Abstract
Bacterial biofilms are typically more tolerant to antimicrobials compared to bacteria in the planktonic phase and therefore require alternative treatment approaches. Mechanical biofilm disruption from ultrasound may be such an alternative by circumventing rapid biofilm adaptation to antimicrobial agents. 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 Pseudomonas 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, 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. Cyclic-di-GMP overproducing mutant strains were also more resilient to disruption from HIFU at these pressures. 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. Our results, therefore, suggest that both physical and biological effects of ultrasound on bacterial biofilms must be considered in future studies.
Highlights
IntroductionThese bacterial cells display a high degree of physiological and topographical heterogeneity and grow on abiotic surfaces (e.g., biomedical devices) or biological surfaces (e.g., lung tissue) (Flemming et al, 2016)
To determine if high intensity focused ultrasound (HIFU) induced a microstructural response in the biofilm, we quantified the biofilms formed by PAO1 mutant labeled with cyan fluorescent protein (Cfp)
Our findings revealed that HIFU was able to remove the bacterial cells and the α-polysaccharides linked to them, whereas removal of β-polysaccharides located at the top of the biofilm did not depend on the biovolume removal
Summary
These bacterial cells display a high degree of physiological and topographical heterogeneity and grow on abiotic surfaces (e.g., biomedical devices) or biological surfaces (e.g., lung tissue) (Flemming et al, 2016). Bacteria within biofilms are more tolerant to antimicrobials compared to planktonic bacterial cells due to self-produced biofilm matrix consisting of extracellular polymeric substance (EPS), high bacterial concentration, Influence of HIFU on P. aeruginosa Biofilms exchange of genetic information in biofilms, differences in growth states of bacteria across the biofilm, and the expression of genes associated with tolerance or resistance to antimicrobial agents (Stewart and Costerton, 2001; Flemming and Wingender, 2010; Høiby et al, 2010). Given the increased tolerance of biofilms to antimicrobials, there remains a growing need for more effective antibiotics and new approaches to target biofilm infections. There have been increased efforts toward alternative approaches to induce cell death and/or promote dispersion of biofilms (Koo et al, 2017; Pinto et al, 2020)
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