Understanding the physical and biological effects of ultrasound on Pseudomonas aeruginosa biofilms

• Main Author: Bharatula, Lakshmi Deepika
• Other Authors: Manojit Pramanik
• Format: Thesis-Doctor of Philosophy
• Language: English
• Published: Nanyang Technological University 2021
• Subjects: Engineering::Bioengineering; Science::Biological sciences::Microbiology::Bacteria
• Online Access: https://hdl.handle.net/10356/151079
• Institution: Nanyang Technological University

Chronic bacterial infections pose a huge threat to global health due to emergence of antimicrobial resistance. While bacterial colonies grow in either a planktonic state or within a microstructure known as a biofilm, the latter accounts for 65% of infections. Biofilms are characterized by the generation of extracellular polymer substrates (EPS) and presence of chemical and genetic heterogeneity. These factors participate in hindering the effect of antibacterial agents favoring the antimicrobial resistance. As resistant strains and persisters surpass novel chemical treatment methods, a potential approach to tackle this issue is mechanically stressing the biofilm to either disrupt the biofilm or enhance the transport of therapeutics to specific targets. Yet, investigations on the interactions between mechanical forces and biofilm are limited. This work proposes to use minimally invasive high intensity focused ultrasound (HIFU) to exert stress on in vitro Pseudomonas aeruginosa biofilms. This project takes a step further from the current literature and stresses upon the need for studying the biological changes that mechanical stress may cause in biofilms. The possibilities, advantages and limitations of such studies are described. In future, this will allow the translation of ultrasound as treatment strategy to clinical research. Another novel aspect is use of well-defined electrochemical sensing system to study the microstructural effects of HIFU on biofilms. Use of this prominent bio-sensing technique will allow real time monitoring of the said effects. Here, the effects of HIFU are characterized using conventional methods, i.e., confocal microscopy, crystal violet assay, and colony forming units. Additionally, an electrochemical based impedimetric sensing system was optimized to rapidly detect the changes in biofilm microstructure. To this effect, indium tin oxide coated polyethylene terephthalate (ITO:PET) was chosen as the biofilm growth substratum due to acoustic impedance match with surrounding medium, optical transparency, and electrical conductivity. Optical characterization of biofilm growth on ITO:PET across four days showed a gradual switch from reversible attachment to biofilm maturation. The biofilm growth was also characterized using electrochemical impedance spectroscopy (EIS). This is an efficient technique to rapidly detect the viability and metabolic activity of the biomass in a non-destructive and low cost setup. To understand the meaning of electrochemical response after HIFU treatment, the optimization was necessary. Since the response of weak electricigens such as P. aeruginosa on ITO:PET is not well understood, the EIS parameters for the system were optimized across four days of biofilm development. A change in the impedance modulus and phase angle was observed as the biofilm developed. Relaxation time analysis showed a dominant time constant at approximately 1 second and confirmed the validity of a two-time constant equivalent circuit model for the biofilm impedance. The interfacial resistance calculated from the equivalent circuit analysis showed a rapid drop after bacterial attachment whereas capacitance of the biofilm was masked by the capacitance of ITO:PET. The trends for interfacial resistance and capacitance were independent to the geometry of the ITO:PET working electrode. EIS across a broad range of potentials with and without inhibitors showed a marked difference between the interfacial resistance of viable and energy inhibited biofilms. Moreover, EIS of exopolysaccharide ∆psl mutant showed a substantial drop in current. Overall, the results indicated that EIS enabled the detection of biofilm formation across large surface areas as early as 24 hours for the weak electroactive species P. aeruginosa using a flexible polymeric substrate. Next, the effects of acoustic exposure on biofilm were investigated using confocal microscopy, crystal violet assay, and electrochemical monitoring. Although ultrasound facilitates biofilm dispersal and may enhance the effectiveness of antimicrobial agents, the resulting biological response of bacteria within the biofilms remains poorly understood. To address this question, we investigated the microstructural effects of P. aeruginosa biofilms exposed to high intensity focused ultrasound (HIFU) at different acoustic pressures and the subsequent biological response. Confocal microscopy images indicated a clear microstructural response at peak negative pressures equal to or greater than 3.5 MPa. In this pressure amplitude range (greater than 3.5 MPa), HIFU partially reduced the biomass of cells and eroded exopolysaccharides from the biofilm. These pressures also elicited a biological response; we observed an increase in a biomarker for biofilm development (cyclic-di-GMP) proportional to ultrasound-induced biofilm removal. The biological response was further evidenced by an increase in the relative abundance of cyclic-di-GMP overproducing variants present in the biofilm after exposure to HIFU. The results, therefore, suggest that both physical and biological effects of ultrasound on bacterial biofilms must be considered in future studies. Furthermore, the findings revealed that cyclic-di-GMP overproducing mutant strains were more resilient to disruption from HIFU at these pressures, suggesting that a biofilm may develop resistance towards ultrasound. Therefore, successive ultrasound treatments were studied to investigate the potential for biofilm resistance to HIFU. We studied planktonic cultures grown from untreated and HIFU-treated samples and observed subsequent biofilm growth. After the first HIFU-treatment, we measured an increase in c-di-GMP activity. Further HIFU-treatment resulted in a weakening of the biofilm followed by a strengthening. However, after three HIFU treatments, HIFU was still able to mechanically remove the cells from the surface. Based on all the previous observations, a continuous and real-time monitoring system was designed to aid the future studies on the effects of HIFU on biofilms.