# Fosfomycin at sub-minimum inhibitory concentration impairs biofilm and efflux pump activity in multidrug-resistant Klebsiella pneumoniae isolates

## TL;DR

Fosfomycin at low doses can weaken biofilms and reduce efflux pump activity in drug-resistant Klebsiella pneumoniae, suggesting it could help fight antibiotic resistance.

## Contribution

This study shows fosfomycin at sub-MIC levels impairs biofilm and efflux pump activity in MDR Klebsiella pneumoniae.

## Key findings

- Sub-MIC fosfomycin significantly reduced biofilm formation in MDR K. pneumoniae isolates.
- Fosfomycin downregulated biofilm-related genes (fimH, mrkD) and efflux pump genes (acrA, acrB).
- SEM revealed morphological changes, including cauliflower-like deformation in treated isolates.

## Abstract

Klebsiella pneumoniae plays a critical role in hospital-acquired infections, which pose a serious threat globally. Older antibiotics such as fosfomycin are being reconsidered to combat MDR K. pneumoniae. In healthcare settings, bacteria are exposed frequently to antibiotics at sub-inhibitory concentrations (sub-MICs), which may alter their virulent characteristics. The aim of this research was to investigate the impact of fosfomycin at sub-MICs on the virulence determinants of MDR K. pneumoniae.

The effect of sub-MIC fosfomycin treatment on biofilm formation was evaluated by crystal violet assay and confocal laser scanning microscopy (CLSM). The cartwheel technique was employed to study efflux pump activity phenotypically. The expression of genes of biofilm (fimH, mrkD) and efflux pump (acrA, acrB) was determined using reverse transcription quantitative PCR (RT-qPCR). Scanning electron microscopy (SEM) was also utilized to visualize the morphological changes.

The MIC of fosfomycin against K. pneumoniae MDR isolates ranged from 256 to 1024 µg/mL. The biofilm formation ability of isolates (n = 50) was reduced significantly (P < 0.05) following the exposure to ¼ MIC of fosfomycin. CLSM analysis revealed disruption of biofilm structure, reduced thickness and density, and increased percentages of dead cells. RT-qPCR revealed 20–60% downregulation of fimH and mrkD and downexpression of acrA and acrB. SEM analysis showed pronounced morphological changes, including a characteristic cauliflower-like deformation.

Fosfomycin at sub-inhibitory levels is able to disrupt biofilm architecture and the efflux pump activity in MDR K. pneumoniae, pointing to its possible role as an adjunct agent in combating antibiotic resistance.

The online version contains supplementary material available at 10.1186/s12866-026-04720-6.