Live 3D Visualization Uncovers Powerful Antibacterial and Antibiofilm Effects Against Superbugs

A groundbreaking discovery from a South Korean research consortium has unveiled a novel antimicrobial peptide, Hirunipin-2, derived from the salivary glands of the medicinal leech Hirudo nipponia. This innovation is poised to revolutionize the fight against multidrug-resistant bacteria, commonly termed superbacteria, which pose a formidable global health crisis. By harnessing advanced imaging modalities coupled with cutting-edge bioinformatics, the research team has pioneered a multi-faceted approach in identifying and validating novel therapeutic agents that may overcome longstanding challenges in antibiotic resistance.

The cornerstone technology employed in this study is three-dimensional holotomography (3D HT), an optical diffraction tomography (ODT) technique that facilitates high-resolution, label-free imaging of bacterial cells and biofilms. Unlike traditional microscopy methods that require staining or other sample preparations potentially altering biological functions, 3D HT offers real-time acquisition of refractive index tomograms. This approach allows precise quantitative assessments of bacterial morphology, growth dynamics, and response to antimicrobial agents at the single-cell level, pushing the boundaries of microbial diagnostics and drug efficacy evaluation.

Dr. Lee Seongsoo’s team at the Korea Basic Science Institute (KBSI) leveraged this technology to monitor the antibacterial and antibiofilm activities of potential agents in multidrug-resistant bacterial populations. The research showcased the ability of 3D HT to vividly visualize the inhibitory processes on bacterial proliferation and biofilm disruption in real-time, marking a significant advancement in understanding the mechanistic underpinnings of antimicrobial action. This dynamic observation is crucial, as biofilms notoriously shield bacteria from conventional antibiotics, leading to persistent infections.

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In parallel, collaborative efforts with Professor Shin Song Yub’s group from Chosun University and Professor Cho Sung-Jin’s group from Chungbuk National University incorporated artificial intelligence-driven bioinformatic analyses to mine the transcriptome database of leech salivary glands. This integrative approach evaluated the structural stability, antibacterial potency, and anti-inflammatory potential of multiple naturally occurring peptides. The combination of big data analytics and molecular biology expedited the identification of nineteen promising antimicrobial candidates, among which Hirunipin-2 demonstrated remarkable efficacy.

Subsequent validation using high-throughput 3D HT screening (3D HT-HTS) enabled the simultaneous quantitative analysis of numerous peptide candidates. This innovative methodology addressed previous limitations where single-substance or single-cell assessments restricted throughput and comprehensive bacterial population analysis. By utilizing the 3D HT-HTS platform, the team was able to rapidly scrutinize the bioactive peptides’ interactions with superbacteria and their biofilm matrices, thereby accelerating the preclinical evaluation pipeline for antimicrobial agents.

Multidrug-resistant bacteria remain a pressing global health threat as their resistance mechanisms undermine the effectiveness of existing antibiotics, rendering many infections difficult to treat and substantially increasing mortality rates. The World Health Organization has highlighted antibiotic resistance as a paramount health issue requiring urgent action and novel therapeutics development. Naturally derived antimicrobial peptides such as Hirunipin-2 represent a promising frontier due to their unique modes of action, reduced likelihood of resistance development, and minimal toxicity profiles.

3D HT imaging not only facilitates visualization but also provides detailed quantitative cellular information without the necessity for labels or dyes. This characteristic is transformative for the field, enabling researchers to study the spatiotemporal dynamics of bacterial populations and their responses to treatments under near-native conditions. The application of this technology in monitoring antimicrobial action at the microscale reveals insights into multitarget mechanisms, crucial for designing agents capable of circumventing bacterial defense strategies effectively.

Previously, the scope of 3D HT was confined mainly to examining single cells or single antimicrobial substances, limiting its applicability in large-scale screening contexts. The present study transcended these limitations through the development and implementation of a 3D HT-HTS pipeline, which could process high volumes of data and sample conditions, thereby facilitating comprehensive antibacterial activity profiling. This approach symbolizes a paradigm shift by marrying high-throughput screening with high-resolution imaging technologies.

Hirunipin-2’s demonstrated antimicrobial and antibiofilm activities were evaluated with exceptional rigor, underscoring the peptide’s potential utility not only as a standalone therapeutic but also as an antibiotic adjuvant. Notably, when combined with conventional antibiotics such as chloramphenicol, ciprofloxacin, tetracycline, and rifampicin, Hirunipin-2 exhibited synergistic effects that enhanced the overall antimicrobial performance. This combinatorial effect could reduce the required dosages of conventional drugs and potentially mitigate side effects and resistance emergence.

The success of this innovative research strategy lies in the seamless integration of indigenous natural product databases, AI-enabled peptide prediction, and advanced 3D HT imaging technologies. Such synergy allows for a more precise and accelerated discovery process of antimicrobial agents tailored against resilient bacterial pathogens. This platform offers a replicable model that could be adapted for diverse infectious diseases where antibiotic resistance remains a crippling challenge.

Dr. Lee Seongsoo emphasized the transformative potential of their work, framing it as a pioneering antimicrobial peptide development strategy that can substantially contribute to resolving the antibiotic resistance dilemma. By leveraging Korea’s native biological resources and sophisticated imaging tools, the team has laid a foundation that is expected to influence novel drug development trajectories globally and inspire subsequent inquiries into antimicrobial resistance mechanisms.

The implications of these findings extend beyond the discovery of a single peptide; the methodology itself heralds a new era in microbiological research and pharmaceutical development. The high-resolution, dynamic visualization of bacterial responses, combined with rapid, large-scale candidate screening, equips researchers with unprecedented tools to outmaneuver evolving bacterial threats. This study thus represents a critical leap forward in the contemporary battle against superbacterial infections.

Supported by the National Research Foundation of Korea, the Commercialization Promotion Agency for R&D Outcomes, and the Korea Basic Science Institute, this milestone research was published in the prestigious journal Advanced Science on March 13, 2025. The article not only delineates the technical prowess of 3D HT-HTS but also establishes Hirunipin-2 as a compelling candidate in the quest for next-generation antimicrobial solutions.

Subject of Research: Novel antimicrobial peptides derived from medicinal leech saliva; real-time 3D imaging and screening of antimicrobial efficacy against multidrug-resistant bacteria.

Article Title: Novel Leech Antimicrobial Peptides, Hirunipins: Real-Time 3D Monitoring of Antimicrobial and Antibiofilm Mechanisms Using Optical Diffraction Tomography

News Publication Date: 13-Mar-2025

Web References: http://dx.doi.org/10.1002/advs.202409803

References: Kim et al., “Real-time monitoring of multi-target antimicrobial mechanisms of peptoids using label-free imaging with optical diffraction tomography,” Advanced Science, vol. 10, 2302483 (2023).

Image Credits: Korea Basic Science Institute (KBSI)

Keywords: antimicrobial peptides, Hirunipin-2, multidrug-resistant bacteria, superbacteria, optical diffraction tomography, 3D holotomography, biofilm disruption, high-throughput screening, antibiotic resistance, synergistic antibiotic adjuvants, AI-driven bioinformatics, natural product drug discovery

Tags: 3D visualization of bacteriaadvanced imaging in microbiologyantibiofilm effects against superbugsantimicrobial peptide Hirunipin-2drug resistance solutionsmedicinal leech-derived therapeuticsmultidrug-resistant bacteria researchnanotechnology in antibiotic researchoptical diffraction tomography applicationsquantitative microbial diagnosticsreal-time bacterial imaging techniquesSouth Korean biomedical innovation