Monitoring in Real Time the Formation and Removal of Biofilms from Clinical Related Pathogens Using an Impedance-Based Technology

Abstract

Bacteria found in diverse ecosystems grow in a community of aggregated cells that favors their survival and colonization. Different extracellular polymeric substances are used to entrap this multispecies community forming a biofilm, which can be associated to biotic and abiotic surfaces. This widespread and successful way of bacterial life, however, can lead to negative effects for human activity since many pathogen and spoiling bacteria form biofilms which are not easy to eradicate. Therefore, the search for novel anti-biofilm bio-active molecules is a very active research area for which simple, reliable, and fast screening methods are demanded. In this work we have successfully validated an impedance-based method, initially developed for the study of adherent eukaryotic cells, to monitor the formation of single-species biofilms of three model bacteria in real time. The xCelligence real time cell analyzer (RTCA) equipment uses specific microtiter E-plates coated with gold-microelectrodes that detect the attachment of adherent cells, thus modifying the impedance signal. In the current study, this technology allowed the distinction between biofilm-producers and non-producers of Staphylococcus aureus and Staphylococcus epidermidis, as well as the formation of Streptococcus mutans biofilms only when sucrose was present in the culture medium. Besides, different impedance values permitted discrimination among the biofilm-producing strains tested regardless of the nature of the polymeric biofilm matrix. Finally, we have continuously monitored the inhibition of staphylococcal biofilm formation by the bacteriophage phi-IPLA7 and the bacteriophage-encoded endolysin LysH5, as well as the removal of a preformed biofilm by this last antimicrobial treatment. Results observed with the impedance-based method showed high correlation with those obtained with standard approaches, such as crystal violet staining and bacteria enumeration, as well as with those obtained upon other abiotic surfaces (polystyrene and stainless steel). Therefore, this RTCA technology opens new opportunities in the biofilm research arena and its application could be further explored for other bacterial genera as well as for different bio-active molecules.

Inhibition ofSteptococcus mutansbiofilm formation by extracts ofTenacibaculumsp. 20J, a bacterium with wide-spectrum quorum quenching activity

“…In this work, we corroborate previous results that indicate the suitability of the xCELLigence® system RTCA to monitor biofilm formation in S. mutans and other Gram-positive bacteria [25–28]. The xCELLigence® system constitutes a low time-consuming method for real-time quantification and continuous scrutiny of bacterial cell adhesion, in contrast to methods based on staining, microscopic visualization, quantification of biofilm-associated cells in situ , or after detachment from the substrate, and indirect methods such as DNA quantification, XTT reduction assay, or dry cell weight assay which are usually end-point techniques.…”

“…The biofilm quantification was done using the xCELLigence® System RTCA (ACEA, Biosciences Inc.) [25–28], in which the increase in the cell adherence on the electrodes causes an increase in the electrode impedance, which is expressed into a dimensionless parameter called cell index. E-plates 16 or E-plate 96 (ACEA, Biosciences Inc.) were inoculated with 180 μl of a 24-h culture of S. mutans in BHI broth supplemented with 0.1% sucrose (OD 600 adjusted to 0.05) and 20 μl of extracts or inhibitors.…”

“…20J against several Gram-positive and marine bacteria prompted us to study the capacity of cell extracts from 20J to interfere with communication mediated by the QS signal AI-2 and biofilm formation in the oral pathogen S. mutans . Surface-associated growth by S. mutans was monitored in real-time using the xCELLigence® technology that measures total biofilm biomass by combining both cellular growth and matrix production [25–28]. Moreover, confocal laser scanning microscopy (CLSM) as well as crystal violet biofilm staining were used to confirm xCELLigence® results.…”

Study of the Relation between the Resonance Behavior of Thickness Shear Mode (TSM) Sensors and the Mechanical Characteristics of Biofilms

“…This behavior with two slopes resembles that obtained for S. epidermidis at the same temperature by Gutiérrez and co-authors [52], using an electric impedance-based method (real time cell analyzer—RTCA). The electrical impedance is related to the mobility of ions in contact with the electrodes.…”

Candida aurisphenotypic heterogeneity determines pathogenicityin vitro

“…Normalised CI values were exported from the RTCA software and analysed in GraphPad Prism (version 8; GraphPad Software Inc., La Jolla, CA). More detailed descriptions for this technology can be found elsewhere [20].…”