<i>In\nSitu</i> Raman Hyperspectral Analysis\nof Microbial Colonies for Secondary Metabolites Screening

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