Antimicrobial resistance gene dynamics in the infant gut microbial ecosystem

The bacteria in the human gut outnumber the total number of cells in the human body, and these microbes play important roles in health and disease. Establishment of the so-called gut microbiome begins during the birthing process and develops throughout infancy to eventually form a mature and stable ecosystem that provides vital services to the host. The infant gut microbiome is known to act as a reservoir for genes that can confer resistance to antibiotic drugs, and the spread of such genes to pathogens is considered one of the main problems in global public health. However, we still lack basic knowledge of the gut microbiomes maturation process during infancy. In this project, we will develop detailed models of how the total bacterial gene repertoire of the infant gut microbiome develops over time, with a special focus on how antibiotic resistance genes accumulate in, and spread among, members of the gut bacterial community. In order to achieve this goal we will employ the latest in high-throughput DNA sequencing technology for describing the bacterial gene content in fecal samples, collected at a high frequency during the first year of life, from a cohort of 12 healthy infants born in Norway. We will refine our models by analyzing a larger number of samples from the comprehensive Norwegian PreventADALL study, in which fecal samples were collected from more than 2000 infants at 3,6 and 12 months of age. This will provide better estimates of natural variability in the timing of key events during early development, as well as impacts of antibiotic use during infancy. A primary goal of the project is to identify resistance profiles associated with different developmental stages. This knowledge will allow clinicians to make better decisions about the types of antibiotics that are best suited for use in young children during specific age windows. So far, we have completed deep metagenomics sequencing of 150 samples from our time series cohort of 12 healthy infants. We have established routines for preparation of large numbers of DNA sequencing libraries for parallel sequencing of the Illumina NovaSeq 6000 platform, the highest throughput technology available as of today. This apparatus can output >10 Tb of sequence data in a single run, and is thus highly appropriate for carrying out a project of this magnitude. We have established bioinformatics pipelines for downstream sequence data processing, including quality control, genome assembly, taxonomic analysis, and annotation of antibiotic resistance marker genes. This has produced a unique data set with regard to temporal resolution and the sheer amount of genetic information available. By analyzing thousands of community-assembled genomes we have identified a diverse pool of genes associated with antimicrobial resistance in healthy Norwegian infants. Along with collaborators at the University of Amsterdam we have started to look into evolutionary patterns in specific microbes in the infant microbiome, and with collaborators in England we are looking into methods for resolving ambiguous genome assemblies into microbial strains. During the summer of 2021 the project hired a PhD student whose main focus will be to look at antimicrobial resistance markers carried on mobile genetic elements, like plasmids or transposons. Such elements are not readily linked with metagenome-assembled genomes, and supplementary technologies, such as single cell techniques, will be required.

The adult human gastrointestinal (GI) microbiome is a stable and complex ecological community comprised of trillions of microorganisms that play vital roles in health and disease. Establishment of this community in the infant is widely recognized a fundamental developmental process with lasting health effects. It is noteworthy that the infant GI microbiome acts as a reservoir for antimicrobial resistance genes (ARGs). The spread of antimicrobial resistance is considered one of the main threats to global public health. The proposed project will provide essential knowledge on the temporal dynamics of ARGs in the developing infant. As part of a recently concluded project funded by the Research Council of Norway, we have analyzed the microbiota of nearly 2700 fecal samples from 12 Norwegian infants during the first year of life. In terms of temporal resolution this sample set is by far the densest of its kind, and our analysis has produced the most detailed picture to date of the human gut colonization process on the microbial population level. There remains, however, a knowledge gap concerning the normal infant gut microbiome maturation process. For example, we still do not understand the ecological mechanisms causing increased ARG carriage in infant microbiomes compared with adults. Our proposed project will drastically extend current knowledge by creating a detailed, time-resolved characterization of the developing infant gut microbiome during the first year of life. To accomplish this, we will employ community level shotgun metagenomic sequencing coupled with emulsion-PCR based techniques. The goal will be to develop conceptual and statistical modes of this vital developmental process with a particular focus on ARG dynamics. Furthermore, we will analyze a large subset of fecal samples (>100) from the comprehensive Norwegian PreventADALL study in order to map natural variability in the timing of key events during early development.