Monoterpenoid-ciprofloxacin hybrids as a strategy to disrupt Staphylococcus aureus biofilms and overcome associated resistance

Abstract

Introduction Staphylococcus aureus is an important opportunistic pathogen whose ability to form biofilms and acquire multidrug resistance limits the effectiveness of conventional antibiotics. Although some antibiotic combinations have shown promise, their clinical use is limited by pharmacokinetic and pharmacodynamic issues, chemical instability and formulation challenges. Objectives This study aimed to develop novel hybrid molecules combining ciprofloxacin with plant-derived monoterpenoids to improve antibacterial and antibiofilm efficacy against S. aureus strains. Methods Based on preliminary antibiotic-terpenoids combination studies in S. aureus CECT 976, citronellol and geraniol were selected for hybrid synthesis. The minimum inhibitory concentration (MIC) of the hybrids was evaluated in ciprofloxacin-susceptible ( S. aureus CECT 976) and resistant strains ( S. aureus 8093). Furthermore, the antibiofilm efficacy was analysed in the prevention and eradication of pre-established biofilms by quantifying biomass, metabolic activity and culturability. Moreover, their impact on biofilm structure and membrane integrity was investigated using optical coherence tomography, flow cytometry and epifluorescence microscopy, respectively. Furthermore, matrix components were analysed by spectrophotometry and confocal laser scanning microscopy (CLSM). The cytotoxic and haemolytic profile of the hybrids were evaluated using mammalian cells and red blood cells, respectively. Results For the resistant strain (ciprofloxacin MIC > 1024 µg mL1), the hybrids showed MICs of 16 µg mL1, indicating a limited shift in activity compared to the susceptible strain (MIC = 8 µg mL1). For pre-established biofilms of the S. aureus resistant strain, where ciprofloxacin was ineffective, the hybrids reduced up to 40 % of biomass, inhibited 90 % of metabolic activity and reduced culturability by ~ 2.3 log(CFU cm2). They also disrupted the biofilm structure, with an average thickness of less than 10 µm in both strains tested. Furthermore, the hybrids caused significant damage to the cell membrane (90 % in the susceptible strain and about 35 % in the resistant strain) and exhibited low toxicity in HepG2 cells. Consistent with confocal observations, alterations in matrix composition were also observed in the presence of the hybrid compounds. Conclusion This study highlights the potential of monoterpenoid-ciprofloxacin hybrids that combine the antibacterial effects of ciprofloxacin with the bioactive properties of monoterpenes, potentially affecting S. aureus biofilms through multiple mechanisms. Copyright (c) 2026. Published by Elsevier B.V.