PhD Thesis defense by Filipa Moutinho Trigo da Roza at the Faculty of Veterinary Medicine of the Complutense University of Madrid

February 14^th, 2025

Integrons are genetic platforms that capture, stockpile, and rearrange integron cassettes (IC), which encode genes with diverse functions, including antimicrobial resistance, phage resistance, or metabolic functions. However—and despite their dissemination to the clinical setting trough plasmids—detecting ICs is still challenging, and many functions they encoded are still unknown.

To address these challenges, in this thesis we have developed a biotechnological tool to identify ICs independently of their sequence, phenotype, or genetic background. We have redesigned a class 1 integron to serve as a capture platform, incorporating ccdB or sacB as counter-selectable markers with an embedded integration site, attI1. These markers enable the selection of recombinants in specific conditions, ensuring viability only if disrupted by incoming ICs, with a low escape mutant rate of 10-6, providing a broad range for detecting cassettes. We first demonstrated that both counter-selectable markers report IC capture in a classical recombination assay. We then developed three distinct applications for the tool: 1) a plasmidic version for harvesting ICs from large chromosomal integrons, producing libraries of up to 10⁵ colonies per assay, 2) a chromosomal version generating a conjugation-sentinel E. coli that allowed us to detect ICs from conjugative plasmids in clinical isolates, and 3) a chromosomal version implemented in naturally competent, genetically modified V. cholerae, enabling the efficient IC capture directly from exogenous DNA, including samples with plasmidic and chromosomal integrons.

This tool uncovers integron content from diverse samples with high specificity. Its sequence-independent design represents a groundbreaking advance for understanding integron cassette ecology.