Combination effects of baicalin with levofloxacin against biofilm-related infections

Abstract

It is important to improve the existing techniques and develop new strategies to prevent bacterial biofilm formation. In this in vitro study, biofilms were established by a clinically isolated strain of Staphylococcus aureus 17546 (t037). Different concentrations of baicalin were added to 3- and 7-day biofilms. Based on colony counts and quantitative analysis of the biomass, sub-minimum inhibitory concentrations (sub-MICs) (1024, 512 or 256 μg/mL) of baicalin clearly decreased the number of bacterial colonies and biomass in vitro. Fluorescence microscopy revealed that sub-MICs (1024, 512, or 256 μg/mL) of baicalin inhibited bacterial adherence to the carrier surface and decreased polysaccharide production. Moreover, baicalin disrupted biofilms and exhibited synergistic effects with levofloxacin. Virulence factors were assessed by western blotting and real-time quantitative polymerase chain reaction, confirming that staphylococcal enterotoxin A, α-haemolysin and coagulase production decreased after baicalin treatment. Additionally, baicalin increased production of thermonuclease in S. aureus, and baicalin at 1024 and 512 μg/mL downregulated agrA expression. Based on these findings, the combination of baicalin with levofloxacin might be a new, feasible strategy for treating S. aureus biofilm-related infections. Baicalin may serve as a new inhibitor that modulates S. aureus virulence factors.

Keywords: Biofilm; baicalin; staphylococcus aureus; virulence factors.

Figure 1

Growth curves of S. aureus at sub-MIC concentrations of baicalin. S. aureus with different concentrations of baicalin (1024-128 µg/ml) or without baicalin (control) was incubated at 37°C for 24 hours, and OD600 was measured every 2 hours. Experiments were performed in triplicate. Mean values are shown. Baicalin concentrations < 1024 μg/mL did not inhibit S. aureus growth.

Figure 2

Inhibitory effects of baicalin on S. aureus biofilm formation. S. aureus was grown on polystyrene carriers with baicalin (1024 μg/mL, 512 μg/mL, 256 μg/mL and 128 μg/mL) for 3 days and 7 days. A. Biofilm bacterial counts were obtained after exposure to baicalin. Biofilm mass formation and bacterial counts were quantified in triplicate (P < 0.05; *compared to the control group). B. Biofilms were stained with crystal violet, and OD595 was measured (P < 0.05; *compared to the control group). C. Static biofilms after exposure to baicalin for 3 days (top) and 7 days (bottom) were stained with SYTO-9. S. aureus within biofilms on polystyrene carriers display green fluorescence (200 ×).

Figure 3

Quantitative analysis of the effects of baicalin with levofloxacin on 3- and 7-day-old biofilms. 3- and 7-day-old biofilms were established on polystyrene carriers by growing S. aureus in TSB-G and then treating with CLR, baicalin, or LEV alone or LEV in combination with baicalin or CLR for 12 h (*compared to the control, P < 0.05; **compared to the LEV, P < 0.05; average of 3 experiments).

Figure 4

Synergistic effects of baicalin with levofloxacin on 3- (A-F) and 7-day (G-L)-old biofilms, as determined by SEM (8000 ×). 3- and 7-day-old biofilms were established on polystyrene carriers by growing S. aureus in TSB-G and treating with CLR, baicalin, and LEV alone or LEV in combination with baicalin or CLR for 12 h. Static biofilms were dehydrated in increasing concentrations of ethanol (70%, 80%, 90%, and 100%) and coated with gold. (A/G) control group, (B/H) 16 μg/mL CLR, (C/I) 256 μg/mL baicalin, (D/J) 32 μg/mL LEV, (E/K) 16 μg/mL CLR + 32 μg/mL LEV, (F/L) 256 μg/mL baicalin + 32 μg/mL LEV.

Figure 5

Synergistic effects of baicalin with levofloxacin on 3- (A-F) and 7-day (G-L)-old biofilms, as determined by CLSM (400 ×). 3- and 7-day-old biofilms were established on polystyrene carriers by growing S. aureus in TSB-G and treating with CLR, baicalin, and LEV alone or LEV combination with baicalin or CLR for 12 h. Static biofilms were stained with SYTO-9 and propidium iodide. (A/G) control group, (B/H) 16 μg/mL CLR, (C/I) 256 μg/mL baicalin, (D/J) 32 μg/mL LEV, (E/K) 16 μg/mL CLR + 32 μg/mL LEV, (F/L) 256 μg/mL baicalin + 32 μg/mL LEV. Green represents live biomass; red represents dead biomass.

Figure 6

Detection of SEA. A. Western blot analysis of SEA for control group and groups treated with 1024, 512, 256 and 128 μg/ml baicalin. B. Quantitative analysis of SEA expression. (*P < 0.05, compared to the control). Data shown are the average of 3 experiments.

Figure 7

Effect of sub-MIC concentration of baicalin on HLA. After treatment of bacterial suspensions with 1024-128 μg/mL baicalin, the levels of specific RNA transcripts in different samples were measured. Total RNA extracts were used as a template for cDNA synthesis, and cDNA was assessed by RTQ-PCR. Relative gene expression levels of hla were calculated. Comparative measurements of relative expression are shown as an average of 3 experiments. *P < 0.05, compared to the control.

Figure 8

Effect of sub-MIC concentration of baicalin on agrA gene expression. After S. aureus 17546 was cultured with different sub-MIC concentrations of baicalin, the levels of specific RNA transcripts in different samples were measure. Total RNA extracts were used as a template for cDNA synthesis, and cDNA was assessed by RTQ-PCR. Data shown are the average of 3 experiments, and relative quantitative values were calculated using the 2^-ΔΔCt. *P < 0.05, compared to the control.

Figure 9

Detection of S. aureus thermonuclease. S. aureus 17546 was cultured with 1024-128 μg/mL baicalin. The bacterial suspension was centrifuged, and the supernatants were incubated for 16 hours. The diameters of the different baicalin groups were larger than those of the control group. *P < 0.05, compared to the control.

Figure 10

Detection of S. aureus coagulase. S. aureus 17546 was cultured with 1024-128 μg/mL baicalin; rabbit blood plasma was mixed with diluted bacterial suspensions and incubated. Coagulation indicates a positive effect. Treatments with higher concentrations of baicalin (1024 and 512 μg/mL) showed inhibited activity of S. aureus coagulase. *P < 0.05, compared to the control.