Abstract

1. The Destruction of Biofilms Using Ultrasound TreatmentBen Rusk1, Dr. Timothy Bigelow2,3, Dr. Larry Halverson4 and Jin Xu31 Physics Department, Truman State University2Department of Electrical and Computer Engineering, 3Department of Mechanical Engineering, 4Department of Plant Pathology, Iowa State University Abstract Experimental Methods Results • Medical implants can sometimes become infected by biofilms, or bacteria communities that show a high level of resistance to antibiotics. There is a need for non-invasive techniques to treat these infections, and one method that displays potential is the use of high intensity focused ultrasound (HIFU). To explore this concept further, biofilms were created in a flow chamber system by Pseudomonas aeruginosa. The biofilms were created on each of three graphite disks located in the chamber. Graphite was chosen because of its common use in medical implants. Multiple trials were run until creating consistent biofilms on each disk was possible. A different flow cell system containing an exposure chamber was then used for the ultrasound treatment of the disks. After the disks were subjected to a pulsed 1.1 MHz signal at 13 MPa,they were examined to determine whether the treatment had removed the biofilms. Examining the disks after the treatment showed evidence that the ultrasound had removed most of the biofilms from the surface. • Growing Biofilms • Use bacteria Pseudomonas aeruginosa with green fluorescent protein (gfp) • Medium is1/10th strength trypticase soy broth (TSB) • Grow on three separate graphite disks, similar to medical implants • Keep in incubator at 37°C • Inject 0.05 concentration of bacteria into flow cell chamber • Let bacteria adhere to disk surface for one hour • Begin flow of TSB solution at 1-2 mL/minute • Continue steady flow for 1 to 4 days • Calibrating the Transducer • │ΓG │ = │VG│/ │VA│ = 128mV / 196mV ≈ 0.65 • The ratio of the two peak to peak voltages gave a value of 0.65 for the graphite reflection coefficient. The desired pressure on the graphite plate to remove biofilms is 13 MPa1. To achieve this pressure the input signal required was determined to be 480 mVp-p. The transducer sent a signal at 1.1 MHz, with 20 cycles and a burst period of 5 ms. • Ultrasound Treatment • The images below show three disks from the same flow chamber post treatment. The disks were allowed to grow for 24 hours in the flow system, and then stored overnight in purified water Each disk is labeled according to its position in the chamber, with front being closest to the medium inlet and back the outlet. All three disks had similar biofilm structures before the treatment, regardless of position. The three disks underwent different treatment, with one disk receiving the HIFU for 56 minutes total (30 seconds/point), one for 28 minutes total (15 seconds/point), and another receiving no HIFU. The green cells are alive while the red are dead/dying cells. • High Intensity Focused Ultrasound Treatment • Graphite disk with biofilm sits in exposure chamber sealed with clear saran wrap • Tank filled with de-gassed water, while purified water is pumped through system during treatment • Disk found using oscilloscope to measure the reflection • Motorized stage moves the transducer • Treatment performed by a computer program designed to scan across the surface of the disk • Ultrasound causes cavitation, or the formation of small micro-bubbles which mechanically destroy the biofilms • Disk stained with live/dead stain post treatment • Fluorescence microscopy is used to visualize live (green) and dead (red) cells to identify biofilm properties and cell health Objectives • Short Term Goals • Prepare flow cell system • Grow consistent, healthy biofilms • Create exposure chamber system • Calibrate transducer for treatment • Perform HIFU treatments • Long Term Goals • Determine if the effects of HIFU treatment are consistent for different types of biofilms • Test whether the treatment destroys or just dislodges the biofilms • Research the biofilms susceptibility to antibiotics after the HIFU treatment What is a Biofilm? • Calibrating the Transducer • │VG│= ΓG ∙ Pinc ∙ k and │VA│= ΓA ∙ Pinc ∙ k • V is the peak to peak voltage of the reflected signal and Γ is the reflection coefficients for the graphite disk and the water. The incident pressure and constant k are equal in both equations giving the relationship: • │VG│/ │VA│ = │ΓG │/ │ ΓA │ • Since: │ΓG │ = (Z2 – Z1) / (Z2 + Z1) and ZAir ≈ 0 the reflection coefficient for the graphite disk is the ratio of the two voltages which can easily be measured: • │ΓG │ = │VG│/ │VA│ • This reflection coefficient is related to the pressures on the graphite and water: • PGraphite = PWater (1 + ΓG) • To achieve the desired pressure on the graphite disk, the system had to be calibrated to have a certain value of pressure on the water (PWater). This pressure is related to the input voltage level. By running a code containing the measured reflection coefficient (│ΓG │) and the desired pressure on the graphite disk (PGraphite ), the necessary input voltage was determined. Conclusions Although no quantitative results were obtained from the experiment, the images show a definitive difference between the untreated graphite disk and the two disks that received HIFU treatment. The control disk shows a mostly healthy and robust biofilm, while the other two disks show very little biofilm and most of the remaining cells are dead. There is no clear difference between the two disks receiving 56 versus 28 minutes of treatment. Before a formal conclusion can be made there needs to be many more trials. It will also be important to determine if the biofilms were completely destroyed or just detached from the disk surface. This initial trial gives evidence that high intensity focused ultrasound can be effective in removing bacterial biofilms from the surface of graphite disks. Further exploration into this phenomena may lead to a major discovery in medicine and a successful treatment for medical implant infections. http://biofilmbook.hypertextbookshop.com Biofilms consist of bacteria that adhere to a surface and each other. These bacteria become dormant, or sessile, and form a protective outer matrix. Inside the structure of the biofilm, fully functional planktonic cells are created. These cells are eventually released and free to form other biofilms and infections. Antibiotics are effective in killing planktonic bacteria, but unsuccessful against biofilms because of the protective matrix and their slow metabolism. As a consequence, implants such as catheters, stints, and heart valves must be removed and replaced in order to fully cure the infection. Above shows the biofilm life cycle and a picture of a biofilm grown in our lab. The bacteria used contains a green fluorescent protein from jelly fish that allows us to readily identify cells. 1T. Bigelow et al., Ultrasound in Med. And Biol., 35(6) (2009) 1026-31