Infectious Diseases: Using xCELLigence for Bacterial Biofilm Studies

Tim Howell

[Real-time monitoring of] antibiotic resistance patterns of biofilms… revealed features that would pass unnoticed by end-point methods.Ferrer et al. Journal of Applied Microbiology 2017 Mar; 122(3): 640-650.
Using xCELLigence for Bacterial Biofilm Studies

Electron micrograph of E. coli biofilm.

Besides playing critical roles in human dental plaques and cavities, chronic infections, rejection of artificial implants, and food poisoning, bacterial biofilms are also responsible for a large percentage of livestock diseases and cause fouling of industrial air and water handling systems, further increasing their economic impact. Though developing drugs to treat biofilms – or prevent their formation in the first place – is of critical importance, the colorimetric assays traditionally used for studying biofilms are inefficient/low throughput, are incompatible with orthogonal assays (i.e. samples are destroyed by the analysis process), and only provide end point data.

Studying biofilms using impedance monitoring with xCELLigence® Real-Time Cell Analysis (RTCA) instruments overcomes each of these limitations, enabling a quantitative and continuous evaluation of biofilms via an assay that is both label-free and totally automated.

RTCA technology opens new opportunities in the biofilm research arena.” “[Our results] support that impedance-based RTCA monitoring is a fast, reliable, and non-time consuming method that can be easily used…to search for bio-actives that interfere with biofilm formation, or remove preformed ones.Guitierrezet al. PLOS ONE 2016 Oct 3; 11(10); e0163966.

View of a single E-Plate well.

ACEA’s electronic microtiter plates (E-Plates®) contain gold microelectrodes that are integrated into the bottom surface of each well. In the photo to the right, which is looking down into a single well from a 96-well plate, the array of gold electrodes is seen to cover ~75% of the well bottom’s surface area.

By applying a weak (~22 mV) electric potential to the E-Plate, the xCELLigence instrument causes a miniscule electric current to flow between the electrodes.  The ease with which this current flows is directly dependent on the total electrode surface area that is covered by bacterial cells and extracellular polymeric substance (EPS), and on how tightly cells and EPS adhere to the electrodes. This principle is represented schematically below, where a single well is shown at two different time points.  Note that, for clarity, only two electrodes are shown in the well bottom.  Compounds that prevent biofilm formation, or that cause the disruption of previously established biofilms, can readily be identified by changes in the impedance signal.

By including antibiotic in the growth media at the time that Staphylococcus aureus 240 was seeded into E-Plate® wells, the capacity of RTCA to identify biofilm blocking activity was evaluated. As seen in Figure A, though each of the 10 antibiotics that was tested displayed prophylactic activity, they did this with differing levels of activity. While cefotaxime completely destroyed the biofilm-associated signal at a concentration of 0.25 μg/mL, linezolid required a 128-fold higher concentration to accomplish this. As a proof of principle, this experiment demonstrates the efficacy of RTCA as a tool for drug screens aimed at preventing biofilms from forming in the first place.

Of very high clinical relevance is the finding that within particular concentration ranges some antibiotics can actually promote biofilm growth. Being able to characterize this unwanted behavior is critical for preventing physicians from unwittingly exacerbating the very infection they are trying to treat. Importantly, this bifurcated behavior was readily detectable, and quantifiable, using RTCA. While at concentrations of 4-32 μg/mL vancomycin is found to suppress Staphylococcus epidermidis 43040 biofilm growth, at concentrations of 62.5 ng/mL-1 μg/mL biofilm growth is stimulated (Figure B).Using RTCA to screen for drugs that prevent biofilm formation. (A) Ten different antibiotics (each represented by a different colored line) were evaluated for their ability to prevent S. aureus 240 from forming biofilm. Antibiotics were present at different concentrations from the moment that bacteria were seeded into wells. 20 hours after seeding, the cell index was measured and compared to the untreated control. The % Cell Index plotted here is simply [(Cell Index)with drug/(Cell Index)without drug]x100. (B) Depending on its concentration, vancomycin either inhibits or stimulates the growth of S. epidermidis 43040 biofilm.

Key Benefits of Using xCELLigence for Bacterial Biofilm Studies:
  1. Continuous monitoring: Track biofilm formation and/or dissipation in real time to reveal behavioral details that are missed by end point assays.
  2. Reduced workload: Once cells are seeded and data acquisition has been initiated, no further involvement is required. Data is continuously recorded for anywhere from minutes to days/weeks.
  3. Diverse applications: Quorum sensing, clinical theranostics, screening for biofilm preventing or disrupting compounds, etc.
Download Biofilm Application Note
Bacterial Biofilms Supporting Information:

  • Bacterial Biofilms Application Note:
  1. Studying Bacterial Biofilms Using Cellular Impedance
  • Bacterial Biofilms Publications:
  1. Bacterial quorum-sensing network architectures. Ng WL, Bassler BL.  Annu Rev Genet. 2009;43:197-222.
  2. Use of the Real Time xCelligence System for Purposes of Medical Microbiology. Junka AF, Janczura A, Smutnicka D, Mączyńska B, Secewicz A, Nowicka J, Bartoszewicz M, Gościniak G. Polish Journal of Microbiol. 2012, 61(3), 191-197.
  3. Staphylococcus aureus and MRSA Growth and Biofilm Formation after Treatment with Antibiotics and SeNPsCihalova K, Chudobova D, Michalek P, Moulick A, Guran R, Kopel P, Adam V, Kizek R. Int J Mol Sci. 2015 Oct 16;16(10):24656-72.
  4. Specificity and complexity in bacterial quorum-sensing systems. Hawver LA, Jung SA, Ng WL.  FEMS Microbiol Rev. 2016 Sep;40(5):738-52.
  5. Monitoring in Real Time the Formation and Removal of Biofilms from Clinical Related Pathogens Using an Impedance-Based Technology. Gutiérrez D, Hidalgo-Cantabrana C, Rodríguez A, García P, Ruas-Madiedo P. PLoS One. 2016 Oct 3;11(10):e0163966.
  6. Effect of Antibiotics on Biofilm Inhibition and Induction measured by Real-Time Cell Analysis.  Ferrer MD, Rodriguez JC, Álvarez L, Artacho A, Royo G, Mira A.  J Appl Microbiol. 2016 Dec 8.
  7. Bacillus subtilis from Soybean Food Shows Antimicrobial Activity for Multidrug-Resistant Acinetobacter baumannii by Affecting the adeS Gene. Wang T, Su J. J Microbiol Biotechnol. 2016 Dec 28;26(12):2043-2050.
  8. Use of Single-Frequency Impedance Spectroscopy to Characterize the Growth Dynamics of Biofilm Formation in Pseudomonas aeruginosa. Van Duuren JBJH, Müsken M, Karge B, Tomasch J, Wittmann C, Häussler S, Brönstrup M. Sci Rep. 2017 Jul 12;7(1):5223