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. 2018 Aug 14;18(1):84.
doi: 10.1186/s12866-018-1224-6.

Real-time Monitoring of Pseudomonas Aeruginosa Biofilm Formation on Endotracheal Tubes in Vitro

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Free PMC article

Real-time Monitoring of Pseudomonas Aeruginosa Biofilm Formation on Endotracheal Tubes in Vitro

Eva Pericolini et al. BMC Microbiol. .
Free PMC article

Abstract

Background: Pseudomonas aeruginosa is an opportunistic bacterial pathogen responsible for both acute and chronic infections in humans. In particular, its ability to form biofilm, on biotic and abiotic surfaces, makes it particularly resistant to host's immune defenses and current antibiotic therapies as well. Innovative antimicrobial materials, like hydrogel, silver salts or nanoparticles have been used to cover new generation catheters with promising results. Nevertheless, biofilm remains a major health problem. For instance, biofilm produced onto endotracheal tubes (ETT) of ventilated patients plays a relevant role in the onset of ventilation-associated pneumonia. Most of our knowledge on Pseudomonas aeruginosa biofilm derives from in vitro studies carried out on abiotic surfaces, such as polystyrene microplates or plastic materials used for ETT manufacturing. However, these approaches often provide underestimated results since other parameters, in addition to bacterial features (i.e. shape and material composition of ETT) might strongly influence biofilm formation.

Results: We used an already established biofilm development assay on medically-relevant foreign devices (CVC catheters) by a stably transformed bioluminescent (BLI)-Pseudomonas aeruginosa strain, in order to follow up biofilm formation on ETT by bioluminescence detection. Our results demonstrated that it is possible: i) to monitor BLI-Pseudomonas aeruginosa biofilm development on ETT pieces in real-time, ii) to evaluate the three-dimensional structure of biofilm directly on ETT, iii) to assess metabolic behavior and the production of microbial virulence traits of bacteria embedded on ETT-biofilm.

Conclusions: Overall, we were able to standardize a rapid and easy-to-perform in vitro model for real-time monitoring Pseudomonas aeruginosa biofilm formation directly onto ETT pieces, taking into account not only microbial factors, but also ETT shape and material. Our study provides a rapid method for future screening and validation of novel antimicrobial drugs as well as for the evaluation of novel biomaterials employed in the production of new classes of ETT.

Keywords: Biofilm; Bioluminescence; Endotracheal tube; Pseudomonas aeruginosa; Real-time monitoring.

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Figures

Fig. 1
Fig. 1
Real-time evaluation of biofilm formation on ETT pieces and CFU counts. Box plots of RLU (a) and of CFU/ml (c) of BLI-Pseudomonas biofilm on ETT pieces, untreated (white box plots) or treated (grey box plots) with gentamicin after 12, 24 or 48 h of incubation. Mean +/− SEM of RLU (b) or CFU/ml (d) analysis from 12 different ETT pieces. Circles or squares indicate the ETT pieces untreated or treated with gentamicin, respectively. Each ETT piece in panel b has a different colour: this allows us to follow the development of biofilm over time. Values of p < 0.05 (*), p < 0.01 (**), p < 0.001 (***) and p < 0.0001 (****) were considered significant
Fig. 2
Fig. 2
Confocal analysis of ETT biofilm. a, c, e: real black and white images acquired by transmitted-illumination confocal microscope of a 0, 24 and 48 h-old biofilm, respectively. These images result from the multiple acquisitions of several focal planes, which are ultimately combined. b, d, f: topography of the surface of a 0, 24 and 48 h-old biofilm, respectively, obtained by means of a false color scale. Specifically, the confocal microscope software allows us to see the differences in sample structure and thickness by assigning different colors to different areas. For each image, its own scale is given on the right side of the figure. The colored figures are tilted with respect to black and white correspondent images to highlight structure and thickness differences. C-inset and E-inset: 3D reconstruction of a 24 and 48 h-old biofilm, respectively, obtained by means of a mathematical linearization of the ETT surfaces. The images shown are representative of 2 independent experiments with the same pattern of results
Fig. 3
Fig. 3
Crystal violet staining of ETT biofilm. Mean +/− SEM of biofilm mass analysed by CV staining (OD570). ETT pieces cultured for 12, 24 and 48 h with BLI-Pseudomonas in the presence (grey columns) or absence (black columns) of gentamicin were assessed. Values of p < 0.05 (*), p < 0.001 (***) and p < 0.0001 (****) were considered significant
Fig. 4
Fig. 4
Quantification of alive/dead cells embedded in ETT biofilm. Relative Fluorescence Units (RFU) mean (a) and percentage (b) of alive (grey) and dead (black) cells into ETT 48 h-old BLI-Pseudomonas biofilm and planktonic cultures, treated or not with gentamicin. The data shown are representative of three independent experiments, which provided similar pattern of results
Fig. 5
Fig. 5
eDNA and pyoverdine determination in cell-free supernatants from ETT biofilm RFU mean +/− SEM of eDNA (a) and pyoverdine (b) production in cell-free supernatants of BLI-Pseudomonas biofilm on ETT pieces and planktonic cultures, untreated or treated with gentamicin, after 0, 24 or 48 h of incubation. 1: untreated planktonic cells 2: gentamicin-treated planktonic cells 3: untreated biofilm 4: gentamicin-treated biofilm ***p < 0.001 and *p < 0.05; gentamicin-treated samples vs untreated samples

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