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.

Conflict of interest statement

The authors have declared that no competing interests exist. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Figures

Fig 1. Schematic drawing of the basis to monitor bacterial biofilm formation by the xCellingence equipment.

Standard E-plates (16-wells) and magnification of one of the wells coated with gold microelectrodes (A). Evolution of the “cell index” (CI), which derived from the electric impedance, throughout the different steps of bacterial biofilm formation by the strain S. aureus 15981 (B).

Fig 2. Variation in the cell index (CI) during S. mutans NCTC10449 biofilm formation, at 37°C under 5% CO2, depending on the carbon source added to the culture medium.

At 8 h of incubation time, those values that have not a common letter are statistically different according to the Duncan mean comparison test (p

Fig 3. Variation in the cell index (CI) during biofilm formation, at 37°C under 5% CO2, of S. mutans NCTC10449 (type strain) and CI2366 (clinical isolate) depending on the carbon source added to the culture medium.

At 8 h of incubation, the presence of the asterisk indicates that CI values were statistically different according to one-way ANOVA tests (pA). Crystal violet staining of the biofilms NCTC10449 and CI2366 formed in the E-plate (B). Counts (Log CFU/ml) of planktonic cultures of both strains in different culture media. Within each strain, the bars that do not share a common letter are statistically different according to ANOVA and Duncan mean comparison tests (p<0.05) (C).

Fig 4. Variation in the cell index (CI) during biofilm formation at 37°C of different S. aureus and S. epidermidis biofilm producers (ISP479r, 15981, 132 or V320 and F12, respectively) and no-biofilm producers (CH1368 and CH48, respectively).

TSB+0.25% glucose was the culture medium used in the experiment (A). Absorbance (595 nm) measured after crystal violet staining of samples collected at different times during the biofilm formation in E-plates of the strains under study (B). Counts (Log CFU/ml) of cells collected from the biofilms formed in the E-plates by the strains under study (C). Statistical differences among strains at three sampling points (8, 16 and 24 h) are collected in S1 Table, which also shows representative mean and SD values.

Fig 5

Inhibition of the biofilm formation by S. aureus 15981 due to the addition of endolysin LysH5 (0.15 μM) to the culture medium TSBG, and by S. epidermidis F12 due to the addition of the bacteriophage phi-IPLA7 (MOI 100) to TSBG expressed as variation of the CI during biofilm treatment; asterisks show the first incubation time after which two or more consecutive values of CI were statistically different (p<0.05), according to one-way ANOVA tests (S2 Table) (A). Counts (Log CFU/ml) of cells collected, after 24 h of treatment, from the biofilms formed in the E-plates; within each strain, the asterisks (p<0.001) show statistical differences according to ANOVA (B).

Fig 6. Removal of 8 h preformed S. aureus 15981 biofilms by endolysin LysH5 (added to TSBG from 0.05 to 2.88 μM) reported as variation of the normalized cell index (CI).

At the final time, values having distinct letter are statistically different (pA). Absorbance (595 nm) measured after crystal violet staining of biofilms after 6 h of LysH5 treatment. Values with asterisks are statistically different (p<0.05) from the reference control (without LysH5 addition) according to one-way ANOVA tests. The photograph on the top shows the biofilms formed in the E-plates, in the presence of different concentrations of LysH5, stained with crystal violet (B).