<i>In Situ</i> Raman Hyperspectral Analysis of Microbial Colonies for Secondary Metabolites Screening

Since the discovery of penicillin, a vast array of microbial antibiotics has been identified and applied in the medical field. Globally, the search for drug candidates <i>via</i> microbial screening is ongoing. Traditional screening methods, however, are time-consuming and require labor-intensive sample processing, significantly reducing throughput. This research introduces a Raman spectroscopy-based screening system tailored to the <i>in situ</i> analysis of microbial colonies on solid culture media. Employing multivariate curve resolution-alternating least-squares (MCR-ALS) for spectral decomposition, our approach reveals the production of secondary metabolites at the single colony level. We enhanced the microbial culture method, enabling direct, high signal-to-noise (S/N) ratio Raman spectroscopic measurements of colonies of <i>Escherichia coli</i> and actinomycetes species. Through semisupervised MCR analysis using the known spectra of actinorhodin and undecylprodigiosin as references, we accurately assessed the production of these compounds by <i>Streptomyces coelicolor</i> A3(2). Furthermore, we herein successfully detected the production of amphotericin B by <i>Streptomyces nodosus</i>, even in the absence of prior spectral information. This demonstrates the potential of our technique in the discovery of secondary metabolites. In addition to enabling the detection of the above-mentioned compounds, this analysis revealed the heterogeneity of the spatial distribution of their production in each colony. Our technique makes a significant contribution to the advancement of microbial screening, offering a rapid, efficient alternative to conventional methods and opening avenues for secondary metabolites discovery.