Table 1.
Susceptibility of P. aeruginosa PAO1 to antimicrobial agents.
Fig 1.
Effects of baicalin on the growth of P. aeruginosa PAO1.
Growth curves (A) and bacterial colony counts (B) of P. aeruginosa PAO1 treated with varying concentrations of baicalin (64–1024 μg/mL) in Luria-Bertani broth at 37°C for 24 h. Experiments were performed in triplicate; the error bars represent the standard error of the OD600 value for each time point in the growth curves and the standard error for the number of CFUs after 24 h treatment. * and ** indicate P-values <0.05 and <0.01, respectively, with respect to the control (no baicalin).
Fig 2.
Inhibitory effects of baicalin on P. aeruginosa PAO1 biofilm formation.
In the dose-dependent analysis (A), bacterial suspensions were seeded in 96-well flat-bottomed polystyrene microtiter plates exposed to sub-MICs of baicalin (16, 32, 64, 128, and 256 μg/mL) for 24 h, and biofilm mass formation and bacterial counts were quantified in triplicate. Values represent the mean ± standard deviation. *P<0.05 and **P<0.01 compared with the control group. In the time-dependent analysis (B), biofilm mass formation and bacterial counts were monitored after exposing biofilms to sub-MICs (64, 128, and 256 μg/mL) of baicalin for 6, 12, 24 or 96 h. Biofilm architecture after 24 h and 96 h of baicalin inhibition was examined by fluorescence microscopy (200× magnification) (C) and SEM (10,000× magnification) (D).
Fig 3.
Baicalin as a co-treatment in combination with three conventional antibiotics to treat 24-h P. aeruginosa PAO1 biofilms.
(A) P. aeruginosa PAO1 biofilm mass and bacterial counts were quantified after treating pre-existing 24-h biofilms with levofloxacin (1 μg/mL), tobramycin (8 μg/mL) and ceftazidime (2 μg/mL) alone or in combination with various sub-MICs (64, 128, and 256 μg/mL) of baicalin for 24 h. (B) Biofilms were exposed to baicalin (256 μg/mL), levofloxacin (1 μg/mL), tobramycin (8 μg/mL), ceftazidime (2 μg/mL) or a baicalin/antibiotic mixture for 6, 12 and 24 h, and changes in biofilm mass formation and bacterial counts were monitored over time. Experiments were performed in triplicate, and values represent the mean ± standard deviation. *P<0.05 and **P<0.01 compared with the vehicle control group. (C) Images show 24-h biofilms observed by SEM (10,000× magnification). (D) Three-dimensional reconstructions of 24-h P. aeruginosa PAO1 biofilms after staining with the LIVE/DEAD viability kit were created using CLSM (200× magnification).
Fig 4.
Baicalin as a co-treatment in combination with three conventional antibiotics to treat 96-h P. aeruginosa PAO1 biofilms.
(A) P. aeruginosa PAO1 biofilm mass and bacterial counts were quantified after treating pre-existing 96-h biofilms with sub-MIC (256 μg/mL) of baicalin for another 24 h. Experiments were performed in triplicate, and values represent the mean ± standard deviation. **P<0.01 compared with the vehicle control group. (B) Images show 96-h biofilms observed by SEM (10000× magnification). (C) Three-dimensional reconstructions of 96-h P. aeruginosa PAO1 biofilms were created using CLSM (200× magnification).
Fig 5.
Inhibitory effects of sub-MICs of baicalin on the production of QS-regulated extracellular virulence factors in Pseudomonas aeruginosa PAO1 culture supernatants.
Virulence factor activity, including LasA protease (A), LasB elastase (B), pyocyanin (C), and rhamnolipid activities (D), was analyzed. Error bars indicate the standard deviations of three measurements. **P<0.01 compared with the untreated control group. $ $P<0.01 compared with the same concentration of baicalin alone.
Fig 6.
Effects of baicalin on PEA production detected by western blotting.
Top image: Western blot analysis of PEA in the control group and in groups treated with sub-MICs of baicalin (64, 128, and 256 μg/mL) for 24 h. Bottom image: band intensity quantitation based on densitometry. **P<0.01 versus the control group. Data are shown as the average of three experiments.
Fig 7.
Effects of baicalin on P. aeruginosa PAO1 motility.
Three motility assays were conducted on Plates Containing Different Concentrations of Agar in the Absence of Baicalin (Untreated Control) or Containing 256 μg/mL Baicalin. (A) Swimming, (B) swarming, and (C) twitching motility diameters were measured using a caliper. The data represent the average values from three independent experiments performed in duplicate. Values are the mean ± standard deviation. *P<0.05 and **P<0.01 compared with the untreated control group.
Fig 8.
Effects of Sub-MICs of Baicalin on the Production of QS Signaling Molecules [3-oxo-C12-HSL (A) and C4-HSL (B)] Extracted from P. aeruginosa PAO1 Culture Supernatants and Analyzed by HPLC/MS. The experiment shown is representative of three independent tests. **P<0.01 versus untreated control. Error bars indicate the standard deviation of three parallel measurements.
Fig 9.
Relative expression levels of QS-regulated genes in the presence of sub-MICs of baicalin (64, 128, and 256 μg/mL) as determined by real-time quantitative PCR.
*P<0.05 and **P<0.01 versus the untreated control. Error bars indicate the standard deviation of three parallel measurements.
Fig 10.
Effects of baicalin on the survival of C. elegans infected with P. aeruginosa PAO1.
(A). Kaplan-Meier survival curve of P. aeruginosa-infected C. elegans. (B). Colony counts of P. aeruginosa PAO1 recovered from worms fed untreated and 256 μg/mL baicalin-treated P. aeruginosa. After 24 h, the nematodes were washed to remove bacteria from the integument and ruptured to recover bacteria from the digestive tracts. Bacterial loads were determined by plating on the appropriate selective medium and counting colonies. Data were obtained from three experiments.
Table 2.
Clearance of implants pre-colonized with wild-type P. aeruginosa PAO1 inserted in the peritoneal cavities of BALB/c mice treated with various agents alone or in combination for 3 days.
Results are expressed as the mean ± standard deviation. *P<0.01 compared with the vehicle control group without baicalin treatment. &P<0.05 and ▲P<0.01 when the combination treatment group was compared with the antibiotic treatment alone.
Fig 11.
Comparison of Gross (A) and Microscopic (B) Pathological Changes in the Peritoneal Tissue after Treatment with Vehicle or Baicalin. Histopathology was performed using an upright microscope at 200× magnification. The white arrow indicates the implant.
Fig 12.
Cytokine levels of IFN-γ and IL-4 in fluid obtained by flushing the peritoneal cavities of mice treated with either baicalin or vehicle, measured by ELISA.
The data show the average values of three independent experiments performed in duplicate. Values are the mean ± standard error. &P<0.05 and &&P<0.01 compared with the vehicle control group, **P<0.01 compared with the group in which the implant was pre-colonized with P. aeruginosa PAO1 but no baicalin was administered.