. Prospective bio-sampling and data collection will allow studying the association of the microbiota with common childhood conditions concerning allergies, obesity, neuro-developmental outcomes , and behaviour. Trial registration The trial has been registered at www. CLINICALTRIALS: gov , Identifier: NCT04447742.

[Insights into the Pathogenesis of Inflammatory Bowel Diseases: Genetics and Microbiota]. / Einblicke in die Pathogenese der chronisch entzündlichen Darmerkrankungen: Genetik und Mikrobiota.

Insights into the Pathogenesis of Inflammatory Bowel Diseases: Genetics and Microbiota Abstract. An inadequate immune response against bacteria of the gastrointestinal tract is the basic mechanism mediating the pathophysiology of inflammatory bowel diseases (IBD). The risk of IBD is partially heritable and approximately 12 % of patients have a family history of IBD. Large genome-wide association studies (GWAS) were able to identify 240 genetic regions associated with IBD. Many of the implicated genes have a function in the immune system, are associated with primary immunodeficiencies or the defense against mycobacteria. Together these 240 genetic regions form an excellent framework for further investigations into the pathogenesis and therapy of IBD. However, GWAS so far were able to unravel only a fraction of the genetic IBD risk. New strategies like genome wide sequencing are currently used to identify additional (rare) genetic variants. In rare cases, IBD is also inherited as a monogenetic disease. Moreover, there likely is significant interaction between genes and environmental factors which can only be unraveled if both, genes and the environment are simultaneously considered. Interestingly, the information provided by genetic risk factors for IBD is unable to predict the clinical course of IBD. New GWAS therefore focus on IBD prognosis and first insights have already been made. The gastrointestinal tract harbors a huge number of microorganisms (microbiota). It remains an enormous challenge for the immune system to contain this bacterial load while enabling the host to benefit from the many essential contributions of the microbiota. In IBD, the microbiota is altered to a dysfunctional (dysbiotic) state showing reduced diversity and a higher amount of potential pathogenic Proteobacteriae, such as Escherichia coli. In IBD, the microbiota is also more dynamic in its composition over time compared to health. Further, IBD dysbiosis is more pronounced in Crohn's disease than in ulcerative colitis. In animal experiments, dysbiosis could be transferred by fecal microbiota transplantation from one mouse to another, triggering inflammation in the recipient. In contrast, a healthy microbiota can downregulate the immune response of the host, for instance by bacterial short chain fatty acids (SCFA) synthesis. In addition, some bacteria with close physical contact to the intestinal wall also have specific immunosuppressive properties. So far, the highly complex network of microbiota, genetics, immune system and environment is only partially understood. The microbiota is a potential therapeutic target which up to now can only be non-specifically influenced by antibiotics, probiotics, prebiotics or fecal microbiota transplantation. A better understanding of the microbiota will likely yield in the discovery of new therapeutic options in the future.

Benchmarking microbial DNA enrichment protocols from human intestinal biopsies.

Shotgun metagenomic sequencing is a powerful tool for studying bacterial communities in their natural habitats or sites of infection, without the need for cultivation. However, low microbial signals in metagenomic sequencing can be overwhelmed by host DNA contamination, resulting in decreased sensitivity for microbial read detection. Several commercial kits and other methods have been developed to enrich bacterial sequences; however, these assays have not been tested extensively for human intestinal tissues yet. Therefore, the objective of this study was to assess the effectiveness of various wet-lab and software-based approaches for depleting host DNA from microbiome samples. Four different microbiome DNA enrichment methods, namely the NEBNext Microbiome DNA Enrichment kit, Molzym Ultra-Deep Microbiome Prep, QIAamp DNA Microbiome kit, and Zymo HostZERO microbial DNA kit, were evaluated, along with a software-controlled adaptive sampling (AS) approach by Oxford Nanopore Technologies (ONT) providing microbial signal enrichment by aborting unwanted host DNA sequencing. The NEBNext and QIAamp kits proved to be effective in shotgun metagenomic sequencing studies, as they efficiently reduced host DNA contamination, resulting in 24% and 28% bacterial DNA sequences, respectively, compared to <1% in the AllPrep controls. Additional optimization steps using further detergents and bead-beating steps improved the efficacy of less efficient protocols but not of the QIAamp kit. In contrast, ONT AS increased the overall number of bacterial reads resulting in a better bacterial metagenomic assembly with more bacterial contigs with greater completeness compared to non-AS approaches. Additionally, AS also allowed for the recovery of antimicrobial resistance markers and the identification of plasmids, demonstrating the potential utility of AS for targeted sequencing of microbial signals in complex samples with high amounts of host DNA. However, ONT AS resulted in relevant shifts in the observed bacterial abundance, including 2 to 5 times more Escherichia coli reads. Furthermore, a modest enrichment of Bacteroides fragilis and Bacteroides thetaiotaomicron was also observed with AS. Overall, this study provides insight into the efficacy and limitations of various methods for reducing host DNA contamination in human intestinal samples to improve the utility of metagenomic sequencing.