In vitro Antibacterial and Antibiofilm Activities of Hibiscus sabdariffa L. Extract and Apple Vinegar against Bacteria Isolated from Diabetic Foot Infections

 

Hisham A. Abbas^1*, Islam M. Abdo², Mahmoud Z. Moustafa²

¹Department of Microbiology and Immunology-Faculty of Pharmacy-Zagazig University- Zagazig- Egypt

²Department of Pharmacognosy and Medicinal Plants-Faculty of Pharmacy-Zagazig University- Zagazig- Egypt

.*Corresponding Author E-mail: h_abdelmonem@yahoo.com

 

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.    

 

 

INTRODUCTION:

Diabetic foot infection is a common and major complication affecting diabetic patients. The underlying causes are the impaired peripheral circulation, neuropathy and low immunity of diabetic patients. These infections, unless properly treated, can lead to disastrous complications including gangrene and lower limbs amputation ^1,2. Staphylococcus aureus, Escherichia coli, Proteus mirabilis, Klebsiella pneumoniae and Pseudomonas aeruginosa are common etiologic agents of diabetic foot ulcers ^3-5. The treatment of such infections is problematic due to both the multidrug resistance of infecting bacteria and their assembly as biofilms. Biofilm is a community of sessile cells housed within a matrix and shows much higher resistance to antibiotics as compared to the planktonic cells ^6-9. The emergence of resistance to antibiotics and the poor supply of new antibiotics necessitates the search for new antimicrobial agents ^10,11. Medicinal plants produce a variety of bioactive compounds, some of which can exert antimicrobial activity ¹². One of these plants is Hibiscus sabdariffa L.  

 

Hibiscus sabdariffa L. is an annual herb that can grow in tropical and sub-tropical areas¹³. Hibiscus sabdariffa can be used as antiseptic, astringent, diuretic, emollient, sedative, and tonic in addition to its use in treatment of abscesses, cough, dysuria, and scurvy¹⁴. Its dried flowers contain anthocyanins pigments which exert cardioprotective, hypocholesterolemic, antioxidative, and hepatoprotective effects in animals^15-17. The wound healing activity of the methanolic extract of Hibiscus sabdariffa L. was previously reported ¹⁸. As a result, it is supposed that it may be beneficial in treating diabetic foot ulcers.

 

Vinegar is an acidic liquid that is produced by the fermentation of many carbohydrates¹⁹. The acidity of vinegar is attributed mainly to acetic acid. In addition to acetic acid, vinegar also contains vitamins, mineral salts, amino acids, polyphenolic compounds (galic acid, catechin, caffeic acid, ferulic acid), and nonvolatile organic acids (tartaric, citric, malic, lactic)^20,21.

 

The antimicrobial activity of acetic acid, as an organic weak acid, is mainly related to its undissociated form²². It can passively diffuse through the bacterial cell wall and dissociate into anions and protons. Protons decrease the internal pH and this pH reduction leads to inhibition of bacterial growth²³.

 

Vinegar was used in ancient medicine for treating infections. Hippocrates recommended a vinegar preparation for treatment of ulcerations and sores²⁴.  Recent research described the antimicrobial properties of vinegar, mainly in food preservation^25-28.

 

The aim of the present study is to investigate the antimicrobial and antibiofilm activities of Hibiscus sabdariffa extract and apple vinegar alone and in combination against bacteria isolated from diabetic foot ulcers.

MATERIALS AND METHODS:                                                                                    

Plant materialThe dried calyx and epicalyx of Hibiscus sabdariffa L. (family Malvaceae) were purchased from local herbal market in Zagazig City, Egypt.

 

Media and chemicals:

Tryptone soya broth, Tryptone soya agar and Mueller Hinton broth were obtained from Oxoid, Hampshire, England. Dimethyl sulphoxide (DMSO) was purchased from Sigma, St. Louis, USA. Apple vinegar was obtained from a local market in Zagazig, Egypt. Other chemicals were of pharmaceutical grade.

 

Bacterial strains:

One clinical isolate of each of Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa, Proteus mirabilis, Escherichia coli and Klebsiella pneumoniae isolated from diabetic foot ulcers were obtained from the stock culture collection of the Department of Microbiology and Immunology, Faculty of Pharmacy, Zagazig University.

 

Extraction of Hibiscus sabdariffa L:

The dried calyx and epicalyx of H. sabdariffa (250 g) was extracted with ethyl alcohol. The ethanolic extract was filtered and then concentrated under vacuum to yield 26.6 g of a viscous reddish residue.

 

Antibacterial activity of Hibiscus extract and apple vinegar:

The antibacterial activities of Hibiscus extract and apple vinegar were determined by the agar well diffusion method as modified from NCCLS²⁹. Mueller-Hinton agar plates were surface-inoculated with the tested strains suspensions adjusted to match 0.5 McFarland standard and the inocula were spread over the surfaces of plates using sterile cotton swabs. After drying of the plates, wells (6 mm diameter) were punched in the agar and 100 μl of Hibiscus extract in DMSO or apple vinegar were added into the wells. The plates were incubated at 37 ºC for 24 h. The antibacterial activity was determined by measuring the diameter of the zone of inhibition. The test was repeated twice and the mean inhibition zones were calculated.

 

Minimum inhibitory concentrations of Hibiscus sabdariffa L. and apple vinegar were determined by the agar dilution method according to Clinical Laboratory and Standards Institute Guidelines ³⁰. Mueller-Hinton agar plates containing different dilutions of Hibiscus extract and apple vinegar were prepared and dried. The bacterial strains were inoculated into Mueller-Hinton broth and incubated at 35 °C until it reaches a turbidity equivalent to or greater than that of the 0.5 McFarland standard.  The turbidity of the suspension was adjusted with sterile saline to achieve a turbidity equivalent to that of a 0.5 McFarland standard. The bacterial suspension was ten-fold diluted in sterile saline to achieve an approximate cell density of 10⁷ CFU/ml. A standardized micropipette was used to deliver 1 μl of the suspension to the surface of Mueller-Hinton agar containing the tested agents dilutions and the control plates, so that the final inoculum on the agar contains approximately 10⁴ CFU per spot. Antimicrobial-free plates were used as growth control. The inoculated agar plates were allowed to stand at room temperature until the liquid was absorbed into the agar. The plates were inverted and incubated at 35-37 °C for 16–20 hours, and the MIC was considered as the lowest concentration of antimicrobial agent that completely inhibited growth.

 

Detection of biofilm formation by tube method:

Biofilm formation was assessed as modified from Christensen et al.³¹ A loopful of pure overnight culture on Tryptone soya agar (TSA) was inoculated into 2 ml of Tryptone soya broth (TSB) in Falcon tubes and incubated for 24 hours at 37°C. The tubes were gently aspirated and washed with phosphate buffered saline (PBS, pH 7.3) and left to dry. The tubes were stained by adding 2 ml crystal violet (1%) for 15 minutes and the excess dye was removed and washed with water. After drying in an inverted position, the tubes were examined and when a stained film appears on the walls of the tubes, this was indicative of biofilm formation.

 

Effect of Hibiscus extract and apple vinegar on established biofilms:

The effect of apple vinegar and Hibiscus extract on established biofilms was investigated according to Ceri et al. ³² with some modifications. Suspensions of the isolates were prepared in Tryptone soya broth (TSB) to a cell density of 1x10⁸ CFU/ml and diluted 1:50 in TSB. Aliquots of 100 μl were delivered into the wells of polystyrene microtiter plates and allowed to grow as biofilms for 24 h at 37 ºC in polystyrene microtiter plates. The planktonic cells were gently removed and the wells were washed three times with phosphate buffered saline (PBS). The wells were filled with 100 μl of different dilutions of Hibiscus extract or apple vinegar (0.25MIC-32 MIC) and the plates were incubated for 24 h at 37 ºC. The wells were again aspirated, washed. The biofilms in the wells were resuspended in the same wells by adding 100 μl of sterile phosphate buff­ered saline and scraping of the sides of the wells with a pipette tip. To calculate MBEC, 10 μl from each well was transferred onto Tryptone soya gar plates (TSA), incubated at 37°C for 24 h MBECs were calculated as the lowest concentrations that inhibited growth on TSA.               

 

Statistical analysis:

The inhibition zones produced by Hibiscus extract, apple vinegar alone or in combination against different isolates were compared by Two Way ANOVA (Bonferroni posttests). P<0.001, P<0.05 were used to detect the significance of differences.

 

RESULTS:

Antibacterial activity of Hibiscus extract:

The extract of Hibiscus at a concentration of 200 mg/ml showed 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 7.75±0.35, each (Table 1). At 100 mg/ml, Hibiscus extract produced inhibition zones of 6.25±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. On the other hand apple vinegar at a concentration of 5% exhibited 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). More or less similar results were obtained at 2.5% apple vinegar. Inhibition zones of 7±0.71, 6.75±0.35, 6±0.71 and 6±0 were reported 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).

 

The combination of apple vinegar and Hibiscus extract was synergistic. At 2.5% of apple vinegar combined with 200 mg/ml of Hibiscus 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).

 

The MIC of Hibiscus 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 (Table 2) Synergistic effect was observed for apple vinegar-Hibiscus extract combination. In the presence of 1/2 MIC of apple vinegar, the MICs of Hibiscus 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 extract was detected at 1/4 MIC of apple vinegar against all tested isolates.

 

Biofilm formation and antibiofilm activity of Hibiscus extract:

All tested isolates were found to be strong biofilm-forming. 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). 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).

 

DISCUSSION:

Diabetic foot ulcers represent a therapeutic challenge because of antibiotic resistance and biofilm formation^33,34. New therapeutic agents are urgently needed to solve this problem. Hibiscus sabdariffa extract is a natural agent that was investigated for its antibacterial and antibiofilm activities. It was found that Hibiscus sabdariffa extract has antibacterial activity against the tested bacteria. The difference of Hibiscus sabdariffa extract (200 mg/ml) and (100 mg/ml) was not statistically significant (P<0.05).  The antibacterial activity of Hibiscus extract was reported in previous studies. Aqueous methanolic extract of Hibiscus sabdariffa calyces showed antibacterial activities against Staphylococcus aureus, Escherichia coli and Klebsiella pneumoniae³⁵. Moreover, Rogger et al.³⁶ reported the antimicrobial activity of Hibiscus sabdariffa against E. coli, Pseudomonas aeruginosa and Staphylococcus aureus. The antimicrobial activity may be attributed to phenolic compounds (falvonoids) that complex the cell walls in addition to disruption of cell membrane permeability and leakage of ions outside the cell^37,38.

 

In this study, apple vinegar showed antibacterial activity against tested bacteria. The antibacterial activity of 5% vinegar was not statistically significant from that of 2.5% vinegar except against Pseudomonas aeruginosa (P<0.05). Zasshi et al.³⁹ reported the antibacterial activity of apple cider vinegar against E. coli. Moreover, the antibacterial activity of Hibiscus sabdariffa extract (200 mg/ml) or 100 mg/ml was not statistically significant from that of 5% vinegar or 2.5% vinegar (P<0.05).

 

Table 1. Antibacterial activity of ethanolic Hibiscus sabdariffa extract and apple vinegar alone and in combination by agar well diffusion method.

Isolates	Inhibition zone diameter in mm (mean±SD)
	Hibiscus Extract 200 mg/ml	Hibiscus Extract 100 mg/ml	Apple vinegar 5%	Apple vinegar 2.5%	Hibiscus extract /Apple vinegar
					200/2.5	100/2.5
Pseudomonas aeruginosa Proteus mirabilis Klebsiella pneumoniae Escherichia coli Staphylococcus aureus Staphylococcus epidermidis	7.75±1.06 7.75±0.35 6.25±0.35 7.75±0.35 7.75±1.06 9.5±0.71	6.75±0.35 6.25±0.35 5.75±0.35 7.25±0.35 7.5±0.71 7.25±0.35	9.5±0.71 8.75±0.35 7.25±1.06 8.25±0.35 8.25±0.35 10±1.41	6±0.71 6.75±0.35 5.25±0.35 6±0 5±0 7±1.41	14±0.71 18.5±0.71 16.75±0.35 16.5±0.71 17±0.71 19±0	12.25±0.35 16.5±0.71 14.25±0.35 15±0 15.25±0.35 16.75±0.35

 

Table 2. Determination of minimum inhibitory concentrations of Hibiscus sabdariffa extract and apple vinegar alone and in combination

Isolate No.	MIC
	Hibiscus (mg/ml)	Vinegar %	Hibiscus /0.5 MIC apple vinegar	Hibiscus /0.25 MIC apple vinegar
Pseudomonas aeruginosa Staphylococcus aureus Klebsiella pneumoniae Proteus mirabilis Escherichia coli Staphylococcus epidermidis	12.5 12.5 25 12.5 12.5 12.5	0.078 0.078 0.078 0.156 0.156 0.078	1.56 3.125 6.25 1.56 1.56 3.125	3.125 3.125 6.25 3.125 3.125 3.125

 

Table 3. Antibiofilm activity of Hibiscus sabdariffa extract and apple Vinegar

Isolate No.	MBEC
	Hibiscus extract mg/ml	Vinegar %
Pseudomonas aeruginosa Staphylococcus aureus Klebsiella pneumoniae Proteus mirabilis Escherichia coli Staphylococcus epidermidis	100 25 50 25 50 25	1.25 2.5 0.625 5 1.25 2.5

 

There was a statistically significant difference between the antibacterial activity of each of the following pairs; Hibiscus sabdariffa extract (200 mg/ml) and Hibiscus sabdariffa extract (200 mg/ml) combined with 2.5 % vinegar (P<0.001), Hibiscus sabdariffa 200 mg/ml and Hibiscus sabdariffa extract/vinegar combination (100/2.5%) (P<0.01 against Pseudomonas aeruginosa, P<0.05 against Proteus mirabilis and P<0.001 against each of Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus and Staphylococcus epidermidis, Hibiscus sabdariffa extract (100 mg/ml) and Hibiscus sabdariffa extract/vinegar combination (100/2.5%) (P<0.001), 5% vinegar and Hibiscus sabdariffa extract/ vinegar combination (200/2.5%) (P<0.01 against Pseudomonas aeruginosa and P<0.001 against other tested strains, 5% vinegar and Hibiscus sabdariffa extract/vinegar combination (100/2.5%) (P<0.05 against Pseudomonas aeruginosa and Proteus mirabilis and P<0.001 against other tested strains, 2.5% vinegar and Hibiscus sabdariffa extract/vinegar combination (200/2.5%) (P<0.001) and 2.5% vinegar and Hibiscus sabdariffa extract/ vinegar combination (100/2.5%) (P< 0.01 against Proteus mirabilis and P<0.001 against other tested strains.

 

In this study biofilm cells of Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa, Proteus mirabilis, E.coli and Klebsiella pneumoniae were eradicated by Hibiscus sabdariffa extract and apple vinegar. To the best of my knowledge, the antibiofilm activity of Hibiscus sabdariffa extract was not investigated. Apple vinegar was previously reported to eradicate Streptococcus pyogenes biofilm⁴⁰.  In addition to the acetic acid content, the antimicrobial effect of apple vinegar may be due to high content of phenolic compounds in apple as reported by Alberto et al.⁴¹. Sendamangalam⁴² showed that polyphenols could decrease streptococcal biofilm formation due to the ability to inhibit the enzymes responsible for production of exopolymers which are a major component in biofilm formation.

 

In summary, Hibiscus sabdariffa extract and apple vinegar are promising natural tools that can be used for the treatment of diabetic foot infections.

 

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Received on 12.12.2013       Modified on 02.01.2014

Accepted on 20.01.2014      © RJPT All right reserved