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

SIMPLE SUMMARY: Antibiotics are widely used for the treatment and control of bovine mastitis. However, the treatment has only been partially effective, as the cure percentage only ranging from 10–30%. Infection by Staphylococcus aureus (S. aureus) is particularly difficult to treat due to the bacteria’s ability to enter and resides inside the host cells. Most antibiotics are ineffective against intracellular bacterial due to the poor penetration into host cells to achieve optimal intracellular bactericidal bioavailability levels. There is therefore, an increasing need to evaluate candidate active substances and develop novel antibiotics effective against intracellular persistence infection. In this study, we examine the potential antibacterial properties of ikarugamycin compound as an alternative drug candidate to be explored for treating persistent bovine mastitis caused by intracellular S. aureus using bovine mammary cell line as an in vitro infection model. We also assessed the potential cytotoxicity effect of ikarugamycin in the infection model. We found that, the ikarugamycin possessed intracellular killing activity against S. aureus within the mammary epithelial cell. This finding highlights the potential application of ikarugamycin as a novel antimicrobial for the treatment of S. aureus mastitis. ABSTRACT: Staphylococcus aureus 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 S. aureus 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 S. aureus 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 S. aureus using gentamicin protection assay. IKA intracellular antibacterial activity assays were used to determine the bactericidal activity of IKA against intracellular S. aureus. The cytotoxicity of IKA against Mac-T cells was evaluated using the resazurin assay. We showed that, S. aureus 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 logs(10) CFU/mL of S. aureus in the first six-hour of treatment respectively. In addition, IKA demonstrated intracellular killing activity by killing 90% of intracellular S. aureus at 5 μg/mL. This level is comparatively lower than 9.2 μg/mL determined as the half-maximal inhibitory concentration (IC(50)) 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 S. aureus mastitis.