UC Berkeley

Microbial commensals can stably colonize the adult gut for years and play key roles in host metabolism and pathogenic defense. However, much less is known about persisting early colonizing strains in infants. Given their early-arrival time, it is plausible that they play critical roles in shaping the trajectory of the assembly of the infant gut microbiome and the maturation of the immune system. The importance of persisting early colonizers in the early life gut microbiome and overall infant health, thus, motivated us to identify those strains and to investigate factors that contribute to their persistence. In my first two chapters, I leveraged longitudinal metagenomics data to explore the topic of persistence in two separate yet evolutionarily intertwined entities: bacteria (Chapter 1) and viruses that predate bacteria, bacteriophages (Chapter 2).

In Chapter 1, I performed strain-resolved analyses of fecal samples of premature and full-term infants collected throughout their first year of life and identified that ~11% of the bacterial colonizers persisted in the infant gut for nearly one year. I then evaluated factors associated with strain persistence and found that bacterial persisters, particularly Bacteroides and Bifidobacterium, were more prevalent in full-term than in preterm infants, and were primarily maternally transmitted. Comparative genomic analyses revealed that strains encoding genes for surface adhesion, iron acquisition, and carbohydrate degradation are more likely to persist in the infant gut than strains lacking these genomic traits.

Bacteriophages (phages) prey on bacteria for survival. Despite their early detection in the infant gut, little is known about the roles they play in gut microbiome assembly. In Chapter 2, I applied strain-resolved metagenomics to examine phage persistence in pre- and full-term infants during their first three years of life. The collection of maternal fecal samples that were three years apart enabled me to not only identify persisting phage strains in mothers but also evaluate the influence of maternal virome on the development of infant gut viromes. I found that maternal gut viromes were more stable than those of infants, with ~20% of phage strain persisting for three years, whereas only ~9% of phages persisted in infants. Similar to bacterial persisters during infants’ first year of life, I also observed that maternally transmitted phages were more likely to colonize the infant gut long-term. Population genetic analyses suggested that phage persisters had a significantly higher nucleotide diversity than non-persisters during the initial colonization phase. This could be partly attributed to the presence of diversity-generating retroelements (DGRs), as these genetic elements were enriched in phage persisters. Further, we found that phages that used an alternative genetic code (recoding of either the TAG or TGA stop codon) were more likely to persist than phages that used standard code. Overall, insights from the first two chapters have implications for developing more effective microbiome interventions such as probiotics and/or phage-based microbiome engineering.

High-throughput metagenomics studies have significantly advanced our understanding of microbial communities from all natural ecosystems. However, this type of analysis is susceptible to contamination, especially those that are internally derived. We noticed signs of within-study, cross-sample contamination when analyzing data for my first chapter, which motivated an in-depth investigation of contamination in metagenomics datasets. In my Third Chapter, I developed a strain-based framework for the detection of internally- and externally-derived contaminants and applied it to two large-scale clinical studies. I demonstrated insidious contamination in metagenomics datasets and flagged the necessity of routinely assessing contamination beyond negative and positive controls. The research provided a devised strain-resolved workflow that is generalizable and can be applied to any microbiome dataset.

Gut microbial interactions directly influence human health. However, the complexity and individuality of the gut microbiomes, as well as the lack of tractable gut microbiome models, have impeded us from linking pivotal gut consortium interactions to resulting host-associated phenotypes of interest. To decipher microbial interactions in their native communities, in my Fourth and Final Chapter, I established stable, reproducible, and person-specific laboratory gut consortia using clinically relevant infant stool samples. I then used these laboratory microbiomes, along with metagenomics functional predictions, to investigate mechanisms explaining infant-specific responses to 2'-fucosyllactose (2’FL), a prevalent human milk oligosaccharide (HMO) and a common infant formula additive. I focused on the growth of Bifidobacterium breve, a prevalent infant gut commensal that plays key roles in early-life gut microbiome assembly and immune system development. B. breve typically cannot metabolize 2’FL on its own, yet in some infants, it demonstrated 2’FL-induced growth. Leveraging infant gut microbiome cultivation and genome-resolved functional analyses, I found that the presence of community members encoding extracellular fucosidases was critical for the enrichment of B. breve when 2’FL was supplemented. Together, this work uncovered key, yet infant-specific, microbial interactions in metabolizing a common dietary component. Given the significant health impacts of Bifidobacterium in infant development, this work also provided strategies for enhancing the colonization of autochthonous B. breve and possibly other Bifidobacterium species. Over time, insights gained from these individualized in vitro microbiomes can lead to the development of more precise and effective personalized microbiome treatments.