Antimicrobial effects of microwave-induced plasma torch (MiniMIP) treatment on Candida albicans biofilms

Abstract

The susceptibility of Candida albicans biofilms to a non-thermal plasma treatment has been investigated in terms of growth, survival and cell viability by a series of in vitro experiments. For different time periods, the C. albicans strain SC5314 was treated with a microwave-induced plasma torch (MiniMIP). The MiniMIP treatment had a strong effect (reduction factor (RF) = 2.97 after 50 s treatment) at a distance of 3 cm between the nozzle and the superior regions of the biofilms. In addition, a viability reduction of 77% after a 20 s plasma treatment and a metabolism reduction of 90% after a 40 s plasma treatment time were observed for C. albicans. After such a treatment, the biofilms revealed an altered morphology of their cells by atomic force microscopy (AFM). Additionally, fluorescence microscopy and confocal laser scanning microscopy (CLSM) analyses of plasma-treated biofilms showed that an inactivation of cells mainly appeared on the bottom side of the biofilms. Thus, the plasma inactivation of the overgrown surface reveals a new possibility to combat biofilms.

Figure 1

CFU, fluorescence and XTT assay of C. albicans biofilms after treatment with the MiniMIP. (A) CFU measurements of the MiniMIP‐treated C. albicans biofilms. The line with ● represents the detection limit. The line with ▪ shows the reduction factor (RF) of the different plasma treatment times. The error bars were calculated using the propagation of error and the weighted error. The RF for the 10 s treatment with the MiniMIP shown to intersect the x‐axis was not statistically significant. (B) Fluorescence LIVE/DEAD assay of the MiniMIP‐treated C. albicans biofilms. The ratio G/R is defined as the division of the emission of green fluorescence by the emission of red fluorescence. (C) XTT measurements of the MiniMIP‐treated C. albicans biofilms. The data points of all measurements represent the weighted mean value of the total population of the treatment time from the quadruple repetition n = 6.

Figure 2

Overview with the Operetta CLS fluorescence microscope of MiniMIP‐treated biofilms. (A) Control; (B) 10 s plasma treatment time; (C) 20 s plasma treatment time; (D) 30 s plasma treatment time; (E) 40 s plasma treatment time; (F) 50 s plasma treatment time; (G) 60 s plasma treatment time. The biofilms were stained with SYTO 9 (green fluorescence for all cells) and propidium iodide (red fluorescence of dead cells). The pictures show inverse images. The scale bar indicates 1 mm.

Figure 3

Confocal laser scanning microscopy (CLSM) images of LIVE/DEAD‐stained C. albicans biofilms after plasma treatment with the MiniMIP. Left panels show an orthogonal view of the top biofilm layer (horizontal optical sections in the centre and vertical optical sections in the flanking pictures). Central and right panels show 3D images with a top and a bottom view of the biofilms. For each biofilm, an area of 1272.2 µm × 1272.2 µm was visualized.

Figure 4

Atomic force microscopy (AFM) images of the MiniMIP‐treated biofilms and untreated controls. The left side shows the topographic image and the right side the error‐signal image of the same spot. (A) Untreated control; (B) 30 s plasma treatment time; (C) 60 s plasma treatment time. The images were acquired in contact mode with a cantilever spring constant of k = 0.1–0.6 N/m² and a frequency of 0.4 Hz, the set point at 8 N/m² and an area of 20 µm².

Figure 5

Optical emission spectroscopy (OES) of the radiofrequency plasma jet kINPen09 and the microwave‐induced plasma torch MiniMIP. (A) Emission spectra of the radiofrequency plasma jet kINPen09. (B) Emission spectra of the MiniMIP. The inset boxes represent the respective wavelength region at higher resolution. The emission spectra represent the respective molecules. The measurement results were obtained with LabVIEW and evaluated with MATLAB.

Figure 6

Structure of the microwave‐driven plasma torch MiniMIP. The ceramic tube leads completely into the inside of the housing, where it is encased by an aluminium tube. The ignition of the plasma takes place at the front edge of the aluminium tube, and plasma propagation is driven by the surrounding microwave field and the gas flow along the longitudinal axis of the ceramic tube leading to a small plasma plume of about 10 mm in length and 3 mm in diameter outside the device. Left: microwave‐driven plasma torch MiniMIP; right: schematic structure of the MiniMIP.