Eradication of Pseudomonas aeruginosa biofilms by atmospheric pressure non-thermal plasma

Abstract

Bacteria exist, in most environments, as complex, organised communities of sessile cells embedded within a matrix of self-produced, hydrated extracellular polymeric substances known as biofilms. Bacterial biofilms represent a ubiquitous and predominant cause of both chronic infections and infections associated with the use of indwelling medical devices such as catheters and prostheses. Such infections typically exhibit significantly enhanced tolerance to antimicrobial, biocidal and immunological challenge. This renders them difficult, sometimes impossible, to treat using conventional chemotherapeutic agents. Effective alternative approaches for prevention and eradication of biofilm associated chronic and device-associated infections are therefore urgently required. Atmospheric pressure non-thermal plasmas are gaining increasing attention as a potential approach for the eradication and control of bacterial infection and contamination. To date, however, the majority of studies have been conducted with reference to planktonic bacteria and rather less attention has been directed towards bacteria in the biofilm mode of growth. In this study, the activity of a kilohertz-driven atmospheric pressure non-thermal plasma jet, operated in a helium oxygen mixture, against Pseudomonas aeruginosa in vitro biofilms was evaluated. Pseudomonas aeruginosa biofilms exhibit marked susceptibility to exposure of the plasma jet effluent, following even relatively short (≈ 10's s) exposure times. Manipulation of plasma operating conditions, for example, plasma operating frequency, had a significant effect on the bacterial inactivation rate. Survival curves exhibit a rapid decline in the number of surviving cells in the first 60 seconds followed by slower rate of cell number reduction. Excellent anti-biofilm activity of the plasma jet was also demonstrated by both confocal scanning laser microscopy and metabolism of the tetrazolium salt, XTT, a measure of bactericidal activity.

Figure 1. The plasma jet used in this study.

(A) Schematic diagram of the plasma jet. (B) Photograph of the plasma jet interacting with a biofilm sample.

Figure 2. Bacterial growth inhibition zones.

P. aeruginosa cell suspensions were spread over MHA plates (9 cm in diameter). The seeded plates were exposed to the 20 kHz plasma jet for (A) 0 s, (B) 120 s, and (C) 240 s and then incubated at 37°C for 24 hours. After incubation, photographs of agar plates, showing bacterial growth inhibition zones, were taken using a digital camera.

Figure 3. Survival curve of biofilm treated with 20 kHz plasma.

48-hour P. aeruginosa biofilms, grown on Calgary Biofilm Device, were exposed to the 20 kHz plasma jet for up to 4 minutes. The number of biofilm surviving cells in each sample was then calculated using colony count method and used to construct the log survival curve. (Each point represents the mean of 3 values ± SE).

Figure 4. Survival curves of biofilms treated with 20 kHz vs. 40 kHz plasma.

Comparison between log survival curves of P. aeruginosa biofilm cells, constructed as described above, upon exposure to both 20 kHz and 40 kHz plasma jet. (Each point represents the mean of 3 values ± SE).

Figure 5. Absorbance of XTT-assay product.

48-hour P. aeruginosa biofilms, grown on Calgary Biofilm Device, were exposed to the 20 kHz plasma jet for up to 4 minutes. After plasma exposure, bacterial cells were dislodged off the pegs into PBS buffer by sonication. 50 µl aliquots of the recovered bacterial suspensions were then mixed with 50 µl of MHB and 20 µl of XTT stock solution and incubated at 37°C for 5 hours. After incubation, the absorbance at 450 nm was measured to quantify XTT metabolic product, the intensity of which is proportional to the number of viable (respiring) cells. (Each point represents the mean of 3 values ± SE).

Figure 6. Percentage cell reduction curves based on colony count method vs. XTT assay.

Percentage cell reduction curves of P. aeruginosa biofilm cells upon exposure to the 20 kHz plasma jet. The dotted line is based on the standard colony count method whereas the solid line is based on the XTT assay. (Each point represents the mean of 3 values ± SE).

Figure 7. CLSM images of the plasma treated biofilms.

3D rendered confocal laser scanning micrographs of 3-day P. aeruginosa biofilms, grown on polycarbonate coupons, exposed to the 20 kHz plasma jet for 0s (A and D), 60 s (B and E), and 240 s (C and F). Green colour indicates surviving cells whereas red colour indicates dead cells. Magnification power is 200x (a-c) and 600x (d–f).