Chassis de captura de cassettes de integron

• Autores: Filipa Moutinho Trigo da Roza
• Directores de la Tesis: José Antonio Escudero García-Calderón (dir. tes.)
• Lectura: En la Universidad Complutense de Madrid ( España ) en 2025
• Idioma: español
• Número de páginas: 214
• Títulos paralelos:
  • Integron Cassette capture chassis
• Tribunal Calificador de la Tesis: Julio Álvarez Sánchez (presid.), Lucía García Pastor (secret.), Fernando de la Cruz (voc.), María del Rocío López Igual (voc.), Didier Mazel (voc.)
• Programa de doctorado: Programa de Doctorado en Veterinaria por la Universidad Complutense de Madrid
• Materias:
  • Ciencias de la vida
    • Microbiología
      • Antibióticos
• Enlaces
  • Tesis en acceso abierto en: Docta Complutense
• Resumen
  • español

    Los integrones son elementos genéticos capaces de captar, reorganizar y diseminar cassettes de integrones (IC), incluyendo ICs que confieren resistencia a antimicrobianos. Estas plataformas genéticas han contribuido significativamente al aumento y dispersión global de patógenos multirresistentes al reclutar más de 170 genes de resistencia frente a la mayoría de las familias de antibióticos. Sin embargo, a pesar de su importancia, la detección de cassettes de integrón sigue siendo un desafío. Los métodos convencionales, como la PCR, suelen introducir sesgos, mientras que las técnicas más avanzadas, como la secuenciación masiva, aún no están establecidas en el uso rutinario de los laboratorios de diagnóstico debido a su coste y complejidad...

  • English

    Integrons are genetic elements that capture, rearrange, and disseminate integron cassettes (IC), which often encode antimicrobial resistance (AR). With over 170 AR genes recruited, integrons play a key role in the global spread of multidrug-resistant pathogens. However, detecting integron cassettes remains challenging. PCR-based methods can introduce biases, while advanced sequencing approaches are still not applicable for routine laboratory use.

    This thesis introduces a novel biotechnological tool for detecting integron cassettes in a sequence-independent manner, addressing the limitations of conventional methods. The tool utilizes a re-engineered class 1 integron with an integron integration site (attI1) embedded within a counter-selectable marker (CSM). Two CSMs were tested: the ccdB toxin, part of a type II toxin-antitoxin system, and sacB, which encodes levansucrase¿a lethal enzyme in the presence of sucrose. In this system, cassette insertion into attI1 disrupts the CSM, enabling bacterial survival as a readout of successful cassette capture. Pre-selection growth ensures bacterial viability. Several tool versions were tested, and the CSM::attI1 fusion protein was optimized for functionality, achieving low escape mutant rates and a broad detection range. Initial validations using a classical recombination assay confirmed reliable cassette capture, independently of the phenotype of the capture IC. The platform was then expanded into three distinct applications.

    First, we developed the "cassette harvester," a plasmid-based tool to create cassette libraries from sedentary chromosomal integrons (SCIs). Using Vibrio cholerae's superintegron (SI), we generated a plasmidic library. Sequencing confirmed the method's high specificity and efficient representation of cassettes, enabling high-throughput studies of unknown cassette functions.

    Second, a chromosome-based version was created to develop a sentinel Escherichia coli strain capable of detecting integron cassettes from mobile plasmids in co-culture systems. This strain successfully identified integrons and characterized cassette content, offering a robust tool for monitoring genetic platforms in microbial communities.

    Third, the Integron Cassette Capture Chassis (I3C) was designed for Vibrio cholerae to capture cassettes from exogenous DNA samples. The superintegron was removed from the genome, and the natural transformation protocol was optimized to enhance integron recombination. Combining the modified strain with DNA enrichment, the tool detected cassettes in diverse samples, highlighting its potential for environmental and clinical applications.

    Overall, this thesis presents a groundbreaking detection platform for detecting integrons without prior sequence knowledge, offering high specificity and versatility. These innovations provide a valuable diagnostic tool and a means to explore integrons and the functional roles of their chromosomal arrays.