# Quantifying host-microbe interactions with bacterial lineage tracing

## TL;DR

Genomic barcoding tracks bacterial lineages in hosts to reveal infection dynamics, including bottlenecks, dissemination, and evolution.

## Contribution

A quantitative framework using DNA barcoding to study host-microbe interactions and infection processes.

## Key findings

- Barcoding reveals the size of the founding bacterial population and the impact of infection bottlenecks.
- Comparing barcodes between tissues identifies routes of bacterial dissemination.
- Dominant barcoded lineages indicate within-host bacterial evolution over time.

## Abstract

Using genomic barcodes to trace bacterial lineages within a host reveals previously unobservable dynamics of infection, including the impact of infection bottlenecks, routes of bacterial dissemination, and patterns of within-host evolution. Barcoding introduces trackable diversity to otherwise isogenic bacterial populations. Comparing the barcodes within an inoculum to those within the host quantifies the ‘founding population’, which reveals the magnitude of population collapse caused by host bottlenecks. Furthermore, comparisons of the founders between tissues can reveal the patterns of pathogen dissemination. On longer timescales, the emergence of dominant barcoded lineages can also be used to detect within-host evolution. Collectively, barcoding studies quantify the hidden parameters that underlie bacterial colonization and creates a quantitative framework for modeling and preventing infectious disease.

Following exposure to bacteria, individuals can become infected if some of the encountered microbes persist and replicate in the host. These ‘founders’ are the organisms that survive host defense processes, and their subsequent adaptation, replication, dissemination, and evolution govern the outcomes of infection. Despite their central importance in establishing infections, the identity of the founding organisms in a tissue is unobservable by traditional colony counting because such bulk measurements of bacterial numbers do not monitor individual clones. DNA barcoding overcomes this observational limitation by introducing genetic diversity without altering bacterial fitness. Analyses of barcode abundances determined by DNA sequencing reveal the identity of the underlying founding population and facilitate tracing the fates of the founders within and between hosts. Barcode-based approaches enable the monitoring of individual clones and the quantification of otherwise hidden facets of host-microbe interactions.

DNA sequencing-based lineage tracing offers a method for analyzing the dynamics of bacterial populations within the host, providing insights into the nature of infection bottlenecks, routes of bacterial dissemination, and patterns of within-host microbial evolution. The relationship between the dose of infectious challenge and the size of the founding population revealed by barcoding studies directly quantifies infection bottlenecks, which eliminate inoculated microorganisms prior to the establishment of founding populations. By quantifying the infection bottleneck, barcoding studies have uncovered host and microbial factors that determine susceptibility to infection, including the roles of stomach acid, innate immunity, vaccination, microbiota, bacterial motility, and microbial quorum sensing. Furthermore, by comparing the identity of founders isolated from different tissues, barcoded bacteria can reveal the paths of pathogen spread and provide high-resolution maps of when and where bacteria disseminate within the host. These studies have identified host and bacterial factors that control dissemination and host compartments that efficiently exchange bacteria; for instance, the gallbladder is a common reservoir for dissemination. Over longer time scales, barcodes can reveal the emergence of dominant lineages, which indicate the evolution of bacterial genotypes within the host in response to factors, including stress, inflammation, and pressure from the microbiota. Collectively, lineage tracing studies quantify the hidden parameters that underlie bacterial colonization and infection, thereby defining new roles for host and bacterial processes in the control of host-microbe interactions.

By quantifying bottlenecks and monitoring the trajectories of individual bacterial clones, barcoding and sequencing-based lineage tracing offer a new approach to address longstanding and fundamental questions in host-microbe interactions. Such questions include understanding why some individuals are more susceptible to infection and deciphering how microbes spread within and between hosts. We anticipate that the capacity to quantify founding lineages will uncover hidden patterns underlying infection, providing a new quantitative framework for modeling and preventing disease. These insights will facilitate the development of live microbial therapeutics by providing knowledge of the host bottlenecks that impede colonization of beneficial microbes. Furthermore, barcoding studies will guide strategies to prevent disseminated bacterial infections by unveiling the paths and timing of bacterial spread. These studies will also accelerate our understanding of how within-host evolution shapes the outcome of infection, which is particularly relevant in the era of increasing antimicrobial resistance. Other promising applications of bacterial barcoding include investigating the dynamics of the microbiome, defining the impacts of microbial heterogeneity, deepening mechanistic understandings of polymicrobial infections, and using barcoded challenge strains for human studies. Additional technological advancements, such as combining barcoding with imaging or temporal control of barcode diversity, will further enhance the utility and precision of these approaches. As high-throughput sequencing technologies become increasingly accessible, we anticipate that DNA barcoding will be a routine method for studying host-microbe interactions.

Bacterial lineage tracing decodes infection bottlenecks, dissemination patterns, and within-host evolution. Determining the size of the bacterial population within the host doesn’t capture the population’s history. Tracing bacteria with genomic barcodes and amplicon sequencing reveals the number of cells that initiate infection (the ‘founding population’) and quantifies bottlenecks. Comparisons of barcodes between organs reveal patterns of dissemination, and the bloom of a single lineage can reflect bacterial evolution. Created in BioRender.