In recent decades, the widespread and often indiscriminate use of antimicrobial agents, particularly in clinical and agricultural settings, has led to the emergence and proliferation of multidrug-resistant pathogens. The growing antimicrobial resistance crisis is further complicated by the broad-spectrum activity of most conventional antibiotics, which not only act on target bacteria but also affect other components of the microbial community. This disruption results in ecological imbalances and facilitates horizontal transfer of resistance genes, accelerating the evolution and acquisition of antimicrobial resistance mechanisms (AMR). As antibiotics are one of the cornerstones of modern medicine, their decreasing efficacy poses one of the greatest threats to public health. In fact, annually, in the European Union (EU) alone, AMR causes around 25,000 deaths and imposes an estimated economic burden of 1.5 billion euros.
Despite the need for solutions, the discovery and development of new classes of antibiotics has been in constant decline for years. As a result, alternative approaches, such as the use of monoclonal antibodies, antimicrobial peptides, bacteriophages, lysins and CRISPR-Cas-based technologies, are receiving increasing attention. While many of these strategies show promise in terms of precision and specificity, challenges remain in terms of delivery, host range and the risk of resistance development.
Previous work from our laboratory demonstrated that genetic modules based on intein-split toxins delivered via conjugation can serve as highly effective antimicrobial agents9. In these systems, each half of a toxin is fused to one half of a split intein, and the expression of the toxin-intein pair is driven by transcription factors specific to the target organism. A key advantage of this strategy lies in the additional layer of post-translational regulation provided by the intein, which prevents the reconstitution of an active toxin in non-target species. This approach is also modular and adaptable, as illustrated by its successful application in selectively eliminating either all Vibrio cholerae or only their antibiotic-resistant derivatives from mixed bacterial populations.
Building on these findings, this study seeks to broaden the applicability and toolkit of this technology as a step toward the development of the next generation of targeted antimicrobials. Specifically, we sought to: (1) identify the optimal platform for disseminating antibacterial modules; (2) develop engineered derivatives of the delivery system to enhance its functionality in complex microbial populations; (3) adapt the toxin-intein modules to target priority pathogens and clinically relevant mobile genetic elements; and (4) validate the performance of these systems in both in vitro and in vivo models.
