Advanced imaging technology, combined with artificial intelligence (AI)-assisted analysis, enabled the discovery of a natural antimicrobial peptide (AMP), called Hirunipin-2, that could help scientists address the global challenge of antibiotic resistance. AMPs show the potential to successfully combat the rise in multidrug-resistant (MDR) bacteria. Of particular interest are AMPs derived from natural sources because they exhibit low cytotoxicity and are unlikely to develop resistance. A joint research team from the Korea Basic Science Institute (KBSI), Chosun University, and Chungbuk National University used an AI-based screening strategy with 3D holotomography (3D HT), also known as optical diffraction tomography (ODT), to analyze antibacterial mechanisms and quickly identify candidate AMP substances. ODT is a label-free imaging technology that produces 3D volumetric images with quantitative measurements of cellular properties. It enables real-time visualization of bacterial morphological changes, membrane damage, and biofilm formation over time. Acinetobacter baumannii (MDRAB) biofilms, untreated as (b) control or treated with (c) Hirunipin-2 over 12 hours. Images for each time are shown in isometric view at the top and Y-axis view at the bottom. d,e): Segmented 3D HT images of MDRAB biofilm for (d) control or (e) Hirunipin-2 for insets of Figure 4b,c. Images are shown in specific RI range (top, RI = 1.340-1.350; bottom, RI = 1.351-1.380). Courtesy of Korea Basic Science Institute." style="width: 400px; height: 262px; float: left; margin-top: 7px; margin-right: 10px; margin-bottom: 7px;" /> a): Experimental design of real-time 3D holotomography (HT) imaging of biofilm formation and color map of refractive index (RI). b,c): Representative 3D HT images of multidrug-resistant Acinetobacter baumannii (MDRAB) biofilms, untreated as (b) control or treated with (c) Hirunipin-2 over 12 hours. Images for each time are shown in isometric view at the top and Y-axis view at the bottom. d,e): Segmented 3D HT images of MDRAB biofilm. Courtesy of Korea Basic Science Institute. To date, ODT imaging has been used to track individual bacterial cells in real time to identify antimicrobial mechanisms such as membrane disruption caused by AMPs. While single-cell imaging allows for the detailed observation of individual cells, it cannot capture the collective dynamics and interactions within a larger bacterial community. To overcome this issue, the researchers attempted time-lapse monitoring using ODT with a wider field of view to simultaneously capture the bulk of bacterial cells. Using AI-based refractive index (RI) analysis, the researchers calculated and segmented the RI values in multiple bacterial cells. This enabled them to distinguish numerous bacterial cells and monitor their changes over time. AI-based bioinformatic analytical techniques enabled the researchers to explore the transcriptome database of the medicinal leech and evaluate the structural stability of substances and their antibacterial and anti-inflammatory functions. Through a combination of real-time imaging and quantitative analysis, the researchers identified 19 potential new AMPs. A 3D high-throughput screening technology was developed to evaluate the candidate substances simultaneously. This technology allowed the researchers to analyze the antibacterial mechanisms and perform rapid antibacterial substance screening at the same time. The researchers performed ODT with high-throughput screening (ODT-HTS) to validate the efficacy of the AMPs in multiple bacterial cells. They found that the AMP Hirunipin-2 had the antimicrobial and antibiofilm properties necessary to inhibit the growth of superbacteria and destroy biofilm. Hirunipin-2 inhibited biofilm formation, eradicated preformed biofilms, and interacted with antibiotics against multidrug-resistant Acinetobacter baumannii (MDRAB) by attacking and rupturing the cell membranes. The synergistic effects of Hirunipin-2 when used with conventional antibiotics, along with its exceptional properties, including drug resistance, antibiofilm activity, and anti-inflammatory activity, established its potential as an AMP against antibiotic-resistant infections. To effectively respond to superbacteria, it is essential to develop technologies that enable precise analysis of antibacterial mechanisms and rapid identification of candidate substances. The joint research team created a tool to perform a real-time quantitative analysis of a large number of antimicrobial peptide candidates, and simultaneously observe the formation and disruption process of biofilms produced by MDR bacteria in a label-free manner. The ODT-HTS platform could potentially be used to study various antibacterial mechanisms, which makes it a promising strategy for many applications, from AMP discovery to MDR research. In the future, the researchers plan to uncover new insights into the antimicrobial mechanisms, such as changes in pH. “We are the first research team that presented an innovative antimicrobial peptide development strategy that can contribute to overcoming the antibiotic resistance problem by combining Korea’s indigenous natural product database and 3D HT-HTS — the cutting-edge imaging technology,” researcher Seongsoo Lee said. “We expect that our strategy can be widely employed in the development of new drugs for treating superbacterial infections and in future research on antibiotic resistance.” The research was published in Advanced Science (www.doi.org/10.1002/advs.202409803).