Simultaneous elucidation of antibiotic mechanism of action and potency with high-throughput Fourier-transform infrared (FTIR) spectroscopy and machine learning.

The low rate of discovery and rapid spread of resistant pathogens have made antibiotic discovery a worldwide priority. In cell-based screening, the mechanism of action (MOA) is identified after antimicrobial activity. This increases rediscovery, impairs low potency candidate detection, and does not guide lead optimization. In this study, high-throughput Fourier-transform infrared (FTIR) spectroscopy was used to discriminate the MOA of 14 antibiotics at pathway, class, and individual antibiotic level. For that, the optimal combinations and parametrizations of spectral preprocessing were selected with cross-validated partial least squares discriminant analysis, to which various machine learning algorithms were applied. This coherently resulted in very good accuracies, independently of the algorithms, and at all levels of MOA. Particularly, an ensemble of subspace discriminants predicted the known pathway (98.6%), antibiotic classes (100%), and individual antibiotics (97.8%) with exceptional accuracy, and similar results were obtained for simulated novel MOA. Even at very low concentrations (1 μg/mL) and growth inhibition (15%), over 70% pathway and class accuracy was achieved, suggesting FTIR spectroscopy can probe the grey chemical matter. Prediction of inhibitory effect was also examined, for which a squared exponential Gaussian process regression yielded a root mean square error of 0.33 and a R of 0.92, indicating that metabolic alterations leading to growth inhibition are intrinsically reflected on FTIR spectra beyond cell density. KEY POINTS: • Antibiotic MOA and potency estimated with high-throughput FTIR spectroscopy • Sub-inhibitory MOA identification suggests ability to explore grey chemical matter • Data analysis optimization improved MOA identification at antibiotic level by 38.