Zataria multiflora essential oil: a potent agent with antibacterial, anti-NorA efflux pump and anti-biofilm activity against ciprofloxacin-resistant Staphylococcus aureus isolates from bovine mastitis.

Bovine mastitis is a prevalent and economically significant disease in dairy cattle, leading to reduced milk production and quality. Staphylococcus aureus is a major causative agent of bovine mastitis, often complicating treatment due to its ability to develop antibiotic resistance. This resistance is frequently mediated by efflux pumps, such as the NorA pump. The aim of this study was to investigate the potential of Zataria multiflora essential oil (EO) to inhibit biofilm formation and disrupt the NorA efflux pump in ciprofloxacin-resistant S. aureus isolates from bovine mastitis.The plant EO was extracted using the hydrodistillation method, and its composition was determined by Gas Chromatography-Mass Spectrometry (GC-MS) analysis. The antibacterial and anti-biofilm activities of the EO were assessed against 7 clinical ciprofloxacin-resistant S. aureus isolates from bovine mastitis. The NorA efflux pump inhibitory (EPI) potential of the EO was assessed using the checkerboard assay against S. aureus norA overexpressed (SA1199B strain) and clinical isolates. Then, the expression of the norA gene at sub-minimum inhibitory (sub-MICs) concentrations levels was also determined. Finally, the cytotoxicity of the Z. multiflora essential oil was assessed using the MTT assay on the Vero cell line. The potent antibacterial activity of Z. multiflora EO against clinical and standard strains was found, with MIC values ranging from 0.25 to 64 µg.ml. The tested EO demonstrated significant anti-biofilm activity at MIC/2 and MIC/4 concentrations. In all clinical and norA overexpressed strains, Z. multiflora EO had a total or partial synergistic effects with ciprofloxacin and ethidium bromide with fractional inhibitory concentration index (FIC) < 2, indicating EPI activity. At MIC/2 and MIC/4 concentrations, Z. multiflora EO significantly downregulated the norA gene (P < 0.01). Z. multiflora EO showed no significant cytotoxicity towards human cells at MIC and 2MIC concentrations. In conclusion, Zataria multiflora EO exhibited promising antibacterial, anti-biofilm and EPI properties against ciprofloxacin-resistant S. aureus isolated from bovine mastitis. Moreover, it showed no significant cytotoxicity towards mammalian cells, suggesting its potential as a safe and effective therapeutic agent for bovine mastitis. Further investigations are warranted to explore its efficacy in vivo.