DNase inhibits early biofilm formation in Pseudomonas aeruginosa- or Staphylococcus aureus-induced empyema models

Abstract

Anti-infection strategies against pleural empyema include the use of antibiotics and drainage treatments, but bacterial eradication rates remain low. A major challenge is the formation of biofilms in the pleural cavity. DNase has antibiofilm efficacy in vitro, and intrapleural therapy with DNase is recommended to treat pleural empyema, but the relevant mechanisms remain limited. Our aim was to investigate whether DNase I inhibit the early biofilm formation in Pseudomonas aeruginosa- or Staphylococcus aureus-induced empyema models. We used various assays, such as crystal violet staining, confocal laser scanning microscopy (CLSM) analysis, peptide nucleic acid-fluorescence in situ hybridization (PNA-FISH), and scanning electron microscopy (SEM) analysis. Our results suggested that DNase I significantly inhibited early biofilm formation in a dose-dependent manner, without affecting the growth of P. aeruginosa or S. aureus in vitro. CLSM analysis confirmed that DNase I decreased the biomass and thickness of both bacterial biofilms. The PNA-FISH and SEM analyses also revealed that DNase I inhibited early (24h) biofilm formation in two empyema models. Thus, the results indicated that DNase inhibited early (24h) biofilm formation in P. aeruginosa- or S. aureus-induced rabbit empyema models and showed its therapeutic potential against empyema biofilms.

Keywords: DNase; Pseudomonas aeruginosa; Staphylococcus aureus; biofilm; pleural empyema.

Figure 1

Growth curves of P. aeruginosa or S. aureus incubated with different DNase I concentrations or with drug solvent (negative control group). Three wells were set up for each group. The mean values and standard error are shown; DNase I has no effect on the growth of P. aeruginosa or S. aureus. The error bars represent the standard error of the OD600 value for each time point in the growth curves (n = 3).

Figure 2

DNase I inhibition of biofilm formation by P. aeruginosa or S. aureus strains. The biofilms were stained with crystal violet and detected by OD₅₉₅ measurements. The results are presented as mean ± SD. An asterisk indicates a significant difference, ** indicates p < 0.01 and **** indicates p < 0.0001, compared with the results in the drug-free control group using one-way ANOVA (n = 3). ANOVA tests. LB, Luria-Bertani; TSB, tryptic soy broth.

Figure 3

Biofilm bacterial counts were obtained after exposure to DNase I for 24 h. The results are presented as mean ± SD. NS indicates p-values > 0.05, compared with the results of the drug-free control group using one-way ANOVA (n = 3).

Figure 4

Fluorescence microscopy images of P. aeruginosa or S. aureus eDNA (100×). Propidium iodide (PI)-stained eDNA in red. In the DNase I group, the extent of PI staining decreased compared with the extent in the drug-free control group.

Figure 5

Relative expression levels of QS system-related genes of P. aeruginosa and S. aureus in the presence or absence of DNase I, as determined using real-time polymerase chain reaction. The data are presented as mean ± SD. An asterisk indicates a significant difference; * indicates p < 0.05 and NS indicates p-values > 0.05, compared with the expression values in the drug-free control group using t-tests (n = 3).

Figure 6

Analysis of DNase I effect on the P. aeruginosa and S. aureus biofilm structures. CLSM analysis of the biofilms formed in the presence or absence of DNase I. The bi-dimensional and three-dimensional biofilm structures were obtained using the LIVE/DEAD^® Biofilm Viability Kit. Viable bacteria cells with intact membranes are stained fluorescent green. (A) Represents the percentage of live cells. (B) The biofilm biomass of CLSM analysis. (C) Mean thickness of biofilm using CLSM analysis. The data are presented as mean ± SD. An asterisk indicates a significant difference; * indicates p < 0.05, ** indicates p < 0.01, and NS indicates p-values > 0.05, compared with the values in the drug-free control group using t-tests (n = 3).

Figure 7

Gross pathology specimens of rabbit pleural cavities 24 h after infection and treatment. The control rabbits had more pleural adhesions and fibrin depositions between the visceral and parietal pleura than the DNase I-treated rabbits. The blue arrows indicate adhesion bands and the red arrows indicate fibrin deposition. The empyema scores are presented as mean ± SD. An asterisk indicates a significant difference; ** indicates p < 0.01, compared with the values in the drug-free control group using t-tests (n = 4).

Figure 8

Morphological changes of the parietal pleura (H&E staining, 100×). The area between the two black arrows is the pleura. In the control rabbits, we observed parietal pleura thickening, accompanied with many inflammatory cell infiltrations. In the DNase-I treated rabbits, the parietal pleura was slightly thickened with only mild inflammation. The pleural thicknesses are presented as mean ± SD. An asterisk indicates significant difference; ** indicates p < 0.01 and *** indicates p < 0.001, compared with the values in the drug-free control group using t-tests (n = 4).

Figure 9

Results of the crystal violet staining of the indwelling catheters. OD values of crystal violet staining for indwelling catheters in two rabbit groups. The results are presented as mean ± SD. An asterisk indicates a significant difference; **** indicate p < 0.0001, compared with the values in the drug-free control group using t-tests (n = 4).

Figure 10

Colony counts of the indwelling catheters. Colony counts of P. aeruginosa or S. aureus on the surface of the indwelling catheters in the two rabbit groups. The results are presented as mean ± SD. NS indicates a p value > 0.05, compared with the values in the drug-free control group using t-tests (n = 4).

Figure 11

PNA-FISH of fibrinous depositions in purulent exudates (100×). The PNA-FISH kit contained a P. aeruginosa-specific probe (red), an S. aureus-specific probe (green), and an unspecific nucleic acid stain DAPI (blue) to show the biofilms. The figure shows that the mass bacteria in the control rabbits were surrounded by host cells. The red and yellow portions are the bacterial aggregates surrounded by polymorphonuclear leukocytes. In the DNase I-treated rabbits, few bacteria were surrounded by host cells. We determined the biofilm sizes using PNA-FISH. The results are presented as mean ± SD. An asterisk indicates a significant difference; *** indicates p < 0.001 and **** indicates p < 0.0001, compared with the values in the drug-free control group using t-tests (n = 4).

Figure 12

Pleural surface as seen using SEM (10,000×). In the control rabbits, we observed biofilm-like structures on the pleural surface. The P. aeruginosa or S. aureus strains were embedded in an electron-dense extracellular matrix (red box) in apparent biofilm-like structures. In the DNase-I treated rabbits, we observed only a few bacteria surrounded by a loose matrix.