Modulators of gene amplification alter evolution of antibiotic resistance in Staphylococcus aureus
Kalinga Pavan T. Silva, Anthony M. Martini, Anupama Khare
Abstract
Gene amplification is thought to be common in bacterial populations, providing a rapid and reversible mode of adaptation to diverse stresses, including the acquisition of antibiotic resistance. We previously showed that the opportunistic pathogen Staphylococcus aureus evolves resistance to the dual-targeting fluoroquinolone delafloxacin (DLX) that inhibits both the DNA gyrase and DNA topoisomerase IV via amplification of an efflux pump encoding gene sdrM.
Introduction
Many antibiotics used to treat bacterial infections are growing increasingly ineffective as clinical and environmental bacteria acquire resistance via horizontal gene transfer or de novo mutation-mediated evolution [1,2]. Such antimicrobial resistance is a major threat to global public health [3,4]. Staphylococcus aureus, especially methicillin-resistant S. aureus (MRSA), commonly acquires resistance to a broad range of antibiotics, and is a major cause of antibiotic resistant infections world-wide [5,6].
Materials and methods
Strains and growth conditions
All strains and plasmids used in this study are listed in S9 Table.
For experiments in liquid media, bacteria were grown at 37oC, shaking at 300 rpm in modified M63 media (13.6 g/L KH2PO4, 2g/L (NH4)2SO4, 0.4 μM ferric citrate, 1mM MgSO4; pH adjusted to 7.0 with KOH) supplemented with 0.3% glucose, 0.1 μg/mL biotin, 2 μg/mL nicotinic acid, 1 × Supplement EZ (Teknova) and 1 × ACGU solution (Teknova) [16]. For experiments with 96-well or deep well plates, the plates were incubated in a Titramax 1000 (Heidolph) incubator at 37oC shaking at 900 rpm.
Results
Resistance evolution via gene amplification requires the recombinase RecA
Gene amplification is thought to be a two-step process where an initial duplication event is mediated by either homologous or non-homologous recombination, and is followed by higher order amplification via RecA-mediated homologous recombination [8]. Thus, while the initial duplication may be RecA-dependent or independent, RecA is thought to be essential for the subsequent gene amplification [20].
Discussion
Gene amplification can lead to resistance against many distinct antibiotics in S. aureus [8], but modulators of amplification in S. aureus have not been described. In this study we used adaptive evolution to investigate the genetic effectors involved in sdrM gene amplification, and the selection and maintenance of amplified segments, that lead to DLX resistance in MRSA (Fig 8).
Acknowledgments
We would like to acknowledge the Center for Cancer Research (CCR) Genomics Core for whole-genome sequencing. This work used the computational resources of the NIH High Performance Computing Biowulf Cluster (http://hpc.nih.gov).
Citation: Silva KPT, Martini AM, Khare A (2025) Modulators of gene amplification alter evolution of antibiotic resistance in Staphylococcus aureus. PLoS Genet 21(12): e1012011. https://doi.org/10.1371/journal.pgen.1012011
Editor: Sean Crosson, Michigan State University, UNITED STATES OF AMERICA
Received: November 5, 2025; Accepted: December 22, 2025; Published: December 31, 2025
This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.
Data Availability: The whole genome sequencing (WGS) data have been deposited at NCBI Short Read Archive (SRA) under the bioproject PRJNA1242835. Raw data for figures is either in specific Supporting Tables, or in S1 Data.
Funding: This work was supported by the Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research. The contributions of the NIH authors are considered Works of the United States Government. The findings and conclusions presented in this paper are those of the authors and do not necessarily reflect the views of the NIH or the U.S. Department of Health and Human Services. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
