Flavaspidic acid BB combined with mupirocin improves its anti-bacterial and anti-biofilm activities against Staphylococcus epidermidis

Abstract

Background: The increase in drug-resistant opportunistic pathogenic bacteria, especially of antibiotic-resistant Staphylococcus epidermidis (S. epidermidis), has led to difficulties in the treatment of skin and soft tissue infections (SSTI). The major reason for bacterial resistance is the formation of bacterial biofilm. Here, we report a promising combination therapy of flavaspidic acid BB (BB) and mupirocin, which can effectively eradicate the biofilm of S. epidermidis and eliminate its drug resistance.

Result: The susceptibility test showed that the combination of BB and mupirocin has good antibacterial and antibiofilm activities, and the fractional inhibitory concentration index (FICI) of BB combined with mupirocin was 0.51 ± 0.00 ~ 0.75 ± 0.05, showing synergistic effect. Moreover, the time-kill curve assay results indicated that the combination of drugs can effectively inhibit the planktonic S. epidermidis. After drugs treatment, the drug-combination showed significantly inhibitory effects on the metabolic activity and total biomass in each stage of biofilm formation. The synergistic effect is likely related to the adhesion between bacteria, which is confirmed by field emission scanning electron microscope. And the expression level of aap, sarA and agrA genes were detected by real-time quantitative PCR (qRT-PCR).

Conclusion: Our study provides the experimental data for the use of BB for the clinical treatment of skin infections and further demonstrate the potential of BB as a novel biofilm inhibitor.

Keywords: Biofilm; Combination therapy; Flavaspidic acid BB; Staphylococcus epidermidis.

Fig. 1

Time-kill curve of S. epidermidis under the combination of BB and mupirocin. The viable bacteria were counted at 0, 2, 4, 6, 8, 12, 24, and 48 h of bacterial growth. The ordinate is the colony count, expressed in the form of log10 (CFU/mL). (A)-(B) SE04; (C)-(D) SE08. Control: Wells with no drugs; BB: flavaspidic acid BB; MUP: mupirocin; B + M: flavaspidic acid BB + mupirocin; 1/2BB: 1/2 MIC of flavaspidic acid BB; 1/2B + M: 1/2 MIC of flavaspidic acid BB combined with 1/2 MIC of mupirocin, and so on. The control and combination groups in panels A and B, C and D are the same

Fig. 2

The metabolic activity of the bacteria at each stage of the formation of S. epidermidis biofilm. The tested strains were incubated for 6, 24 and 48 h to form adhesion, aggregation and maturity stage of biofilms respectively, and treated with BB and mupirocin, followed by the biofilms being treated with XTT solution, and quantified. A SE04 treated for 6 h; B SE04 treated for 24 h; C SE04 treated for 48 h; D SE08 treated for 6 h; E SE08 treated for 24 h; F SE08 treated for 48 h. Compared with the control group, * means P < 0.05, ** means P < 0.01, *** means P < 0.001, **** means P < 0.0001; Compared with the BB group, # means P < 0.05, #### means P < 0.0001; Compared with the mupirocin group, + means P < 0.05, +  +  +  + means P < 0.0001

Fig. 3

The quantity of biomass at each stage of biofilm formation of the S. epidermidis. The tested strains were incubated for 6, 24 and 48 h to form adhesion, aggregation and maturity stage of biofilms respectively, and treated with BB and mupirocin, followed by the biofilms being stained with crystal violet, and quantified. (A) SE04 treated for 6 h; (B) SE04 treated for 24 h; (C) SE04 treated for 48 h; (D) SE08 treated for 6 h; (E) SE08 treated for 24 h; (F) SE08 treated for 48 h. Compared with the control group, ** means P < 0.01, **** means P < 0.0001; Compared with the BB group, # means P < 0.05, ## means P < 0.01, ### means P < 0.001; Compared with the mupirocin group, + means P < 0.05, +  + means P < 0.01, +  +  + means P < 0.001, +  +  +  + means P < 0.0001

Fig. 4

Effect on BB and mupirocin on S. epidermidis (SE04) using FESEM analysis. BB: 20 μg/mL flavaspidic acid BB; MUP: 640 μg/mL mupirocin; Combination: 20 μg/mL flavaspidic acid BB + 640 μg/mL mupirocin. Magnification: × 5000

Fig. 5

Effect on BB and mupirocin on S. epidermidis (SE08) using FESEM analysis. BB: 20 μg/mL flavaspidic acid BB; MUP: 5 μg/mL mupirocin; Combination: 20 μg/mL flavaspidic acid BB + 5 μg/mL mupirocin. Magnification: × 3000

Fig. 6

Gene expression analysis of S. epidermidis. The SE04 and SE08 were incubated for 6, 24, 48 h to form biofilms, and treated with BB and mupirocin, followed by the biofilms being detected the genes expression by qRT-PCR. A The aap gene expression level; B The sarA gene expression level; C The agrA gene expression level. Compared with the control group, * means P < 0.05, ** means P < 0.01, *** means P < 0.001, **** means P < 0.0001; Compared with the BB or mupirocin group, ## means P < 0.01, ### means P < 0.001