Bacterial biofilms are complex microbial communities that contribute to the pathogenesis of chronic infections. Therefore, it is crucial to detect biofilm-associated infections in early stages as their delayed treatment becomes more complicated. Herein, we describe a label-free electrochemical impedance spectroscopy (EIS) method for detecting biofilm formation by Staphylococcus aureus and Staphylococcus epidermidis. Printed circuit board-based biamperometric gold electrodes were modified with poly-L-lysine to enhance bacterial attachment to the sensor surface. Formation and inhibition of biofilms were evaluated based on changes in charge transfer resistance (Rct). The control Rct value increased by ~90 kΩ for S. epidermidis biofilm and by ~60 kΩ for S. aureus biofilms. Antibiotic-treated samples exhibited similar values to those using the control. In addition, biofilm formation was evaluated through optical microscopy using safranin staining, and the micrographs suggest significant biomass on the electrodes, whereas the control appeared clear. Atomic force microscopy was used to visualize the biofilm on the electrode surface, obtain cross-sectional profiles, and evaluate its roughness. The roughness parameters indicate that S. aureus forms a rougher biofilm than S. epidermidis, while S. epidermidis forms a more compact biofilm. These findings suggest that the optimized EIS-based method effectively monitors changes related to biofilms and serves as a promising tool for evaluation of new anti-biofilm agents, such as antibiotics, phages or antibodies.

Central European Institute of Technology Masaryk University Kamenice 5 625 00 Brno Czech Republic

Department of Biochemistry Faculty of Science Masaryk University Kamenice 5 625 00 Brno Czech Republic

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Mahto K.U., Vandana, Priyadarshanee M., Samantaray D.P., Das S. Bacterial Biofilm and Extracellular Polymeric Substances in the Treatment of Environmental Pollutants: Beyond the Protective Role in Survivability. J. Clean. Prod. 2022;379:134759. doi: 10.1016/j.jclepro.2022.134759. DOI

Wang Y., Bian Z., Wang Y. Biofilm Formation and Inhibition Mediated by Bacterial Quorum Sensing. Appl. Microbiol. Biotechnol. 2022;106:6365–6381. doi: 10.1007/s00253-022-12150-3. PubMed DOI

Karygianni L., Ren Z., Koo H., Thurnheer T. Biofilm Matrixome: Extracellular Components in Structured Microbial Communities. Trends Microbiol. 2020;28:668–681. doi: 10.1016/j.tim.2020.03.016. PubMed DOI

Guzmán-Soto I., McTiernan C., Gonzalez-Gomez M., Ross A., Gupta K., Suuronen E.J., Mah T.-F., Griffith M., Alarcon E.I. Mimicking Biofilm Formation and Development: Recent Progress in in vitro and in vivo Biofilm Models. iScience. 2021;24:102443. doi: 10.1016/j.isci.2021.102443. PubMed DOI PMC

Kreve S., Reis A.C.D. Bacterial Adhesion to Biomaterials: What Regulates This Attachment? A Review. Jpn. Dent. Sci. Rev. 2021;57:85–96. doi: 10.1016/j.jdsr.2021.05.003. PubMed DOI PMC

Wang C., Hou J., van der Mei H.C., Busscher H.J., Ren Y. Emergent Properties in Streptococcus mutans Biofilms Are Controlled through Adhesion Force Sensing by Initial Colonizers. mBio. 2019;10:10-1128. doi: 10.1128/mBio.01908-19. PubMed DOI PMC

Palmer J., Flint S., Brooks J. Bacterial Cell Attachment, the Beginning of a Biofilm. J. Ind. Microbiol. Biotechnol. 2007;34:577–588. doi: 10.1007/s10295-007-0234-4. PubMed DOI

Theis T.J., Daubert T.A., Kluthe K.E., Brodd K.L., Nuxoll A.S. Staphylococcus aureus Persisters Are Associated with Reduced Clearance in a Catheter-Associated Biofilm Infection. Front. Cell Infect. Microbiol. 2023;13:1178526. doi: 10.3389/fcimb.2023.1178526. PubMed DOI PMC

Conlon B.P. Staphylococcus aureus Chronic and Relapsing Infections: Evidence of a Role for Persister Cells: An Investigation of Persister Cells, Their Formation and Their Role in S. aureus Disease. BioEssays. 2014;36:991–996. doi: 10.1002/bies.201400080. PubMed DOI

Lewis K. Persister Cells, Dormancy and Infectious Disease. Nat. Rev. Microbiol. 2007;5:48–56. doi: 10.1038/nrmicro1557. PubMed DOI

Zhao A., Sun J., Liu Y. Understanding Bacterial Biofilms: From Definition to Treatment Strategies. Front. Cell Infect. Microbiol. 2023;13:1137947. doi: 10.3389/fcimb.2023.1137947. PubMed DOI PMC

Rather M.A., Gupta K., Mandal M. Microbial Biofilm: Formation, Architecture, Antibiotic Resistance, and Control Strategies. Braz. J. Microbiol. 2021;52:1701–1718. doi: 10.1007/s42770-021-00624-x. PubMed DOI PMC

Del Pozo J.L. Biofilm-Related Disease. Expert. Rev. Anti Infect. Ther. 2018;16:51–65. doi: 10.1080/14787210.2018.1417036. PubMed DOI

Severn M.M., Horswill A.R. Staphylococcus epidermidis and Its Dual Lifestyle in Skin Health and Infection. Nat. Rev. Microbiol. 2023;21:97–111. doi: 10.1038/s41579-022-00780-3. PubMed DOI PMC

Idrees M., Sawant S., Karodia N., Rahman A. Staphylococcus aureus Biofilm: Morphology, Genetics, Pathogenesis and Treatment Strategies. Int. J. Environ. Res. Public Health. 2021;18:7602. doi: 10.3390/ijerph18147602. PubMed DOI PMC

Diepoltová A., Konečná K., Janďourek O., Nachtigal P. Study of the Impact of Cultivation Conditions and Peg Surface Modification on the in vitro Biofilm Formation of Staphylococcus aureus and Staphylococcus epidermidis in a System Analogous to the Calgary Biofilm Device. J. Med. Microbiol. 2021;70:001371. doi: 10.1099/jmm.0.001371. PubMed DOI

Arciola C.R., Campoccia D., Montanaro L. Detection of Biofilm-Forming Strains of Staphylococcus epidermidis and S. aureus. Expert. Rev. Mol. Diagn. 2002;2:478–484. doi: 10.1586/14737159.2.5.478. PubMed DOI

Khatoon Z., McTiernan C.D., Suuronen E.J., Mah T.-F., Alarcon E.I., Alarcon Bacterial E.I. Bacterial Biofilm Formation on Implantable Devices and Approaches to Its Treatment and Prevention. Heliyon. 2018;4:1067. doi: 10.1016/j.heliyon.2018.e01067. PubMed DOI PMC

Li G., Wu Y., Li Y., Hong Y., Zhao X., Reyes P.I., Lu Y. Early Stage Detection of Staphylococcus epidermidis Biofilm Formation Using MgZnO Dual-Gate TFT Biosensor. Biosens. Bioelectron. 2020;151:111993. doi: 10.1016/j.bios.2019.111993. PubMed DOI

Guła G., Szymanowska P., Piasecki T., Góras S., Gotszalk T., Drulis-Kawa Z. The Application of Impedance Spectroscopy for Pseudomonas Biofilm Monitoring during Phage Infection. Viruses. 2020;12:407. doi: 10.3390/v12040407. PubMed DOI PMC

Achinas S., Yska S.K., Charalampogiannis N., Krooneman J., Euverink G.J.W. A Technological Understanding of Biofilm Detection Techniques: A Review. Materials. 2020;13:3147. doi: 10.3390/ma13143147. PubMed DOI PMC

Ameer S., Ibrahim H., Yaseen M.U., Kulsoom F., Cinti S., Sher M. Electrochemical Impedance Spectroscopy-Based Sensing of Biofilms: A Comprehensive Review. Biosensors. 2023;13:777. doi: 10.3390/bios13080777. PubMed DOI PMC

Nag M., Lahiri D. Analytical Methodologies for Biofilm Research. 1st ed. Springer; New York, NY, USA: 2021.

Kretzschmar J., Harnisch F. Electrochemical Impedance Spectroscopy on Biofilm Electrodes—Conclusive or Euphonious? Curr. Opin. Electrochem. 2021;29:100757. doi: 10.1016/j.coelec.2021.100757. DOI

Romero M.C., Méndez-Tovar M. Impedance Analysis for the Study of Biofilm Formation on Electrodes: An Overview. J. Mex. Chem. Soc. 2023;67:547–565. doi: 10.29356/jmcs.v67i4.2005. DOI

Xu W., Ceylan Koydemir H. Non-Invasive Biomedical Sensors for Early Detection and Monitoring of Bacterial Biofilm Growth at the Point of Care. Lab. Chip. 2022;22:4758–4773. doi: 10.1039/D2LC00776B. PubMed DOI

Swami P., Anand S., Holani A., Gupta S. Impedance Spectroscopy for Bacterial Cell Monitoring, Analysis, and Antibiotic Susceptibility Testing. Langmuir. 2024;40:21907–21930. doi: 10.1021/acs.langmuir.4c01907. PubMed DOI

Paredes J., Becerro S., Arana S. Comparison of Real Time Impedance Monitoring of Bacterial Biofilm Cultures in Different Experimental Setups Mimicking Real Field Environments. Sens. Actuators B Chem. 2014;195:667–676. doi: 10.1016/j.snb.2014.01.098. DOI

McGlennen M., Dieser M., Foreman C.M., Warnat S. Monitoring Biofilm Growth and Dispersal in Real-Time with Impedance Biosensors. J. Ind. Microbiol. Biotechnol. 2023;50:kuad022. doi: 10.1093/jimb/kuad022. PubMed DOI PMC

Ward A.C., Hannah A.J., Kendrick S.L., Tucker N.P., MacGregor G., Connolly P. Identification and Characterisation of Staphylococcus aureus on Low Cost Screen Printed Carbon Electrodes Using Impedance Spectroscopy. Biosens. Bioelectron. 2018;110:65–70. doi: 10.1016/j.bios.2018.03.048. PubMed DOI

Kim T., Kang J., Lee J.H., Yoon J. Influence of Attached Bacteria and Biofilm on Double-Layer Capacitance during Biofilm Monitoring by Electrochemical Impedance Spectroscopy. Water Res. 2011;45:4615–4622. doi: 10.1016/j.watres.2011.06.010. PubMed DOI

Lacina K., Věžník J., Sopoušek J., Farka Z., Lacinová V., Skládal P. Concentration and Diffusion of the Redox Probe as Key Parameters for Label-Free Impedimetric Immunosensing. Bioelectrochemistry. 2023;149:108308. doi: 10.1016/j.bioelechem.2022.108308. PubMed DOI

Obořilová R., Šimečková H., Pastucha M., Klimovič Š., Víšová I., Přibyl J., Vaisocherová-Lísalová H., Pantůček R., Skládal P., Mašlaňová I., et al. Atomic Force Microscopy and Surface Plasmon Resonan Ce for Real-Time Single-Cell Monitoring of Bacteriophage-Mediated Lysis of Bacteria. Nanoscale. 2021;13:13538–13549. doi: 10.1039/D1NR02921E. PubMed DOI

Sopoušek J., Věžník J., Houser J., Skládal P., Lacina K. Crucial Factors Governing the Electrochemical Impedance on Protein-Modified Surfaces. Electrochim. Acta. 2021;388:138616. doi: 10.1016/j.electacta.2021.138616. DOI

Standar K., Kreikemeyer B., Redanz S., Münter W.L., Laue M., Podbielski A. Setup of an in vitro Test System for Basic Studies on Biofilm Behavior of Mixed-Species Cultures with Dental and Periodontal Pathogens. PLoS ONE. 2010;5:e13135. doi: 10.1371/journal.pone.0013135. PubMed DOI PMC

Obořilová R., Kučerová E., Botka T., Vaisocherová-Lísalová H., Skládal P., Farka Z. Piezoelectric Biosensor with Dissipation Monitoring Enables the Analysis of Bacterial Lytic Agent Activity. Sci. Rep. 2025;15:3419. doi: 10.1038/s41598-024-85064-x. PubMed DOI PMC

Deng M., Wang Y., Chen G., Liu J., Wang Z., Xu H. Poly-L-Lysine-Functionalized Magnetic Beads Combined with Polymerase Chain Reaction for the Detection of Staphylococcus aureus and Escherichia coli O157:H7 in Milk. J. Dairy Sci. 2021;104:12342–12352. doi: 10.3168/jds.2021-20612. PubMed DOI

Marka S., Zografaki M.E., Papaioannou G.M., Mavrikou S., Flemetakis E., Kintzios S. Impedance In vitro Assessment for the Detection of Salmonella typhimurium Infection in Intestinal Human Cancer Cells. Chemosensors. 2023;11:534. doi: 10.3390/chemosensors11100534. DOI

Kim B.R., Bae Y.M., Lee S.Y. Effect of Environmental Conditions on Biofilm Formation and Related Characteristics of Staphylococcus aureus. J. Food Saf. 2016;36:412–422. doi: 10.1111/jfs.12263. DOI

Oliver L.M., Dunlop P.S.M., Byrne J.A., Blair I.S., Boyle M., Mcguigan K.G., Mcadams E.T. An Impedimetric Sensor for Monitoring the Growth of Staphylococcus epidermidis; Proceedings of the 2006 International Conference of the IEEE Engineering in Medicine and Biology Society; New York, NY, USA. 30 August–3 September 2006; pp. 535–538. PubMed

Lofrumento C., Arci F., Carlesi S., Ricci M., Castellucci E., Becucci M. Safranin-O Dye in the Ground State. A Study by Density Functional Theory, Raman, SERS and Infrared Spectroscopy. Spectrochim. Acta A Mol. Biomol. Spectrosc. 2015;137:677–684. doi: 10.1016/j.saa.2014.07.051. PubMed DOI

Ommen P., Zobek N., Meyer R.L. Quantification of Biofilm Biomass by Staining: Non-Toxic Safranin Can Replace the Popular Crystal Violet. J. Microbiol. Methods. 2017;141:87–89. doi: 10.1016/j.mimet.2017.08.003. PubMed DOI

James S.A., Powell L.C., Wright C.J. Microbial Biofilms—Importance and Applications. InTech; Rijeka, Croatia: 2016. Atomic Force Microscopy of Biofilms—Imaging, Interactions, and Mechanics.

Tollersrud T., Berge T., Andersen S.R., Lund A. Imaging the Surface of Staphylococcus aureus by Atomic Force Microscopy. APMIS. 2001;109:541–545. doi: 10.1111/j.1600-0463.2001.907808.x. PubMed DOI

Chatterjee S., Biswas N., Datta A., Dey R., Maiti P. Atomic Force Microscopy in Biofilm Study. Microscopy. 2014;63:269–278. doi: 10.1093/jmicro/dfu013. PubMed DOI