Exploring Transduction and Adaptation of Plasmids in Antimicrobial Resistance.

• Autores: Emilia Florentine Wedel
• Directores de la Tesis: Bruno González Zorn (dir. tes.)
• Lectura: En la Universidad Complutense de Madrid ( España ) en 2024
• Idioma: inglés
• Títulos paralelos:
  • Explorando la Transducción y la Adaptación de Plásmidos en la Resistencia a los Antimicrobianos.
• Tribunal Calificador de la Tesis: María Molina Martín (presid.), Alicia Aranaz Martín (secret.), Teresa Pellicer (voc.), María Pilar Garcillán Barcia (voc.), Jens Hammerl (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

  • Antimicrobial resistance (AMR) has become a growing threat to public health worldwide. The increasing use and misuse of antibiotics since their introduction into the clinical setting has led to the emergence of bacteria resistant to multiple antimicrobial agents, resulting in infections that are increasingly difficult to treat. Plasmids are an important vehicle for antimicrobial resistance genes, serving as reservoirs and facilitating their spread. In this dissertation, we characterize the small hydrophobic peptide ShyP, which poses an obstacle in plasmid adaptation to Escherichia coli, and we explore the dissemination of plasmids and plasmid-mediated antibiotic resistance through bacteriophages.

    To assess the impact of the ShyP peptide on E. coli, we measured optical density and cell viability in growth curves and carried out fluorescence assays with SYTOX Green dye to identify possible membrane damage after ShyP expression. Electron microscopy was used to analyze further effects of the peptide on E. coli cells. Then, a computational analysis was carried out to compare ShyP and toxins from type I toxin-antitoxin systems. In addition, we tested putative antitoxins that we identified via RNA sequencing for activity against ShyP toxicity. Finally, we compared the shyP promoter function in E. coli and its original host Haemophilus influenzae by replacing the shyP gene with GFP and measuring fluorescence in growth curves.

    To explore the transduction of plasmids and plasmid-mediated AMR, we used a transduction model based on Salmonella Typhimurium LT2 pSLT- and the temperate pac-phage P22 HT105/1 int201. Phage lysates were prepared from nine S. Typhimurium donor strains, each carrying a different plasmid varying in size, copy number and incompatibility group. For transduction we infected an empty S. Typhimurium strain with phage lysate and selected for armA or other resistance genes. After transduction experiments, we studied the genetic environment in which the armA gene was transduced, using polymerase chain reaction for small plasmids and Nanopore long read sequencing for large plasmids. Finally, we sequenced the capsid DNA of two phage lysates, one from a donor with a small plasmid and another from a donor with a large plasmid.

    In our experiments we determined that ShyP has toxic effects on E. coli cells by targeting the cell membrane in addition to causing morphological changes like cell elongation and inflation. Due to its toxicity, expression leads to growth arrest and cell death, which is more severe if expression is induced before the exponential phase. Furthermore, the peptide has the same properties as toxins from type I toxin antitoxin systems, but we could not identify an antitoxin, which means that the function of ShyP is still unclear. We have also found that transduction of AMR genes occurs with higher frequency if encoded on small multicopy plasmids which are transduced as entire plasmids, whereas transduction of large plasmids is more complex and results in entire plasmids, smaller plasmids, and integration of plasmid DNA into the chromosome. This integration is mediated by IS26, which is also the basis for homologous recombination leading to inversions and deletions in the transduced plasmids. In addition, we found a discrepancy between the high encapsidation of a large plasmid and its low transduction frequency, which we attribute to a low production of transducing particles carrying components important for generating viable transductants, such as armA and the origin of replication.

    By exploring the potential of plasmid transduction and characterizing ShyP, this work contributes to a better understanding of the spread and adaptation of plasmids to novel hosts. A deeper insight into these mechanisms can help to develop new strategies and techniques to reduce the dissemination of AMR genes and alleviate the burden of this constantly increasing risk to public health.