Real-time 3D visualization reveals potent antibacterial and antibiofilm activity against superbacteria
From screening to efficacy assessment via real-time 3D-holotomography imaging
image:
Experimental design and workflow of AMP prediction using in silico tools and 3D HT-HTS. Hirunipin 2 as a selected AMP candidate was evaluated on antibacterial, antibiofilm, antiinflammatory activiry and efficacy for antibiotic adjuvant.
view moreCredit: Korea Basic Science Institute (KBSI)
A research group in Korea has discovered a novel natural antimicrobial peptide, ‘Hirunipin-2,’ from the salivary glands of the medicinal leech (Hirudo nipponia) using cutting-edge imaging technology, demonstrating that the material has high potential as a new therapeutic effective against superbacteria. This discovery underscores the significant potential of combining natural resources with advanced imaging technologies to address the global challenge of antibiotic-resistant infections.
Dr. Lee Seongsoo’s research group at the Honam Regional Center of the Korea Basic Science Institute (KBSI) successfully observed and quantitatively analyzed the antibacterial and antibiofilm activity of multidrug-resistant bacteria (MDR-bacteria or superbacteria) in real time using three-dimensional holotomography (3D HT*) technology. Through this technology, the researchers were able to confirm through real-time imaging the process in which the growth of superbacteria was inhibited by the antibiotic material and the biofilm was effectively destroyed.
*three-dimensional holotomography (3D HT) is a technique of optical diffraction tomography (ODT) to measure the 3D refractive index (RI) tomogram of a microscopic sample such as biological cells and tissues.
The joint research team, also including Professor Shin Song Yub’s group from the School of Medicine of Chosun University and Professor Cho Sung-Jin’s group from the Department of Biology of Chungbuk National University, explored the transcriptome database of the salivary gland tissue of the medicinal leech using AI-based bioinformatic analytical techniques to evaluate the structural stability of the substances and their antibacterial and anti-inflammatory functions, thereby deriving 19 new peptide candidates. Thereafter, the joint research team introduced a 3D HT screening technology (a high-throughput screening using 3D HT; 3D HT-HTS or ODT-HTS), which was developed to simultaneously evaluate a large number of candidate substances, and performed an antibacterial mechanism analysis and rapid antibacterial substance screening at the same time. Through this, the researchers finally discovered Hirunipin-2, a novel natural substance-derived antibacterial peptide.
MDR-bacteria, referring to bacteria that do not respond to existing antibiotics, are identified as a major factor in making infection treatment difficult and increasing mortality rates. The World Health Organization (WHO) also warns that antibiotic resistance is a serious threat to human health, and the development of novel antibacterial substances has emerged as an urgent task to address the issue. In particular, antibacterial peptides derived from natural products are drawing much attention as next-generation antibiotic candidates because they have a low possibility of developing resistance and exhibit low toxicity. To effectively respond to superbacteria, it is necessary to develop technologies that enable precise analysis of antibacterial mechanisms in addition to rapid discovery candidate substances.
3D HT imaging is a new analytical technique for observing bacteria in real time and acquiring various quantitative information within cells without preprocessing such as staining, and it has recently attracted attention in the development of therapeutics against superbacteria. In 2023, Dr. Lee Seongsoo’s research group demonstrated the multitarget mechanism of action of antimicrobial agents through real-time visualization at the single bacterial cell level using 3D HT technology. However, at the time, it was mainly limited to single cell or single substance analysis, and there were technical limitations in simultaneously assessing a large number of antimicrobial candidates or comprehensively analyzing the bulk bacterial response within a bacterial population [Kim et. al., “Real-time monitoring of multi-target antimicrobial mechanisms of peptoids using label-free imaging with optical diffraction tomography,” Advanced Science 10, 2302483 (2023)].
This study, however, was conducted by introducing 3D HT-HTS technology, so that the researchers performed a real-time quantitative analysis of a large number of antimicrobial peptide candidates, and simultaneously observed the formation and disruption process of biofilms produced by MDR bacteria in a label-free manner. In particular, the antimicrobial and antibiofilm efficacy of Hirunipin-2, which was newly discovered through this study, was assessed with high reliability, and combined use of the substance with existing antibiotics such as chloramphenicol, ciprofloxacin, tetracycline, and rifampicin showed a synergistic effect of enhancing antimicrobial activity, suggesting its potential use as an antibiotic adjuvant.
In the present study, the KBSI research group developed a 3D HT-HTS analysis technology in the process of discovering antibacterial substances and evaluating their efficacy and focused on the final selection of antibacterial agents and verification of their efficacy using the technology. The research groups from Chosun University and Chungbuk National University predicted new peptide candidates through AI-based bioinformatic analysis and conducted antibacterial and antibiofilm activity evaluations.
Dr. Lee Seongsoo, a principal investigator at KBSI, emphasized the significance of this study, saying, “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.” Dr. Lee added, “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.”
a) Experimental design of real-time 3D HT imaging of biofilm formation and color map of RI. b,c) Representative 3D HT images of MDRAB biofilms untreated as (b) control or treated with (c) hirunipin 2 over 12 h. 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).
Credit
Korea Basic Science Institute (KBSI)
The Korea Basic Science Institute (KBSI), a government-funded research institution established in 1988, conducts research and development, research support and joint research related to high-tech research equipment as well as advanced analytical science technology. With the aid of cutting-edge research equipment and outstanding human resources infrastructure, KBSI aims to provide a global platform for domestic and international researchers to achieve creative and harmonious research outcomes.
This research was supported by the National Research Foundation of Korea (NRF), the Commercialization Promotion Agency for R&D Outcomes (COMPA), and the Korea Basic Science Institute (KBSI). The results of this study were published on March 13, 2025 in the online edition of Advanced Science (IF=14.3), a globally renowned journal.
The image features that the antimicrobial peptide hirunipin 2, secreted from the salivary glands of the leech (Hirudo nipponia), kills multidrug-resistant bacteria by attacking their cell membranes and rupturing the cell membranes, as well as destroying mature biofilms already formed by the multidrug-resistant bacteria.
Credit
Korea Basic Science Institute (KBSI
Journal
Advanced Science
Article Title
Novel Leech Antimicrobial Peptides, Hirunipins: Real-Time 3D Monitoring of Antimicrobial and Antibiofilm Mechanisms Using Optical Diffraction Tomography
