Combination antibiotic therapy is increasingly being employed to reduce the risk of emerging resistance to antibiotics in bacteria during therapy and to increase the antibacterial spectrum. Beta-lactamase inhibitor-penicillin combinations, such as ampicillin-sulbactam, amoxicillin-clavulanate, ticarcillin-clavulanate and piperacillin-tazobactam are used for beta-lactamase resistant bacteria. Of these antibiotic combinations, ticarcillin-clavulanate and piperacillin-tazobactam have been reported as having the broadest spectra of activity, including against Enterobacteriaceae and Pseudomonas aeruginosa1.
A few studies have found that the antibacterial activity of antibiotics against different bacteria can be improved by combining them with crude plant extracts2-4. For example, Indonesian plant extracts such as Kaempferia pandurata have shown a synergistic activity in combination with penicillin or ampicillin against methicillin-sensitive Staphylococcus aureus (MSSA) and methicillin-resistant Staphylococcus aureus (MRSA)2. However, the exact mechanism of these interactions is still under investigation.
Curcuma longa Linn. (Zingiberaceae family), traditionally known as turmeric, is a traditional plant found in Indonesia typically used for spices, colouring, preservative and also for treating bacterial infections. A major compound found in Curcuma longa is curcumin, which has been reported to be responsible for most of the plants biological activities, such as antioxidant, antimicrobial, antitumor and anti inflammatory activities5-6. Curcumin also possesses potent antibacterial activity against a wide range of bacteria7-8 and has synergistic activities with various anti-microbial drugs9-10. However, these studies were only conducted in in vitro experiments. In this study, the in situ antibacterial activity of the combination of curcumin and antibiotics against Staphylococcus aureus caused skin infections was evaluated.
MATERIALS AND METHODS
This study was carried out from January, 2015-December, 2016.
Materials: Mueller Hinton Agar (MHA) (Oxoid Ltd., Hampshire, England), Mueller Hinton Broth (MHB) (Oxoid Ltd., Hampshire, England) and MacConkey agar (Oxoid Ltd., Hampshire, England) were used as growth media for the bacteria. Tetracycline HCl, ciprofloxacin HCl, ethanol 70%, NaCl, glutaraldehyde, phosphate buffer, osmium tetroxide, white vaseline and liquid paraffin were purchased from Brataco, Indonesia. Curcumin was obtained from Curcuma longa (Sigma Aldrich, St. Louis, MO, USA), which contains 80% curcumin. Dimethyl sulfoxide (DMSO) (Merck, Germany) was used for diluting the curcumin.
Test microorganisms and culture: Staphylococcus aureus and Escherichia coli were obtained from the culture collection of the School of Pharmacy, Bandung Institute of Technology and were cultured aerobically in MHA and MHB media.
Determination of the susceptibility of bacteria: In vitro susceptibility tests were performed in a 96-well microtiter plate to determine the minimum inhibitory concentrations (MICs) of the extracts and tetracycline HCl. The tests were carried out against S. aureus and E. coli using standard broth microdilution methods with a bacteria inoculum of 5×105 CFU mL1, according to the guidelines of the Clinical and Laboratory Standards Institute CLSI M7-A8 standard11. The MIC was defined as the lowest concentration of antimicrobial agents that resulted in the complete inhibition of visible bacterial growth. Furthermore, the minimum bactericidal concentrations (MBCs) were also established for each test sample. Approximately 100 μL of media from each well which showed no visible growth were spread on MHA plates. The plates were then incubated at 37°C for 24 h, or until microbial growth was seen in the growth-positive control plates. The MBC was defined as the lowest concentration of antimicrobial agents at which all bacteria in the culture are killed, or the lowest concentration of drugs that kill 99.9% of the total initially viable cells12.
Checkerboard assay: The antibacterial activities of the combination of antibiotics and plant extracts were investigated using the checkerboard broth microdilution method. Two-fold serial dilutions of the antibiotic and the plant extracts were prepared for every combination tested and 50 μL aliquots of each component was placed into the wells of the sterile 96-well microtiter plate. The inoculum was prepared using the above-described MIC determination method. The microtiter plates were then incubated at 37°C and the MIC was determined after 18-20 h of incubation.
The fractional inhibitory concentration (FIC index) for all of the combinations was determined using the following formula13:
Where, FICA and FICB are the fractional inhibitory concentration of drugs A and B, MICA and MICB are the minimum inhibitory concentration of drugs A and B and [A] and [B] are the concentration of drugs A and B, respectively. An FIC index of <0.5 was interpreted as synergistic, between 0.5 and 4 was interpreted as additive or indifferent and a value of >4 was interpreted as antagonistic14.
Animals: Six to seven months old male New Zealand albino rabbits (2-3 kg b.wt.,) were obtained from a rabbit husbandry in Lembang, Indonesia. Rabbits were housed individually and were fed a standard pellet diet, fresh vegetables (carrots and cabbage) and tap water. All of the procedures were performed according to the rules of animal handling experiments using animals, of the School of Pharmacy, Bandung Institute of Technology, adopted from the guidelines of "Institutional Animal Care and Use Committee Guidebook"15.
Curcumin and tetracycline combination ointment: A curcumin and tetracycline combination ointment was made using a base consisting of 90% white vaseline and 10% liquid paraffin. The base was sterilized at 170°C for 1 h. Tetracycline and curcumin were then added to the sterile ointment base and the mixture homogenized.
Acute skin irritation study: An acute skin irritation study was performed for the 1% tetracycline and 2% curcumin combination ointment according to the Organization for Economic Cooperation and Development methodology used for determining the degree of acute dermal irritation/corrosion16. Half a gram of ointment was applied to the skin of each animal at a shaved, dorsal area of approximately 6 cm2. Both of the treated sites on each rabbit were covered by sterile gauze and the backs of the rabbits were wrapped with non-occlusive bandages. After 24 h, the bandage and the test materials were removed and 1 h later the sites were examined for abnormal skin responses, including irritation, redness, itching, inflammation and other related symptoms17.
Ocular irritation test: At least three albino rabbits were used for the ocular irritation test. One Hundred milligram of the combination ointment was placed in the conjunctival sac of the right eye of each rabbit, the left eye of the rabbits was used as a negative control. The eyelids were then gently held together for about 1 sec in order to prevent loss of the test material. Observations were performed 1, 24, 48 and 72 h after application of the drug. The ocular response was assessed by calculating the score of the cornea, iris, conjunctiva and chemosis17.
Determination of in situ antibacterial activity of a combination of curcumin and tetracycline HCl topically on the back of each rabbit: An area of approximately 6 cm2 on the back of each rabbit was shaved and the skin divided into six marked areas designated for the positive control, ointment base control, tetracycline HCl 3% (Trifacyclin® as a reference) and three combinations of tetracycline HCl and curcumin (1:2, 1.5:1.5 and 2:1%). Each area of shaved skin was inoculated with bacteria by an intracutaneous injection of 0.1 mL S. aureus suspension (108 CFU mL1 of bacteria) and was then left for 24 h. The areas were then treated based on their groups by applying half a gram of each sample. After treatment, the treated sites were covered by sterile gauze and wrapped with non-occlusive bandages. The samples were applied every day for 29 days. Observation of skin reactions, including erythema, edema and pus, was carried out every 3 days for 30 days. Evaluation of the skin reactions was performed by calculating the dermal irritation score (DIS)17.
Scanning electron microscopy: Scanning electron microscopy (SEM) was performed on S. aureus treated with curcumin, tetracycline and a combination of curcumin and tetracycline at MIC. Staphylococcus aureus was cultured to reach the mid-log growth phase in MHB before use. The control and treated cells were prepared for morphological observation. The bacterial samples were washed 5 times with fresh media and then fixed with 2.5% glutaraldehyde in phosphate buffer (pH 7.2) at 4°C for 1 h, washed 3 times with phosphate buffer for 10 min and fixed with 1% osmium tetroxide for 2 h. This was followed by three washings in the phosphate buffer for 10 min and subsequently dehydrated in a series of ethanol concentrations (30, 50, 70, 90 and 95%), for 15 min each. The samples were subjected to 100% ethanol and CO2 to achieve the critical point and then coated with gold ion in a pressure metallic chamber. At the end of the process, the samples were submitted for analysis by SEM (Hitachi SU3500, Japan).
Transmission electron microscopy: Bacterial cells treated with a combination of curcumin and tetracycline was observed using transmission electron microscopy (TEM). The cells were harvested after 18 h of incubation and fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer for 2 h. The cells were then washed 3 times with 0.05 M phosphate buffer (pH 7.2) and post fixed for 2 h with 1% osmium tetroxide in a 0.1 M phosphate buffer (pH 7.2) at room temperature. After being washed twice in phosphate buffer, the cells were dehydrated through serial graded concentrations of ethanol (35, 70, 95 and 100%, respectively) for 15 min, then infiltrated and embedded in Spurrs resin. Ultrathin sections were cut with a diamond knife using an ultramicrotome and then mounted on bare copper grids. Finally, specimens were counterstained with 2% (w/v) for 3 min and then with 0.25% (w/v) lead citrate solution for 2 min and examined with a Tecnai G2 electron microscope (FEI, USA) operated at 120 kV.
Statistical analysis: Statistical analysis of the results was performed using one-way ANOVA followed by LSD and Tukey post hoc analysis using SPSS 16.00 software (SPSS Inc., Chicago, IL, USA). The p-values of <0.05 were considered to be statistically significant.
In vitro antibacterial activity of curcumin against S. aureus and E. coli: The antibacterial activity against S. aureus and E. coli of curcumin, tetracycline and ciprofloxacin were determined by measuring its MIC and MBC. The lowest antibacterial activity against S. aureus and E. coli was shown by curcumin, with an MIC value of 64 μg mL1 for both bacteria (Table 1). On the other hand, ciprofloxacin showed the best antibacterial activity against S. aureus and E. coli, with MIC values of 0.125 and 0.0625 μg mL1 and MBC values of 0.5 and 0.0625 μg mL1, respectively. The antibacterial activity of curcumin in combination with tetracycline or ciprofloxacin was further evaluated.
In vitro antibacterial activity of a combination of curcumin and tetracycline or ciprofloxacin against S. aureus and E. coli: This experiment was conducted in order to analyze the potency of curcumin in increasing the antibacterial activity of tetracycline or ciprofloxacin against S. aureus and E. coli. The results showed that the antibacterial activity of curcumin in combination with tetracycline or ciprofloxacin against S. aureus has an FIC index below 0.5, suggesting a synergistic interaction (Table 2). On the other hand, its antibacterial activity against E. coli showed a synergistic activity only in combination with tetracycline (Table 2). Therefore, the combination of curcumin and tetracycline was chosen for further in vivo studies.
Acute skin and dermal irritation study of a combination of curcumin and tetracycline: The study continued evaluating the safety of a combination of curcumin and tetracycline when used topically. The experiment was performed based on the Organization and Economic Cooperation Development (OECD) standard protocol. After 24 h of exposure to the combination ointment, there were no dermal or ocular irritations observed on rabbit skin or eyes. Similar results were observed at 48 and 72 h after ointment exposure (Table 3 and 4). This data show that the combination of 2% curcumin and 1% tetracycline does not cause acute dermal and ocular irritation, suggesting that it is safe to be used topically.
In situ antibacterial activity of curcumin and tetracycline combination ointment against S. aureus-infected rabbit skin: In this study, the in situ antibacterial activity of the curcumin-ciprofloxacin combination was not evaluated because it had less antibacterial activity than the curcumin-tetracycline combination. Moreover, there is no comparable ciprofloxacin ointment dosage form available in the Indonesian market. Therefore, the combination of curcumin and tetracycline was chosen as a further in situ antibacterial activity study. Various concentrations of curcumin-tetracycline combinations were tested on S. aureus infected rabbit skin.
MIC and MBC of curcumin, tetracycline and ciprofloxacin against S. aureus and E. coli
The data are expressed as the means of three independent experiments, MIC: Minimum inhibitory concentration, MBC: Minimum bactericidal concentration
FIC index of combination of curcumin with tetracycline or ciprofloxacin against S. aureus and E. coli
MICCu: MIC of curcumin, MICT: MIC of tetracycline: MIC; MIC of ciprofloxacin
Dermal irritation index of combination 2% curcumin: 1% tetracycline
Ocular irritation index of combination of 2% curcumin: 1% tetracycline
Wound score after curcumin-tetracycline combination treatment against S.aureus infections
The results (Fig. 1) show that the positive control, ointment base and the combination of 1.5% curcumin and 1.5% tetracycline resulted in the highest DIS on the last day of observation and the combination of 2% curcumin and 1% tetracycline resulted in the lowest. This suggests that the latter was able to induce faster healing of S. aureus infected skin. All of the animals used in this study showed the same wound score at day 0 of observation, a slight increase in wound score was observed for all groups at day 3. Furthermore, all of the animal groups showed an overall decrease in wound score, including the positive control group, suggesting that there is a self-recovery mechanism in the animals. However, the group treated with the combination of 2% curcumin and 1% tetracycline showed the lowest wound score, suggesting that this combination has the fastest recovery among the groups (Fig. 1).
Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) observations at 18 h: The effects of the curcumin-tetracycline combination on the surface morphology of S. aureus during its logarithmic phase of growth are presented in Fig. 2 and 3.
Scanning electron micrograph of S. aureus treated with combination of curcumin and tetracycline. The swollen cells were observed after treated for 18 h with regular shape of (a) Control, (b) Curcumin, (c) Tetracycline and (d) Combination of curcumin and tetracycline, Enlargement: For a, c, d 50,000× and for b 40,000×
Transmission electron micrograph of S. aureus treated with combination of curcumin and tetracycline. Damaged cell wall was observed after 18 h of incubation (a) Control and (b) Combination of curcumin and tetracycline, Enlargement: 40,000×
The antimicrobial activityon the surface morphology of individual cells was visualized. It can be observed that exposure to the combination resulted in distortion and disruption of the bacterial wall, resulting in deformation of the cell, while the untreated cells appeared round-shaped.
The present data suggest that the combination of 2% curcumin and 1% tetracycline has in situ antibacterial activity against skin infections caused by S. aureus. This combination showed the fastest healing time among various concentrations of curcumin-tetracycline combinations and is safe to be used topically.
S. aureus contributes to a majority of skin and soft tissue infections (SSTIs),18 while E. coli infections occur exclusively in immunodeficient patients19. Moreover, Meulemans et al.20 showed that the injection of S. aureus into rabbit skin resulted in similar skin infections to those seen in the field. Therefore, for an in situ study, an S. aureus suspension was chosen to be injected into the skin of rabbits to induce SSTIs.
Curcumin has been reported to inhibit the growth of antibiotic-sensitive and resistant bacteria, including S. aureus and methicillin-resistant S. aureus21. In vitro synergistic antibacterial activities of curcumin with various antibiotics and bacteria have also been previously reported9-10. Furthermore, combinations of curcumin with gentamicin, tetracycline and ciprofloxacin against S. aureus showed synergistic antibacterial activity,9-10 which is in line with the present study. In this study, it is demonstrated that a combination of 2% curcumin and 1% tetracycline resulted in the best antibacterial effectiveness against S. aureus infection through the disruption of the bacterial cell wall. Tetracycline was chosen to be combined with curcumin because a tetracycline ointment product is available in the market, while a ciprofloxacin ointment is not. Several studies have reported the mechanism of action of curcumin, Tyagi et al.22 demonstrated that curcumin at higher concentrations can have antibacterial activity against Gram-negative and Gram-positive bacteria by causing membrane permeabilization. Other studies demonstrated that curcumin inhibits the assembly of FtsZ (prokaryotic homologue of eukaryotic cytoskeletal protein tubulin) protofilaments which leads to the inhibition of bacterial cell proliferation23-25. The synergistic interaction observed in the present study is probably due to an increase of tetracycline influx into the bacteria cells caused by membrane hyper permeabilization.
This study implies a pharmacological and toxicological knowledge of a curcumin-tetracycline combination ointment for the development of clinical studies to treat skin infections. The therapeutic use of a curcumin-tetracycline combination can be explored further for other type of infections as it has a wide spectrum of antibacterial activity. However, one of the limitations of this study is that the antibacterial activity of the curcumin-tetracyclin combination ointment was only tested on rabbit skin, it is unknown whether the results can be extrapolated to humans. Moreover, the antibacterial activity was only tested on S. aureus induced skin infections; its activity on other bacterial infections should be investigated further.
In an in vitro study, curcumin has shown antibacterial activity against Staphylococcus aureus and Escherichia coli and has synergistic interaction in combination with tetracycline. The combination of curcumin and tetracycline accelerates the healing process of S. aureus-caused skin infections as observed on rabbit skin; its mechanism of action is by disrupting the bacterial wall. A combination of 2% curcumin and 1% tetracycline does not induce dermal or ocular irritation. Therefore, this combination has the potential to be developed as an alternative drug in the treatment of skin infections.
This study discovered that the combination of 2% curcumin and 1% tetracycline had antibacterial activity against Staphylococcus aureus that can be beneficial for S. aureus-induced skin infection on rabbit. This investigation will facilitate the researcher to obtain an alternative drug for skin infection that many researchers were not able to explore.
This study was supported by the Ministry of Research, Technology and Higher Education, Indonesia (077/SP2H/LT/DRPM/II/2016).