Atomic Force Microscopy (AFM) Imaging and Assessment of Plasma-Treated Bacterial Biofilms

VANDERVOORT, K.; BRELLES MARIÑO, G.

La Plata

Workshop; Imaging Techniques for Biotechnology and Biomedical Applications Workshop; 2016

Bacterial biofilms are microbial communities less susceptible to standard eradication methods than free-living bacteria. The use of gas-discharge plasmas represents a novel alternative because plasmas contain a mixture of charged particles, free radicals, and UV radiation, each individually known as growth-controlling agents. An atmospheric-pressure gas discharge plasma was produced using a capacitively coupled electrode design. A plasma jet was generated in He at a flow of 20.4 L/min. Plasma was used to treat for various exposure times either Chromobacterium violaceum or Pseudomonas aeruginosa biofilms grown under different culture regimes. AFM images were obtained in air using a Quesant Instruments Universal Scanning Probe Microscope in contact mode. Commercial silicon cantilevers from MikroMaschTM with spring constants from 0.1 to 0.5 N/m were used. AFM was used to reveal the sequential changes in cell morphology occurring during plasma treatment and to study micromechanical properties of the plasma-treated biofilm. Chromobacterium violaceum biofilms grown in microtiter platesC. violaceum biofilms were grown in 96-wells microtiter plates and treated with plasma for various exposure times. For imaging, bacteria were disaggregated from the biofilms by sonication and suspended to produce smears onto glass slides. The plasma treatment renders bacterial cells nonculturable after short plasma-exposure times (e.g., 5 min). AFM images show that cells go through sequential morphological changes after plasma treatment. At short exposure times, cell may undergo modifications ranging from minimal changes (such as cells with "rougher" surfaces) to putative loss of cell walls, leading to the production of spheroplasts [1]. We verified the relative ?roughness? of cells by examining image cross sections and analyzing the standard deviation of the surface height. These surface features are consistent with cells undergoing damage and were observed in a small percentage of the 5-minute plasma exposure scans [2]. After 60 minutes of plasma treatment, no recognizably intact cells but only debris or cell remnants were obtained. Longer exposure times result in major cell damage [1, 2]Pseudomonas aeruginosa PAO1 biofilms grown in a CDC biofilm reactorP. aeruginosa biofilms grown on glass coupons either in batch or in continuous culture in a biofilm reactor were treated with gas-discharge plasma for various exposure times and processed for AFM imaging. For each coupon, at least four separated regions were imaged to obtain a representative sample and ensure reproducibility. To assess micromechanical properties of the biofilm, force-displacement curves were obtained at widely separated locations on the coupon. The procedure consisted of bringing the AFM tip in contact with the sample and then moving the sample upward a set distance while monitoring the deflection of the cantilever. At each sampling location where force-displacement curves were obtained, the tip was brought in and out of contact to the maximum set sample deflection and the displacement curve was recorded upon the fifth trial. This technique helped to reduce hysteresis that was often observed in the first few trials. The process was repeated so that at least five force-displacement curves were recorded at each sampling location. For comparing samples with different plasma treatments, all of the force-displacement data were recorded on the same day using the same cantilever. This method ensured control for humidity and cantilever dependent factors that can influence the shape of these curves. Results show that the adhesiveness and thickness of the Pseudomonas biofilms grown on glass coupons in batch culture are reduced upon plasma treatment. Sequential morphological changes upon plasma treatment are also observed [3, 4]. For biofilms grown in continuous culture, we also reported morphological changes upon plasma treatment with modified adhesiveness to glass and a decrease in the thickness of the biofilm matrix. We hypothesized that the decrease and eventual loss of the biofilm matrix would reduce the adhesiveness of the biofilm to the surface to which it is anchored and would lead to disorganization of its tridimensional structure. To test this hypothesis we assessed micromechanical properties of the biofilm through force-displacement curves obtained from two distinct sample locations: areas of mostly matrix material or areas of mostly bacteria. For each sample, the average value of the heights of the adhesive steps was always greater when measured at mostly matrix areas than for areas of mostly bacteria. Also, force-displacement curves were obtained on two of the samples comparing the 0 minute to the 30-minute plasma treatment. For these comparisons, curves were obtained only on areas of predominately cells since none of the 30-minute treatments yielded any areas of mostly matrix. On average, the adhesion to the plasma treated areas is significantly less than for the control. Analyses of the results from about 300 images and approximately 700 force displacement curves, show that plasma treatment of P. aeruginosa biofilms results in little change in cell morphology for short exposure times while longer exposures results in significant loss of the biofilm structure and cell death. The adhesiveness of the biofilm varies across its structure and is higher in areas with larger amounts of matrix. Plasma treatment removes or at least reduces the matrix, presumably by oxidation/peroxidation due to the presence of free radicals, and the areas of predominantly bacteria are less adhesive after the treatment. These findings demonstrate the utility of AFM imaging techniques to understand some of the processes biofilms undergo upon plasma treatment.