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

• Citro_Ciprofloxacin and Ger_ciprofloxacin show a strong activity against S. aureus . • Ciprofloxacin loses antibacterial and antibiofilm activity against the resistant strain. • Hybrids retain potent antibacterial activity against the ciprofloxacin-resistant strain. • Hybrids show strong antibiofilm effect in both susceptible and resistant strains. • Hybrids outperform ciprofloxacin-monoterpenoid combinations while using lower doses. • Hybrids disrupt biofilm structure with a clear reduction in thickness in both strains. • Hybrids damage biofilm-cell membranes while showing no detectable HepG2 toxicity. 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. This study aimed to develop novel hybrid molecules combining ciprofloxacin with plant-derived monoterpenoids to improve antibacterial and antibiofilm efficacy against S. aureus strains. 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. For the resistant strain (ciprofloxacin MIC > 1024 µg mL −1 ), the hybrids showed MICs of 16 µg mL −1 , indicating a limited shift in activity compared to the susceptible strain (MIC = 8 µg mL −1 ). 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 cm −2 ). 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. 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.