# Elucidation of population-based bacterial adaptation to antimicrobial treatment by single-cell sequencing analysis of the gut microbiome of a hospital patient

## TL;DR

This study uses single-cell sequencing to track how gut bacteria adapt to antibiotics in a hospital patient, revealing new resistance genes and horizontal gene transfer.

## Contribution

The study introduces single-cell sequencing as a novel method to detect and trace antibiotic resistance gene evolution and horizontal transfer in unclassified and known bacterial species.

## Key findings

- Single-cell sequencing detected antibiotic resistance genes (ARGs) in 92 bacterial species, including unclassified strains.
- The cfr(C) gene was found in 11 species, with distinct mutation patterns in Klebsiella pneumoniae and other taxa.
- 309 horizontal gene transfer events were identified, involving genes like folE and queE that aid DNA repair in antibiotic-treated bacteria.

## Abstract

In this study, we used single-cell sequencing to analyze the gut microbiome of an adult male patient with acute cerebral hemorrhage undergoing antibiotic treatment. We identified 92 bacterial species, including 23 Firmicutes and one archaeon from Methanobacteriota, along with 69 unclassified strains. Single-cell sequencing effectively detected bacteria carrying antibiotic resistance genes (ARGs), particularly in unclassified species, and traced the evolution of these genes across diverse bacterial taxa. Notably, the cfr(C) gene was detected in 11 bacterial species following antimicrobial treatment, with mutation patterns characterized in Enterococcus faecalis, Klebsiella pneumoniae, Ruthenibacterium UN-1, and four unclassified species. In total, 29 ARG subtypes across eight types were identified in 13 known, five unknown, and 18 unclassified species, allowing us to trace their evolution routes. In addition, we detected a total of 309 horizontal gene transfer (HGT) events, in which several genes like folE and queE were frequently involved. The products of these genes are known to enhance the ability of the recipient bacterial strains to repair DNA damage and maintain genomic stability, especially following prolonged antibiotic treatment. Comparison between isolated strain genomes (IS-KP1) and single-cell analysis confirmed the presence of at least two K. pneumoniae strains in the patient, with one exhibiting a larger extent of involvement in ARG co-evolution. This strain was found to contain the cfr(C) and fosXCC genes, which were absent in IS-KP1. Klebsiella strains were also found to participate actively in HGT events. In conclusion, the study identified a wide range of ARGs and HGT events within the microbiome. The detection of K. pneumoniae strains with distinct ARG evolution patterns underscores the gut microbiome’s adaptability to environmental changes. These findings facilitate the development of novel antimicrobial strategies by fine-tuning the gut microbiome composition.

This study highlights the power of single-cell sequencing to unravel the diversity and dynamics of the gut microbiome during antibiotic treatment in a patient with acute cerebral hemorrhage. By identifying antibiotic resistance genes (ARGs) in both known and unclassified bacterial species, we reveal the intricate evolution and horizontal transfer of resistance traits across taxa. The discovery of distinct ARG patterns, including the emergence of the cfr(C) gene in multiple species and its co-evolution in K. pneumoniae, underscores the gut microbiome’s adaptability to antimicrobial pressures. These findings provide critical insights into the mechanisms driving resistance dissemination and offer potential pathways for developing precision microbiome-based therapies to combat antibiotic resistance.