Antibiofilm efficacy of emodin alone or combined with ampicillin against methicillin-resistant Staphylococcus aureus

Abstract

Methicillin-resistant Staphylococcus aureus (MRSA) is recognized as a significant global health concern. The development of resistance to a broad spectrum of antibiotics, particularly following biofilm formation, renders conventional therapeutic options for MRSA ineffective. Three MRSA clinical isolates were examined in vitro to assess their biofilm-forming capacity and the disruptive effects on pre-established biofilm (via crystal violet staining and scanning electron microscopy), and quantify extracellular DNA (eDNA) release after exposed to emodin alone or in combination with ampicillin. In addition, real-time PCR was employed to investigate the impact of emodin on the expression of biofilm-related genes in MRSA biofilms. The inhibitory effect of emodin on biofilm formation and disruption was observed in a dose dependent manner. The antagonistic activity of emodin in combination with ampicillin against MRSA biofilms was confirmed through adhesion assays. Real-time PCR analysis revealed that emodin, either alone or in combination with ampicillin, effectively downregulated the transcriptional levels of the biofilm-related genes fnbpB, clfA and atlA, but not icaA. In addition, drug treatment resulted in a significant reduction in eDNA release and protein contain in EPS (extracellular polymeric substances), which corresponded to the markedly decreased transcript level of atlA and fnbpB, respectively. These observations suggest that emodin, either alone or in combination with ampicillin, holds potential as a therapeutic approach for MRSA biofilm-related infections.

Keywords: Ampicillin; Antibiofilm activity; Emodin; MRSA.

Fig. 1

Effects of drugs on biofilm formation of MRSA strain. Biofilm formation by MRSA in presence of Amp (A) and emodin (B), in 96-well plates after 40–48 h, was assessed by crystal violet staining. Each bar indicates the mean values ± SE from at least three independent experiments. Control group means no Amp added in (A) and no emodin in (B) added. *p < 0.05, **p < 0.01, ***p < 0.001, compared with their respective control groups.

Fig. 2

Combined effects of drugs on biofilm formation of MRSA strains. Biofilm formation by MRSA in presence of Amp and emodin, in 96-well plates after 40–48 h, was assessed by crystal violet staining. Each bar indicates the mean values ± SE from at least three independent experiments. Control group means no emodin added. *p < 0.05, **p < 0.01, ***p < 0.001, compared with their respective control groups.

Fig. 3

Effects of drugs on biofilm disruption of MRSA strain. Mature biofilm, incubated with fresh TSB + 0.5% glucose media containing Amp (A) and emodin (B) for another 40–48 h, was assessed by crystal violet staining. Each bar indicates the mean values ± SE from at least three independent experiments. Control group means no Amp added in (A) and no Emodin added in (B). *p < 0.05, **p < 0.01, ***p < 0.001, compared with their respective control groups.

Fig. 4

Combined effects of drugs on biofilm disruption of MRSA strains. Mature biofilm, incubated with fresh TSB + 0.5% glucose media containing Amp and emodin for another 40–48 h, was assessed by crystal violet staining. Each bar indicates the mean values ± SE from at least three independent experiments. Control group means no emodin added. *p < 0.05, **p < 0.01, ***p < 0.001, compared with their respective control groups.

Fig. 5

SEM analysis of MRSA 19−10 biofilm. SEM images of biofilm formed by MRSA 19−10 that had been incubated with 1/4 MIC of Amp together with 1/4 to 1 MIC concentrations of emodin for 40–48 h. Scale bars in the figures represented 10 μm.

Fig. 6

Expression of biofilm-related genes in MRSA 19−10 in response to emodin or in combination with Amp. The normalized fold expression changes in biofilm-related genes following exposure to emodin alone or in combination with Amp for 24 h was plotted against control biofilms without exposure to Emodin (A) or with only exposure to Amp (B) using gyrB as the reference gene. Each bar indicates the mean values ± SE from at least three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, compared with their respective control groups.

Fig. 7

Effect of drugs on PIA formation and on extracellular proteins. (A) MRSA 19−10 was grown on Congo red medium and incubated with 1/4 MIC of Amp together with 1/4 to 1 MIC concentrations of emodin; (B) Analysis the effect of drugs on extracellular proteins by micro BCA method. Each bar indicates the mean values ± SE from at least three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, compared with their respective control groups.

Fig. 8

Effects of drugs on eDNA release and autolysis of MRSA strain 19−10. (A-B) The amount of eDNA in the cell-free supernatants from MRSA 19−10 biofilms treated with emodin alone or in combination with Amp was measured by spectrophotometry (upper panel) and agarose gel electrophoresis (down panel); C) Triton X-100 was used to stimulate autolysis in MRSA 19−10 cells grown in the absence or presence of various concentrations of emodin. The data were from a single representative experiment and were reproduced at least three times.