ABSTRACT:
This study aimed to study the antibacterial and antibiofilm activities of the ethanolic extract of Hibiscus sabdariffa L. and apple vinegar alone and in combination against one strong biofilm forming strain of each of Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa, Escherichia coli, Proteus mirabilis and Klebsiella pneumoniae. The antibacterial activity was estimated by agar well diffusion method and determination of minimum inhibitory concentration (MIC) and the antibiofilm activity was evaluated on pre-formed biofilms by determining the minimum biofilm eradication concentrations (MBEC). The ethanolic extract of Hibiscus sabdariffa at 200 mg/ml demonstrated a higher antibacterial activity against Staphylococcus epidermidis, Pseudomonas aeruginosa, Escherichia coli and Staphylococcus aureus with inhibition zones of 9.5±0.71, 7.75±1.06, 7.75±0.35 and 7.75±1.06, respectively than against Proteus mirabilis and Klebsiella pneumoniae with inhibition zone of 6.25±0.35, each. Moreover, Hibiscus sabdariffa extract at 100 mg/ml produced inhibition zones of 7.75±0.35, 7.5±0.71, 6.75±0.35, 5.75±0.35 against Proteus mirabilis, Staphylococcus aureus, Pseudomonas aeruginosa and Klebsiella pneumoniae, respectively, while it produced inhibition zone of 7.25±0.35 against each of Escherichia coli and Staphylococcus epidermidis. There was no significant difference between the antibacterial activities of the two tested concentrations of Hibiscus sabdariffa extract. Apple vinegar at a concentration of 5% showed a stronger antibacterial activity against Staphylococcus epidermidis, Pseudomonas aeruginosa, Proteus mirabilis with inhibition zones of 10±0.35, 9.5±0.71, and 8.75±0.35, respectively as compared to that against Staphylococcus aureus and Escherichia coli (8.25±0.35), each and Klebsiella pneumoniae (7.25±1.06). Furthermore, at a concentration of 2.5% of apple vinegar, inhibition zones of 7±0.71, 6.75±0.35, 6±0.71 and 6±0 were found against Staphylococcus epidermidis, Proteus mirabilis, Pseudomonas aeruginosa and Escherichia coli, respectively. Lower effects were observed against Klebsiella pneumoniae (5.25±0.35) and Staphylococcus aureus (5±0). There was no significant difference between the antibacterial activities of 2.5% and 5% apple vinegar except against Pseudomonas aeruginosa. Comparing the antibacterial activities of Hibiscus sabdariffa extract (200 or 100 mg/ml) and apple vinegar (2.5% or 5%), the differences were not statistically significant. The combination of apple vinegar and Hibiscus sabdariffa extract was synergistic. At 2.5% of apple vinegar combined with 200 mg/ml of Hibiscus sabdariffa extract, Staphylococcus epidermidis and Proteus mirabilis were more sensitive to the combination (19±0 and 18.5±0.71, respectively) than Klebsiella pneumoniae (16.75±0.35), Escherichia coli (16.5±0.71) and Staphylococcus aureus (17±0.71). Pseudomonas aeruginosa was less sensitive to this combination (14±0.71). Moreover, combination of 2.5% apple vinegar and 100 mg/ml Hibiscus extract showed higher activity against Staphylococcus epidermidis and Proteus mirabilis (16.75±0.35 and 16.5±0.71, respectively) than against Staphylococcus aureus, E. coli and Klebsiella pneumoniae (15.25±0.35, 15±0 and 14.25±0.35, respectively). Pseudomonas aeruginosa showed more resistance to this combination (12.25±0.35). Both combinations showed statistically significant higher antibacterial activities than Hibiscus sabdariffa extract (200 or 100 mg/ml) or apple vinegar (2.5% or 5%). The MIC of Hibiscus sabdariffa extract was 12.5 mg/ml against all tested isolates except for Klebsiella pneumoniae (25 mg/ml), while the MIC of apple vinegar was 0.078% against Pseudomonas aeruginosa, Staphylococcus aureus and Klebsiella pneumoniae and 0.156% against Proteus mirabilis and E. coli. Synergistic effect was observed for apple vinegar- Hibiscus sabdariffa extract combination. In the presence of 1/2 MIC of apple vinegar, the MICs of Hibiscus sabdariffa extract decreased by 8 folds in case of Pseudomonas aeruginosa, Proteus mirabilis and E. coli and by 4 folds against Staphylococcus aureus, Staphylococcus epidermidis and Klebsiella pneumoniae. Moreover, a 4-fold drop in MICs of Hibiscus sabdariffa extract was detected when combined with 1/4 MIC of apple vinegar against all tested isolates. Both Hibiscus extract and apple vinegar exerted antibiofilm activities against tested strains. The biofilm eradicating activity of Hibiscus extract was more pronounced against Klebsiella pneumoniae (biofilm eradication at 1X MIC), Staphylococcus aureus, Proteus mirabilis, E.coli and Staphylococcus epidermidis (2X MIC) than against Pseudomonas aeruginosa (8X MIC). Apple vinegar showed lower antibiofilm activity than Hibiscus extract. It could remove biofilms of Klebsiella pneumoniae and E.coli at 8X MIC, Pseudomonas aeruginosa at 16X MIC, Staphylococcus epidermidis, Proteus mirabilis and Staphylococcus aureus (32X MIC). The present study suggests the use of Hibiscus sabdariffa L. extract and apple vinegar to treat diabetic foot infections as a natural inexpensive remedy.