Electroporation as a potentiator of antimicrobial efficacy

Many antibiotics are no longer effective against a growing number of pathogens, and antibiotic resistance is increasingly becoming a global health threat. In addition, chronic bacterial diseases can significantly worsen the outcome of concurrent viral infections, including COVID-19. Thus, development of novel approaches – combining multiple defense strategies with different modes of actions aimed at different targets – will be critical for effective disease control in the »post-antibiotic era«. Among the most promising such approaches is electroporation, in which microorganisms are permeabilized by short electric pulses of sufficient strength to facilitate the uptake of various molecules – including antibiotics and alternative antimicrobials. Electroporation is highly optimizable and reproducible, with permeabilization controlled by adjusting pulse amplitude and duration, and the physical nature of the underlying phenomenon (formation of aqueous pores in the lipid bilayer) prevents the development of resistance. To date, reports on combined use of an antibiotic or alternative antimicrobial and electroporation have shown some improvements in bacterial inactivation. However, a systematic evaluation of the synergistic effect – its qualitative and quantitative dependence on bacterial envelope, types of antimicrobials, their target, and resistance mechanisms – has yet to be performed. This is essential for broader understanding and thus recognition of electroporation as an effective potentiator of antimicrobials, and especially for its application in practice. In this project, we aim to systematically evaluate this synergistic effect by relating it to (i) bacterial envelope (Gram-negative (C–) vs. Gram-positive (G+) bacteria), (ii) resistant vs. susceptible strains, (iii) intracellular target of the antimicrobial, (iv) mechanism of resistance to the antimicrobial, and (v) comparison of conventional antibiotics vs. alternative antimicrobials. The project groups have already collaborated on a small-scale study (Lovšin et al., Front Microbiol 12:722232, 2021), showing that potentiation at the minimum inhibitory concentration is higher for ampicillin (target: wall synthesis) than for ciprofloxacin (DNA replication) and tetracycline (protein synthesis). This testifies to the adequacy and feasibility of our approach, but the comprehensive systematic study proposed here requires a larger-scale effort and dedicated funding. We divide our project into 5 work packages consisting of 12 tasks with 9 deliverables. We will first focus on 5 representative antibiotics and test them alone and in combination with electroporation on 8 representative bacteria (4 G– and 4 G+) and then extend this approach to 5 representative non-toxic alternative antimicrobials. The expertise of the group at the Faculty of Electrical Engineering of the University of Ljubljana (led by Prof. Tadej Kotnik and Prof. Damijan Miklavčič), one of the world’s leading groups in membrane electroporation, will be combined with those at the Biotechnical Faculty of the University of Ljubljana (led by Assoc. Prof. Anja Klančnik), and the Jožef Stefan Institute (led by Assoc. Prof. Jerica Sabotič) providing complementary expertise in bacterial resistance to antibiotics and alternative antimicrobials, respectively, and will form a multidisciplinary team that can successfully implement of the project’s objectives. In our proposal we also elaborate the project’s risk assessment and mitigating measures. The goal of this project is to form the basis for a systematic approach to efficiently combine antimicrobial substances and electroporation, to inactivate both Gram-positive and Gram-negative bacteria at lower concentrations of antibiotics (or even non-toxic alternative antimicrobials) and weaker/fewer electric pulses, reducing environmental and economic costs of bacterial inactivation, particularly in contaminated waters.