The combined antibacterial effects of sodium new houttuyfonate and berberine chloride against growing and persistent methicillin-resistant and vancomycin-intermediate Staphylococcus aureus

Abstract

Background: Infections caused by drug-resistant Staphylococcus aureus, especially vancomycin-intermediate Staphylococcus aureus (VISA), leave clinicians with limited therapeutic options for treatment. Persister cells is a leading cause of recalcitrant infection and antibiotic treatment failure, and there is no drug in clinical use that specifically targets persister cells currently. Here, we report a promising combination therapy of sodium new houttuyfonate (SNH) and berberine chloride (BBR) which is able to eradicate both growing and persistent drug-resistant Staphylococcus aureus.

Results: The susceptibility test showed SNH exhibited anti-MRSA activity with MIC₉₀ at 64 μg/mL, while BBR showed weak anti-MRSA activity with MIC₉₀ at 512 μg/mL. MICs of BBR in combination with 1/2 MIC SNH decreased by 4 to 64 folds compared with MICs of BBR alone. The results of time-killing assays revealed that the combined use of sub-MIC SNH and BBR offered an in vitro synergistic action against growing MRSA (including pathogenic MRSA) and VISA strains. More importantly, the combination of SNH and BBR was able to eradicate VISA Mu50 and pathogenic MRSA persister cells. The synergistic effect is likely related to the interruption of the cell membrane caused by SNH, which is confirmed by scanning electron microscope and membrane potential and permeability analysis.

Conclusions: Our study provide a promising clinical curative strategy for combating drug-resistant S. aureus infections, especially for recalcitrant infections caused by persister cells.

Keywords: Berberine; Combination therapy; MRSA; Persistence; Sodium new houttuyfonate; VISA.

Fig. 1

Cumulative inhibitory rate (%) of MRSA strains at different MIC levels of SNH and BBR

Fig. 2

MICs of BBR alone and in combination with 1/2 MIC SNH

Fig. 3

The combination of SNH and BBR killed growing MRSA and VISA cells. a MRSA ATCC33591 treated with SNH (16 μg/mL, 1/4 MIC), BBR (64 μg/mL, 1/8MIC) or their combination. b MRSA ATCC43300 treated with SNH (32 μg/mL, 1/2 MIC), BBR (128 μg/mL, 1/2MIC) or their combination. c VISA Mu50 treated with SNH (64 μg/mL, MIC), BBR (256 μg/mL, 1/2MIC) or their combination. d MRSA CCPM(A)-P-0116173 treated with SNH (8 μg/mL, 1/4 MIC), BBR (64 μg/mL, 1/2MIC) or their combination. e MRSA CCPM(A)-P-010850 treated with SNH (8 μg/mL, 1/4 MIC), BBR (32 μg/mL, 1/2MIC) or their combination. f MRSA CCPM(A)-P-011012 treated with SNH (16 μg/mL, 1/4 MIC), BBR (32 μg/mL, 1/2MIC) or their combination. The lower limit of detection is indicated by a dotted line

Fig. 4

The combination of SNH and BBR eradicated S. aureus persister cells. a MRSA ATCC33591 persister cells treated with SNH (64 μg/mL, MIC), BBR (512 μg/mL, MIC) or SNH-BBR combination for 24 h. b VISA Mu50 persister cells treated with SNH (64 μg/mL, MIC), BBR (1024 μg/mL, 2MIC) or SNH-BBR combination for 24 h. c Clinical MRSA CCPM(A)-P-0116173 persister cells treated with SNH (64 μg/mL, 2MIC), BBR (256 μg/mL, 2MIC) or SNH-BBR combination for 24 h. d Clinical MRSA CCPM(A)-P-010850 treated with SNH (64 μg/mL, 2MIC), BBR (128 μg/mL, 2MIC) or SNH-BBR combination for 24 h. e Clinical MRSA CCPM(A)-P-01012 treated with SNH (64 μg/mL, MIC), BBR (128 μg/mL, 2MIC) or SNH-BBR combination for 24 h. The y-axis starts at the value of the minimum detection limit. Asterisks (*) denote statistical significance compared with untreated control, while hash marks (#) indicate significant differences between the groups of SNH-BBR combination and BBR alone, as determined by one-way ANOVA followed by Turkey’s multiple-comparison test (****, P < 0.0001; ####, P < 0.0001)

Fig. 5

Effects of SNH and BBR on S. aureus ATCC33591 using SEM analysis. The images were acquired at a magnification of 20,000 (upper panel, a-d) or 50,000 (bottom panel, e-h) times. The bacterial cells were treated with nothing (a, e), 1/4MIC of SNH (b, f), 1/8MIC of BBR (c, g) or combination of 1/4MIC SNH and 1/8MIC BBR (d, h) for 4 h incubation

Fig. 6

SNH led to a decreased potential and an increased permeability of cell membrane in S. aureus ATCC33591. Bacterial membrane potential (a) was represented by the ratio of red/green fluorescence of DiOC₂(3). CCCP (5 μM) was used as the positive control in the DiOC₂(3)-based membrane potential assay. Bacterial membrane permeability (b) was measured by TO-PRO-3. Nisin (25 μg/mL, green) was used as a positive control in the TO-PRO-3-based membrane permeability assay. Untreated ATCC33591 cells were regarded as a negative control (black) in both assays. Cells treated with 1/4 MIC SNH, 1/8 MIC BBR and the SNH-BBR combination were presented as blue, orange and red, respectively. Asterisks denote statistical significance as determined by one-way ANOVA followed by Tukey’s multiple-comparison analysis ****, P < 0.0001