Abstract
Bacterial biofilm prevention and eradication are common treatment problems, hence there is a need for advanced and precise experimental methods for its monitoring. Bacterial resistance to antibiotics has resulted in an interest in using a natural bacterial enemy—bacteriophages. In this study, we present the application of quartz tuning forks (QTF) as impedance sensors to determine in real-time the direct changes in Pseudomonas aeruginosa PAO1 biofilm growth dynamics during Pseudomonas phage LUZ 19 treatment at different multiplicities of infection (MOI). The impedance of the electric equivalent circuit (EEC) allowed us to measure the series resistance (Rs) corresponding to the growth-medium resistance (planktonic culture changes) and the conductance (G) corresponding to the level of QTF sensor surface coverage by bacterial cells and the extracellular polymer structure (EPS) matrix. It was shown that phage impacts on sessile cells (G dynamics) was very similar in the 10-day biofilm development regardless of applied MOI (0.1, 1 or 10). The application of phages at an early stage (at the sixth h) and on three-day biofilm caused a significant slowdown in biofilm dynamics, whereas the two-day biofilm turned out to be insensitive to phage infection. We observed an inhibitory effect of phage infection on the planktonic culture (Rs dynamics) regardless of the MOI applied and the time point of infection. Moreover, the Rs parameter made it possible to detect PAO1 population regrowth at the latest time points of incubation. The number of phage-insensitive forms reached the level of untreated culture at around the sixth day of infection. We conclude that the proposed impedance spectroscopy technique can be used to measure the physiological changes in the biofilm matrix composition, as well as the condition of planktonic cultures in order to evaluate the activity of anti-biofilm compounds.
Highlights
Bacterial biofilm is a complex and dynamically changing structure responding to the external environment
Our previous experience with the application quartz tuning forks (QTF) sensor platform regarded biofilm biomass measurements when P. aeruginosa sessile cells were treated with antibiotics [15,21]
The changes in the resonant frequency of QTFs covered with biofilm structure were measured, which were converted into the mass of bacterial biofilm at selected culture time points
Summary
Bacterial biofilm is a complex and dynamically changing structure responding to the external environment. Microbial cell aggregates are surrounded by complex polymer substances (matrix) composed of proteins, polysaccharides, eDNA (extracellular DNA), glycolipids and fatty acids [1]. Bacteria inside the biofilm are not synchronized in terms of metabolic activity, and biofilm structure is highly heterogeneous in this respect [2,3]. The protection against unfavorable environmental conditions can be implemented by the extracellular polymer structure (EPS) production, increasing. Biofilm is a long-term strategy for bacterial survival under stress conditions. Cells embedded in the consortia cause biomaterials dysfunction, chronic infections, are highly insensitive to treatment and pose a threat in industry or are responsible for biofouling [5]. To evaluate the complex and sublime interactions of antibacterials and biofilm cells it was necessary to develop various measurement techniques
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