Abstract
In this study we investigated the influence of oxygen availability on a phenotypic microtiter screen to identify new, natural product inhibitors of growth for the bovine mastitis-causing microorganisms; Streptococcus uberis, Staphylococcus aureus, and Escherichia coli. Mastitis is a common disease in dairy cattle worldwide and is a major cause of reduced milk yield and antibiotic usage in dairy herds. Prevention of bovine mastitis commonly relies on the application of teat disinfectants that contain either iodine or chlorhexidine. These compounds are used extensively in human clinical settings and increased tolerance to chlorhexidine has been reported in both Gram-positive and Gram-negative microorganisms. As such new, non-human use alternatives are required for the agricultural industry. Our screening was conducted under normoxic (20% oxygen) and hypoxic (<1% oxygen) conditions to mimic the conditions on teat skin and within the mammary gland respectively, against two natural compound libraries. No compounds inhibited E. coli under either oxygen condition. Against the Gram-positive microorganisms, 12 inhibitory compounds were identified under normoxic conditions, and 10 under hypoxic conditions. Data revealed a clear oxygen-dependency amongst compounds inhibiting growth, with only partial overlap between oxygen conditions. The oxygen-dependent inhibitory activity of a naturally occurring quinone, β-lapachone, against S. uberis was subsequently investigated and we demonstrated that this compound is only active under normoxic conditions with a minimum inhibitory concentration and minimum bactericidal concentration of 32 μM and kills via a reactive oxygen species-dependent mechanism as has been demonstrated in other microorganisms. These results demonstrate the importance of considering oxygen-availability in high-throughput inhibitor discovery.
Highlights
Bovine mastitis is the most common reason for antibiotic use in the dairy industry worldwide (Mcdougall, 2014; Gomes and Henriques, 2016)
Growth of S. uberis, S. aureus, and E. coli for high-throughput screening (HTS) was readily achieved in microtiter 96-well plates under both normoxic and hypoxic conditions, where hypoxic conditions are defined as
S. uberis, S. aureus and E. coli were screened against the well-characterized, Natural Products Set II (NPSII) library (120 compounds) and the commercially available Natural Product Library (NPL) (131 compounds)
Summary
Bovine mastitis is the most common reason for antibiotic use in the dairy industry worldwide (Mcdougall, 2014; Gomes and Henriques, 2016). Application of teat antiseptics commonly containing either chlorhexidine or iodine is highly effective in reducing transmission of bacteria controlling the risk of mastitis (Pankey et al, 1984; Williamson and LacyHulbert, 2013). Antibiotic therapy of mastitis may increase the risk of selection of resistant bacteria, and as some of the same classes of medically important antibiotics used in agriculture (e.g., β-lactams) are used in human medicine, the emergence of resistant bacteria is concerning. Discovery of antimicrobial alternatives for animal, but not human use, so-called ‘Green Antimicrobials’ (Toutain et al, 2016), for the prevention, or treatment of bacterial infections, such as bovine mastitis, would greatly assist in reducing the global usage of medically important humanuse antimicrobials
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