Ecological Activities in Childcare Microbiome

Abstract

The field of human and environmental microbiome research has undergone remarkable growth over the past two decades, driven by technological innovations that allow us to characterize complex microbial communities without traditional culture-based approaches. Despite these advances, several fundamental challenges persist: terminology inconsistencies that hinder communication among microbiome researchers, incomplete characterization of microbial transmission dynamics in key developmental settings like childcare centers, and difficulty distinguishing viable from non-viable microorganisms in community samples. This dissertation addresses these interconnected challenges through three distinct yet complementary investigations that collectively advance both the technical foundations and applied aspects of microbiome science, with particular attention to early-life exposures in built environments where children spend significant portions of their developmental years. Chapter 1 examines the evolving lexicon of microbiome research, highlighting how inconsistent terminology has created confusion in the field. Through critical analysis of common terms like "microflora" and "metagenomics," we demonstrate how shifting definitions and misapplications have complicated research interpretation. The chapter provides a structured review of computational strategies for microbiome data analysis, distinguishing between taxonomic and functional approaches derived from various meta'omic methods. By organizing and clarifying vocabulary for major classes of microbiome analysis and their resulting feature tables, this work establishes a framework for more precise communication, enhancing the field's ability to address specific questions about microbial community composition and function with appropriate methodological alignment. Chapter 2 applies advanced multi-omic techniques to investigate microbial transmission in childcare facilities—critical environments where young children experience formative microbial exposures during immune development. This represents the first comprehensive study integrating microbiome samples from childcare environments and children using full-length amplicon sequencing and both short-read and long-read metagenomic approaches. The findings reveal distinct environmental microbial signatures, with human-associated microorganisms predominating on high-touch surfaces while greater taxonomic diversity characterizes low-touch areas. The identification of transmission pathways between children and their environment, including food-associated microbes like Lactococcus lactis and Streptococcus thermophilus, provides insights into microbial exposure routes during early development. Additionally, the improved genomic resolution afforded by paired sequencing technologies enabled detection of lateral gene transfer events and characterization of bacteriophage ecology, highlighting how enhanced methodological approaches can reveal previously understudied aspects of environmental microbiomes. Chapter 3 addresses the fundamental challenge of distinguishing viable from non-viable microorganisms in complex communities. This work develops a novel, high-throughput approach based on sequencing multiple optimized marker gene transcripts to identify actively transcribing microbes. Through rational marker gene selection, in silico primer design, and optimized amplification protocols validated against paired metagenomic and metatranscriptomic datasets, the method provides an accessible alternative to more costly and technically demanding techniques. The resulting protocol demonstrated effectiveness in distinguishing viable microbes across synthetic, human, and built environment samples, offering a scalable solution for identifying and quantifying functional community members in diverse microbiome samples. Together, these chapters represent a comprehensive contribution to microbiome research methodology and application. By addressing terminological precision, environmental context, and viability assessment, this work provides a foundation for more rigorous and biologically meaningful microbiome studies with applications spanning environmental monitoring, clinical diagnostics, and public health interventions.