Exploring Clinical Class 1 Integrons as Valuable Targets for the Re-sensitization of Multidrug Resistant Pathogenic Bacteria Using CRISPR-Cas

Skovmand, A. B.; Paaske, N. J.; Penil-Celis, A.; Schonemann, A. E.; Hansen, L. L.; Kot, W.; Garcillan-Barcia, M. P.; Nielsen, T. K.

doi:10.1101/2025.03.28.645910

bioRxiv · 10.1101/2025.03.28.645910

Exploring Clinical Class 1 Integrons as Valuable Targets for the Re-sensitization of Multidrug Resistant Pathogenic Bacteria Using CRISPR-Cas

Skovmand, A. B.·Paaske, N. J.·Penil-Celis, A.·Schonemann, A. E.·Hansen, L. L.·Kot, W.·Garcillan-Barcia, M. P.·Nielsen, T. K.

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Abstract

The increasing prevalence of bacteria resistant to many or all types of antibiotics poses a major health crisis. Novel classes of antibiotics are only slowly being developed and alternative strategies are needed to tackle the issue. Mobile genetic elements and class 1 integrons are important facilitators for antibiotic resistance genes, with the latter being highly conserved in human pathogens. The growing prevalence of multidrug-resistant bacteria and the paucity in the development of new antibiotics underscore the urgent need for innovative approaches in the treatment of pathogens. Among these, CRISPR-Cas nucleases can be used to cleave acquired resistance genes, leading to either plasmid curing or cell death if the target is on a chromosome. In this study, we investigate the feasibility of using class 1 integrons as a target for Cas9-based cleavage leading to re-sensitizing antibiotic-resistant bacteria. We analyze the conserved and widespread integrase gene intI1 and conclude that it is a suitable target for Cas-based re-sensitization due to its high sequence conservation and its occurrence largely limited to human pathogens, alleviating the risk of targeting benign bacteria. We developed a broad host range conjugative plasmid encoding a class 1 integron-targeting Cas9 system that leads to removal of resistance plasmids in target bacteria with subsequent re-sensitization towards antibiotics. We find that 290 distinct ARGs co-occur on int1-harboring plasmids, showing the potential for re-sensitization towards a very broad range of antibiotics.

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Skovmand, A. B., Paaske, N. J., Penil-Celis, A., Schonemann, A. E., Hansen, L. L., Kot, W., Garcillan-Barcia, M. P., Nielsen, T. K.. 2025-03-28. Exploring Clinical Class 1 Integrons as Valuable Targets for the Re-sensitization of Multidrug Resistant Pathogenic Bacteria Using CRISPR-Cas. https://doi.org/10.1101/2025.03.28.645910

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