Quantitative multi-kingdom profiling of a neonatal ICU cohort gut microbiome samples using cell-based spike-in; plus additional samples to support Multi-Kingdom SpikeSeq and confirm mouse colonization CFU data.

The mammalian microbiota is a dynamic ecosystem – a community that transitions throughout early-life, and is critical to shaping host health. While next-generation sequencing (NGS) has intricately mapped the composition of the microbiome, disentangling the environmental, host and microbial factors driving ecosystem change has been challenging. A major barrier to this has been reliance on single-kingdom, compositional data – studies track the proportion of different bacteria but not their absolute abundances1, nor other community members, confounding our abilities to fully understand ecosystem dynamics. Here, we develop a scalable NGS-based pipeline to sensitively and robustly measure absolute abundances of three microbial kingdoms within a sample. We use this to quantify the dynamics of bacterial, fungal, and archaeal communities in the developing preterm infant gut. We reveal dramatic changes in microbial loads within and between infants during early-life, uncovering dynamics of specific taxa masked by relative abundances and an inverse correlation between bacterial and fungal loads. Applying ecological models, we discover and validate in vitro and in vivo causal drivers of microbiota dynamics. We find that specific pioneer bacteria facilitate the colonization of others, and identify a fungal species that inhibits the Enterobacteriaceae, a major bacterial family encompassing pathogenic and drug-resistant organisms. Together, our work unveils the power of within- and between-kingdom microbial interactions to shape microbial ecosystems, a critical step in engineering microbiomes for human health.