<i>P. aeruginosa</i> virulence tests on lettuce leaves. Lettuce leaves were injected with <i>P. aeruginosa</i> biofilm-forming cells treated with plasma for 0, 1 or 30 minutes (panels “a”, “b” and “c” respectively) as described in Materials and Methods.

<p>A control injected with saline (panel “d”) was included as a negative control.</p

<i>P. aeruginosa</i> biofilm force-displacement curve for the control sample on a glass coupon.

<p>Data for tip-sample approach (blue circles) and tip-sample retraction (red circles) are shown. The negative displacement of the cantilever that occurred due to tip adhesion to the biofilm upon retraction is designated as the adhesive step, and is measured to be 0.288 µm for this curve.</p

<i>P. aeruginosa</i> biofilm force-displacement curve adhesive step data (sample 3), 0 minute (control, hatched bars) and 30 minute plasma-treatment (solid bars).

<p>The height of each bar on the graph corresponds to the mean slope of five curves obtained for each region. The horizontal black bars represent the mean adhesive step values.</p

AFM images. 10×10 µm² area AFM image of <i>P. aeruginosa</i> biofilm for the control sample indicating locations for obtaining force-displacement curve data.

<p>Force-displacement curves obtained at locations similar to point A were designated as areas of predominately matrix material and locations similar to point B were designated as areas of predominately bacteria.</p

Survivor curve for <i>P. aeruginosa</i> plasma-treated biofilms.

<p>Log of the number of <i>P. aeruginosa</i> CFU/mL vs plasma exposure time (0 to 30 minutes). Results are the average of five independent experiments. Each experiment was performed in duplicate. Bars represent the standard error of the mean.</p

Atomic force microscope images of <i>P. aeruginosa</i> biofilms treated with plasma for 0 minute (column a), 1 minute (column b) and 30 minutes (column c).

<p>Data for samples 1, 2, and 3 are displayed in the top, middle, and bottom rows, respectively. Image areas are 10×10 µm².</p

<i>P. aeruginosa</i> biofilm force-displacement curve adhesive step data for 0 minute (control) plasma-treated sample 1.

<p>The height of each bar on the graph corresponds to the mean adhesive step height of five curves obtained for each region. Error bars represent the standard error of the mean. Hatched bars and solid bars represent measurements on mostly bacteria and mostly matrix areas, respectively.</p

Plasma-Mediated Inactivation of Pseudomonas aeruginosa Biofilms Grown on Borosilicate Surfaces under Continuous Culture System

Biofilms are microbial communities attached to a surface and embedded in a matrix composed of exopolysaccharides and excreted nucleic acids. Bacterial biofilms are responsible for undesirable effects such as disease, prostheses colonization, biofouling, equipment damage, and pipe plugging. Biofilms are also more resilient than free-living cells to regular sterilization methods and therefore it is indispensable to develop better ways to control and remove them. The use of gas discharge plasmas is a good alternative since plasmas contain a mixture of reactive agents well-known for their decontamination potential against free microorganisms. We have previously reported that Pseudomonas aeruginosa biofilms were inactivated after a 1-min plasma exposure. We determined that the adhesiveness and the thickness of Pseudomonas biofilms grown on borosilicate were reduced. We also reported sequential morphological changes and loss of viability upon plasma treatment. However, the studies were carried out in batch cultures. The use of a continuous culture results in a more homogenous environment ensuring reproducible biofilm growth. The aim of this work was to study plasma-mediated inactivation of P. aeruginosa biofilms grown on borosilicate in a continuous culture system. In this paper we show that biofilms grown on glass under continuous culture can be inactivated by using gas discharge plasma. Both biofilm architecture and cell culturability are impacted by the plasma treatment. The inactivation kinetics is similar to previously described ones and cells go through sequential changes ranging from minimal modification without loss of viability at short plasma exposure times, to major structure and viability loss at longer exposure times. We report that changes in biofilm structure leading to the loss of culturability and viability are related to a decrease of the biofilm matrix adhesiveness. To our knowledge, there has been no attempt to evaluate the inactivation/sterilization of biofilms grown in a continuous system