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.