Adhesion to and biofilm formation on IB3-1 bronchial cells by Stenotrophomonas maltophilia isolates from cystic fibrosis patients

Abstract

Background: Stenotrophomonas maltophilia has recently gained considerable attention as an important emerging pathogen in cystic fibrosis (CF) patients. However, the role of this microorganism in the pathophysiology of CF lung disease remains largely unexplored. In the present study for the first time we assessed the ability of S. maltophilia CF isolates to adhere to and form biofilm in experimental infection experiments using the CF-derived bronchial epithelial IB3-1cell line. The role of flagella on the adhesiveness of S. maltophilia to IB3-1 cell monolayers was also assessed by using fliI mutant derivative strains.

Results: All S. maltophilia CF isolates tested in the present study were able, although at different levels, to adhere to and form biofilm on IB3-1 cell monolayers. Scanning electron and confocal microscopy revealed S. maltophilia structures typical of biofilm formation on bronchial IB3-1 cells. The loss of flagella significantly (P < 0.001) decreased bacterial adhesiveness, if compared to that of their parental flagellated strains. S. maltophilia CF isolates were also able to invade IB3-1 cells, albeit at a very low level (internalization rate ranged from 0.01 to 4.94%). Pre-exposure of IB3-1 cells to P. aeruginosa PAO1 significantly increased S. maltophilia adhesiveness. Further, the presence of S. maltophilia negatively influenced P. aeruginosa PAO1 adhesiveness.

Conclusions: The main contribution of the present study is the finding that S. maltophilia is able to form biofilm on and invade CF-derived IB3-1 bronchial epithelial cells, thus posing a rationale for the persistence and the systemic spread of this opportunistic pathogen in CF patients. Experiments using in vivo models which more closely mimic CF pulmonary tissues will certainly be needed to validate the relevance of our results.

Figure 1

Adhesion to and biofilm formation on IB3-1 cell monolayer of clinical isolates of S. maltophilia from CF patients. A. Adhesion levels of S. maltophilia to IB3-1 cell monolayers. Strains OBGTC9 and OBGTC10 showed the highest level of adhesiveness, significantly higher than for the other strains (** P < 0.001; ANOVA-test followed by Newman-Keuls multiple comparison post-test). B. Biofilm formed by S. maltophilia on IB3-1 cell monolayers. Strain OBGTC37 formed the highest amount of biofilm, significantly higher (** P < 0.001; ANOVA-test followed by Newman-Keuls multiple comparison post-test) than other strains tested. Results are expressed as means + SDs.

Figure 2

SEM observation of 24 hours-biofilm formed byclinical isolate S. maltophilia OBGTC9 on IB3-1 cell monolayer. Scanning electron micrographs showing cell cluster morphology (microcolony) strongly suggestive of biofilm formation. Bacterial cells lose their outlines for the presence of extracellular matrix (arrows). Magnification: ×2.500 (Figure 2A), ×5.000 (Figure 2B).

Figure 3

CLSM observation of 24 hours-biofilm formed byclinical isolate S. maltophilia OBGTC9 on IB3-1 cell monolayer. A-B. CLSM micrographs of not fixed specimens of unexposed (control; Figure 3A) and OBGTC9-exposed (Figure 3B) IB3-1 cell monolayer stained with Syto-9 (green fluorescence, indicating live cells), propidium iodide (red fluorescence, indcating dead cells), and Con-A (blue fluorescence, indicating extracellular matrix). Image capture was set for visualization of: (a) green fluorescence only; (b) red fluorescence only; (c) blue fluorescence only (3) or; (d) co-localization of all three fluorescence signals. Note the formation of a S. maltophilia microcolony embedded in matrix whose formation is significantly increased in infected vs control IB3-1 cell monolayers. C. CLSM examination of fixed IB3-1 monolayer exposed to S. maltophilia OBGTC9 for 24 hours: three-dimensional representation. Green fluorescence indicates autofluorescence of IB3-1 cytoplasm following exposure to fixation mixture; red fluorescence indicates binding of propidium iodide to nucleic acids of both IB3-1 and S. maltophilia cells. Note the microcolony organization of S. maltophilia on almost all IB3-1 cells. Magnification, ×100.

Figure 4

Adhesion to IB3-1 cell monolayer by S. maltophilia OBGTC9 and OBGTC10 wild type strains, and relative fliI^- mutants. A. The adhesiveness of OBGTC9 and OBGTC10 flagellar mutants fliI- was significantly lower than that of wild type strains (** P < 0.001 vs OBGTC9 fliI^-; °° P < 0.001 vs OBGTC10 fliI^-; ANOVA-test followed by Newman-Keuls multiple comparison post-test). Results are expressed as means + SDs. B. The inactivation of the fliI gene was confirmed by swimming motility assay: OBGTC9 wild type (left), and relative fliI^- mutant (right).

Figure 5

Adhesion to and biofilm formation on polystyrene by 12 S. maltophilia isolates from CF patients. A. Adhesion (grey bars) and biofilm (black bars) levels were assessed by crystal violet colorimetric technique and expressed as optical density read at 492 nm (OD₄₉₂). OBGTC26 strain adhesiveness was significantly higher than OBGTC49, OBGTC50, and OBGTC52 strains (* P < 0.05; Kruskall-Wallis test followed by Dunn's multiple comparison post-test). Biofilm formed by OBGTC20 strain was significantly higher than that produced by OBGTC9 and OBGTC49 strains (** P < 0.01; Kruskall-Wallis test followed by Dunn's multiple comparison post-test). Results are expressed as means + SDs. B. Relationship between adhesion to and biofilm formation levels on polystyrene. A statistically significant positive correlation was found between adhesion and biofilm levels (Pearson r = 0.641; P < 0.05).

Figure 6

IB3-1 cell monolayer co-infection assays. IB3-1 cell monolayers were exposed first to P. aeruginosa PAO1 for 2 hours (PAO1 co), then for a further 2 hours to S. maltophilia OBGTC9 strain (OBGTC9 co). Control infections consisted of exposure for 2 hours to S. maltophilia OBGTC9 (OBGTC9 single 2 h) or P. aeruginosa PAO1 (PAO1 single 2 h). Results are expressed as means + SDs. Pre-exposure of IB3-1 cell monolayer to P. aeruginosa PAO1 significantly improved S. maltophilia OBGTC9 adhesiveness (** P < 0.01 vs OBGTC9 single 2 h; ANOVA-test followed by Newman-Keuls multiple comparison post-test). When IB3-1 cells were concomitantly infected, S. maltophilia OBGTC9 adhesiveness was significantly higher than that showed by P. aeruginosa PAO1 (** P < 0.001 vs PAO1 co; ANOVA-test followed by Newman-Keuls multiple comparison post-test).