Real-time fluorescence microscopy to study bacterial response to antimicrobial

• Autores: Ingrid Vanessa Ortega Rengifo
• Directores de la Tesis: Cristina Flors Ong (dir. tes.)
• Lectura: En la Universidad Autónoma de Madrid ( España ) en 2023
• Idioma: inglés
• Número de páginas: 134
• Títulos paralelos:
  • Microscopía de fluorescencia en tiempo real para estudiar la respuesta bacteriana frente a estrategias antimicrobianas
• Enlaces
  • Tesis en acceso abierto en: Biblos-e Archivo
• Resumen

  • Over the last few decades, advanced imaging techniques have improved our ability to analyze biological systems and observe biological processes in real-time. This thesis focuses on the use of fluorescence microscopy to study the initial response of bacteria exposed to different antimicrobial challenges. To that end, the thesis explores the use of Min oscillations in Escherichia coli as a real-time reporter for bacterial physiological response. The Min system regulates cell division in E. coli, and the thesis shows that monitoring its oscillation dynamics at the single-cell level provides valuable insight into the time scale of events that ultimately lead to cell death. This manuscript is divided into eight chapters. Chapter 1 provides an overview of the problem of bacterial resistance and several antimicrobial approaches used as alternatives to antibiotics. It also introduces the Min protein system and its application as a reporter of the physiological state of bacteria by fluorescence imaging methods. In Chapter 2, the general materials and methods used in this thesis are outlined, as well as the methodology employed to analyze experimental data. Chapter 3 describes the optimization of the experimental conditions for fluorescence imaging experiments. In Chapter 4 the initial response of bacteria exposed to polymyxin B and gentamicin antibiotics is monitored following the changes in the MinD oscillation period. Chapter 5 introduces antimicrobial photodynamic inactivation and analyzes the alteration in the oscillation pattern during treatment. These experiments highlight that Min oscillation is able to report more subtle effects on bacterial physiology compared to traditional methods. The purpose of Chapter 6 is to monitor in situ the antimicrobial activity of photoactivable hydrogels loaded with the photosensitizer Methylene Blue. The loading process of the photosensitizer into hydrogels results in different responses of Min oscillation dynamics, providing a deeper understanding of the underlying mechanism. Chapter 7 aims to study the real-time response of bacteria to weak physical interactions with mechano-bactericidal materials, which is experimentally done by flowing single-wall carbon nanotubes onto bacteria immobilized on a microfluidic system. The aim of Chapter 8 is to provide coherence and perspective to the main results of the thesis, as well as an outlook on how advanced microscopy methods and future experiments may impact the study of Min oscillations to report on bacterial physiology