Characterization of antimicrobial resistance mechanisms in clinically isolated Enterococcus faecalis using whole-genome sequencing and transcriptomic profiling

Project Abstract

Enterococcus faecalis is a leading cause of nosocomial infections and a notable reservoir of antimicrobial resistance (AMR) genes, contributing to treatment failures and increased morbidity. This study integrates whole-genome sequencing (WGS) and transcriptomic profiling to characterize genetic determinants and expression dynamics underlying AMR in clinically isolated E. faecalis strains collected from diverse hospital units over a 24-month period. We performed high-throughput WGS to identify resistance genes, mobile genetic elements, virulence factors, and single-nucleotide polymorphisms associated with resistance phenotypes. Parallel RNA-sequencing under baseline growth conditions and antibiotic exposure (beta-lactams, aminoglycosides, glycopeptides, and fluoroquinolones) elucidated differential gene expression patterns that reflect adaptive responses and potential regulatory networks governing resistance mechanisms. Phenotypic antimicrobial susceptibility testing (AST) and MIC determinations were conducted to correlate genotypic data with observed resistance, while conjugation and plasmid analyses assessed horizontal transfer potential of key resistance determinants. Our results reveal a complex resistome composed of intrinsic and acquired determinants, including genes mediating cell wall synthesis modification, efflux pump regulation, target protection, and enzymatic modification or inactivation of antibiotics. Notably, we identified widespread presence of van operons and a repertoire of aminoglycoside-modifying enzymes embedded in diverse plasmids and transposons, suggesting active dissemination among clinical isolates. WGS also uncovered lineages with mosaic genomes attributable to recombination events that co-localize resistance genes with virulence-associated loci, highlighting the potential for convergent evolution in hospital environments. Transcriptomic data demonstrated condition-dependent upregulation of resistance determinants, such as upregulation of vancomycin resistance machinery in response to glycopeptide exposure and induction of multiple efflux systems during fluoroquinolone challenge. Signaling pathways implicating two-component regulatory systems, global stress responses, and quorum-sensing elements were implicated in orchestrating adaptive transcriptional responses, indicating robust regulatory networks that enhance survival under antimicrobial pressure. Integrative analysis linked specific genetic contexts to distinct expression profiles, enabling the delineation of genotype-phenotype associations and potential biomarkers for rapid AMR detection. Phylogenomic and pan-genomic analyses delineated clonal clusters with heightened resistance burdens, suggesting nosocomial propagation in addition to intrinsic resistance traits. The study provides a comprehensive map of the resistome and its transcriptional dynamics in clinically relevant E. faecalis, identifying candidate targets for therapeutic intervention and informing infection control strategies. The findings underscore the importance of combining genome-scale genotypic and transcriptomic approaches to capture both potential resistance capabilities and actualized responses under therapeutic stress, thereby improving diagnostic accuracy and guiding personalized antimicrobial stewardship in hospital settings. This integrated framework sets the stage for longitudinal surveillance of AMR evolution in E. faecalis and highlights critical determinants that may drive future resistance outbreaks.

Project Overview

What This Project Is About

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A plain-language overview of studying how certain bacteria called Enterococcus faecalis become resistant to antibiotics. The project uses two modern methods: whole-genome sequencing, which reads all of the DNA in a bacteria, and transcriptomic profiling, which looks at which genes are active. By combining these, we aim to understand which genes and pathways help the bacteria survive antibiotic treatment and how these defenses appear in real clinical samples.

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The Problem It Addresses

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Antibiotic-resistant Enterococcus faecalis infections are harder to treat and can lead to longer illness and more deaths. Current knowledge often doesn’t reveal exactly how resistance is built inside individual bacteria. This project tackles the gap by linking genetic information with gene activity to identify real-world resistance mechanisms that could inform better therapies and stewardship.

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Objectives of the Project

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1. Identify the key resistance genes present in clinically isolated Enterococcus faecalis strains.
2. Profile which genes are turned on or off in response to antibiotic exposure.
3. Correlate genomic features with observed resistance patterns in the lab.
4. Map potential gene networks that contribute to resistance.
5. Suggest targets for future diagnostic tests or treatments.

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What You Will Do Step by Step

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1. Collect or obtain clinically isolated strains with known resistance profiles.
2. Extract DNA and perform whole-genome sequencing to identify resistance genes.
3. Expose bacteria to antibiotics and extract RNA for transcriptomic profiling.
4. Analyze gene expression data to see which genes respond to treatment.
5. Integrate genomic and transcriptomic results to identify key resistance pathways.
6. Validate findings with targeted laboratory tests where feasible.

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Expected Outcome

Clear identification of major resistance genes and the active pathways behind them, plus a set of candidate markers for diagnostics and possible ideas for new treatment approaches. The project should yield a practical framework for linking genome data to functional resistance behavior in Enterococcus faecalis.