The effect of Cyclic-di-GMP on biofilm formation by in a novel empyema model.

BACKGROUND

() is a common pathogenic bacterium which causes pleural empyema, and infection of is often associated with biofilm. The aim of this study was to establish a model of rabbit empyema infected by to determine whether it causes the formation of biofilm in the pleural cavity. Furthermore, we investigated the effect of cyclic diguanosine monophosphate (c-di-GMP) on biofilm formation in this empyema model.

METHODS

Twenty rabbits were used and randomly divided into five groups: PAO1, PAO1Δ, and PAO1/p infection groups, and Luria-Bertani (LB) broth and turpentine control groups. A drainage catheter was implanted into the pleural cavity through thoracentesis. The three infection groups were respectively infected with PAO1, PAO1Δ, and PAO1/p strains, which caused empyema. The two control groups were injected with LB or turpentine. After 4 days of infection, we sacrificed the rabbits. We evaluated the pathology of pleura through hematoxylin-eosin staining. Colony count and crystal violet assay were used to analyze the biofilm formation on the surface of catheters. Scanning electron was used to observe the biofilm on the surface of the pleura. Peptide nucleic acids-fluorescence in situ hybridization (PNA-FISH) was used to observe the biofilm in the fibrinous deposition.

RESULTS

By the PNA-FISH assay, biofilms were observed in the fibrinous deposition of the three infection groups. The red fluorescence area of the PAO1Δ infection group was larger than that of the PAO1 and PAO1/p - infection groups. Through electron microscopy, we observed that PAO1 strains were embedded in an electron-dense extracellular matrix on the surface of pleural tissue, and appeared to be biofilm-like structures. For the crystal violet assay, the optical density values of different groups were significantly different: PAO1Δ > PAO1 > PAO1/p > control groups (P<0.05).

CONCLUSIONS

To the best knowledge of the authors, this is the first study to report forming biofilm in a novel animal model of pleural empyema. In addition, c-di-GMP signaling molecules played an important role in biofilm formation in the pleural cavity.