Antibacterial Activity of Ikarugamycin against Intracellular <i>Staphylococcus aureus</i> in Bovine Mammary Epithelial Cells In Vitro Infection Model

<i>Staphylococcus aureus</i> is an ubiquitous and versatile pathogen associated with a wide range of diseases. In animals, this bacterium is one of the causative agents of bovine mastitis, responsible for huge economic losses in the dairy industry. Besides the development of antibiotic resistance, the intracellular survival of <i>S. aureus</i> within udder cells has rendered many antibiotics ineffective, leading to therapeutic failure. Our study therefore aims to investigate the in vitro bactericidal activity of ikarugamycin (IKA) against intracellular <i>S. aureus</i> using a bovine mammary epithelial cells (Mac-T cells) infection model and determine the cytotoxic effect. Minimum inhibitory concentration (MIC) was used to determine the antibacterial activity of IKA, and Mac-T cells were infected with <i>S. aureus</i> using gentamicin protection assay. IKA intracellular antibacterial activity assays were used to determine the bactericidal activity of IKA against intracellular <i>S. aureus</i>. The cytotoxicity of IKA against Mac-T cells was evaluated using the resazurin assay. We showed that, <i>S. aureus</i> is susceptible to IKA with a MIC value of 0.6 μg/mL. IKA at 4 × MIC and 8 × MIC have bactericidal activity by reducing 3 and 5 logs₁₀ CFU/mL of <i>S. aureus</i> in the first six-hour of treatment respectively. In addition, IKA demonstrated intracellular killing activity by killing 90% of intracellular <i>S. aureus</i> at 5 μg/mL. This level is comparatively lower than 9.2 μg/mL determined as the half-maximal inhibitory concentration (IC₅₀) of IKA required to kill 50% of Mac-T cells, highlighting a lower concentration required for bactericidal effect compared to the cytotoxic effect. The study highlighted that importance of IKA as a potential antibiotic candidate to be explored for the in vivo efficacy in treating <i>S. aureus</i> mastitis.