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Inhibitory effect of cinnamaldehyde alone and in combination with thymol, eugenol and thymoquinone against Staphylococcus epidermidis
Accepted Manuscript Title: “Inhibitory effect of cinnamaldehyde alone and in combination with thymol, eugenol and thymoquinone against Staphylococcus epidermidis” Author: Garima Sharma Kiran Raturi Shweta Dang Sanjay Gupta Reema Gabrani PII: DOI: Reference:
S2210-8033(16)30094-X http://dx.doi.org/doi:10.1016/j.hermed.2016.11.001 HERMED 159
To appear in: Received date: Revised date: Accepted date:
19-8-2015 29-2-2016 21-11-2016
Please cite this article as: Sharma, Garima, Raturi, Kiran, Dang, Shweta, Gupta, Sanjay, Gabrani, Reema, “Inhibitory effect of cinnamaldehyde alone and in combination with thymol, eugenol and thymoquinone against Staphylococcus epidermidis”.Journal of Herbal Medicine http://dx.doi.org/10.1016/j.hermed.2016.11.001 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Title: “Inhibitory effect of cinnamaldehyde alone and in combination with thymol, eugenol and thymoquinone against Staphylococcus epidermidis” Garima Sharma#, Kiran Raturi#, Shweta Dang, Sanjay Gupta, Reema Gabrani* Centre for Emerging Diseases, Department of Biotechnology, Jaypee Institute of Information Technology, A-10, Sector-62, NOIDA, India
Garima Sharma: Tel: +91-120-2594536; e-mail: [email protected] Kiran Raturi: Tel: +91-9910677541; e-mail: [email protected] Shweta Dang:Tel: +91-120-2594207; e-mail: [email protected] Sanjay Gupta: Tel: +91-120-2594204; e-mail: [email protected] #
Both authors have contributed equally.
*
Corresponding author. Tel: +91-120-2594210; Email: [email protected]
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Title:
Inhibitory effect of cinnamaldehyde alone and in combination with thymol, eugenol and thymoquinone against Staphylococcus epidermidis
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ABSTRACT Staphylococcus epidermidis has emerged as an important cause of nosocomial infections. These infections are very difficult to cure due to formation of persistent and recurrent biofilm, which decreases the susceptibility of bacteria to antimicrobial agents. Phytochemicals, especially in combination, can lower the dose of antimicrobial agent needed, reduce the toxicity and diminish the probability of developing resistance to microorganisms. The present investigation was undertaken to study the effect of cinnamaldehyde with other essential oil components (thymol, thymoquinone and eugenol) on the growth of S. epidermidis planktonic culture and its biofilm. All compounds used in the current study were found to be effective against the planktonic culture and biofilm of S. epidermidis. Thymol and cinnamaldehyde showed synergistic antimicrobial activity which considerably reduced the dose needed compared with cinnamaldehyde alone. Cinnamaldehyde in combination with other compounds showed time- and concentrationdependent enhanced bacterial membrane permeabilization as determined by β-galactosidase activity on Escherichia coli ML35p strain. Moreover, the synergistic combination was less cytotoxic on the vero cell line compared with cinnamaldehyde alone. The results of the current study indicate that cinnamaldehyde and thymol in combination can be effective against S. epidermidis planktonic culture and biofilm development. KEY WORDS: Antibiofilm, Cytotoxicity, Eugenol, Phytochemical, Thymol, Thymoquinone
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1. INTRODUCTION S. epidermidis, Gram-positive bacteria, is generally found on human skin as a commensal but can be the major cause of severe nosocomial infection in certain patients (Decker et al., 2015). It has been shown to develop resistance to antibiotics, highlighting the necessity for urgent development of novel therapeutics and antimicrobial strategies (Perazzi et al., 2006). Over the past few years, medicinal plants have gained a lot of interest and the attention of the scientific community and have demonstrated the promising potential of antimicrobial plant-derived substances (Mustaffa et al., 2011; Wise et al., 2011). A recent study indicates that essential oil components may target the bacterial cell by disrupting its membrane and interacting with various biochemical and molecular targets (Lopez-Romero et al., 2015). Essential oil components like cinnamaldehyde, thymol, thymoquinone and eugenol have been reported to be active on Gram-positive and Gram-negative bacteria (Hyldgaard et al., 2012; Nazzaro et al., 2013). Trans-cinnamaldehyde is a major constituent of cinnamon essential oil obtained from the bark extract of Cinnamomum zeylanicum. Its antimicrobial activity has been evaluated against various bacteria including food-borne pathogens (Siddiqua et al., 2015). Thymol is a major component of oregano oil, which is retrieved from Origanum glandulosum. Thymol and its derivative have been shown to be effective against various bacteria including Salmonella enterica serovar Typhimurium and Escherichia coli K88 (Levent et al., 2016). Eugenol is an active constituent of clove (Eugenia caryophillis) and is widely reported for its use in dental care due to its analgesic and antibacterial effects (Freires et al., 2015). Thymoquinone, found in the volatile oil of Nigella sativa, has been recently reported for potential antimethicillin-resistant Staphylococcus aureus activity (Hariharan et al., 2016). Considering the interest of researchers and an 5
increase in consumer demand for effective, safe, natural products, there is a need for compiling and generating quantitative data on phytochemical activity and ingenious screening programs. One of the main causes of resistance to antibiotics is the formation of biofilm. Biofilm is a microbial community in which the cells are irreversibly attached to a substratum or to each other embedded in a matrix of self-produced extra polymeric substance (EPS) exhibiting an altered phenotype with respect to growth rate, stress tolerance and gene transcription (Piette et al., 2009; Trulzsch et al., 2007). It has been argued that targeting the organism at multiple sites can facilitate its killing in a synergistic manner and reduces its chance of developing drug resistance. Interaction of components could have synergistic, additive or antagonistic effects on the growth of microorganisms (Van Zyla et al., 2010). Essential oil constituents are divided into terpenes, terpenoids, phenylpropanoids and others. The phenylpropanoids and terpenoid phenols in combination can cooperate in a synergistic manner to enhance the biological activity, probably due to the occurrence of different functional groups (Bassolé et al., 2012). Different components of essential oils often interact in a complex way which leads to varied pharmacological effects and therapeutic outcomes. Hence cinnamaldehyde, an aromatic aldehyde and a phenylpropanoid, was tested for possible synergistic activity with another phenylpropanoid with an alcoholic functional group, eugenol, and the phenolic terpenoids thymol and thymoquinone. Cinnamaldehyde has shown synergistic effects with curcumin against S. epidermidis (Sharma et al., 2014), eugenol against Laetiporus sulphureus (Yen and Chang, 2008), and fluconazole against Aspergillus fumigatus and Trichophyton rubrum (Khan and Ahmad, 2011). Trans-cinnamaldehyde was found to be effective in preventing the growth of E. coli biofilm, which is an uropathogen on plates
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and catheters without any cytotoxic effects on human bladder epithelial cells (Amalaradjou et al., 2010). Cinnamon oil has been reported for antibacterial activity against clinical S. epidermidis strains on both planktonic and biofilm cultures and decrease in biofilm formation was directly correlated to reduction in ica expression (Nuryastuti et al., 2009). In this study, the antimicrobial activities of cinnamaldehyde, thymol, thymoquinone and eugenol, as well as cinnamaldehyde in combination with other essential oil components were evaluated against S. epidermidis planktonic culture and biofilm genesis. The mechanism of action was also explored by assessing their effect on the inner membrane permeability of E. coli by β-galactosidase assay. The cytotoxicity of the synergistic combination was tested on a mammalian cell line.
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2. MATERIALS AND METHODS 2.1 Materials E. coli ML35p was generously gifted by Dr. Dinkar Sahal from ICGEB, New Delhi. Trans-cinnamaldehyde and eugenol were purchased from Sigma-Aldrich (Dorset, UK) and thymol and thymoquinone were purchased from HiMedia, India. 2.2 Preparation of essential oil compounds Cinnamaldehyde (1g/ml), eugenol (1g/ml), thymol (50 mg/ml) and thymoquinone (50 mg/ml) were initially diluted (1:10) in Luria Bertani (LB) broth containing 0.25% DMSO and were further diluted in LB. These compounds were filter sterilized prior to use. 2.3 Preparation of inoculum S. epidermidis (MTCC no.435) was procured from MTCC, Chandigarh, India. S. epidermidis, the indicator organism in the current study was grown overnight at 37°C with shaking at 200 rpm. The inoculum from the secondary culture equivalent to 1 × 106 cfu/ml was used to set up the antimicrobial assay (Wiegand et al., 2008). 2.4 Determination of minimum inhibitory concentration (MIC) of essential oil components against S. epidermidis The MICs of cinnamaldehyde, thymol, thymoquinone and eugenol were determined using a microbroth dilution assay using CLSI guidelines (2009). S. epidermidis suspension (100 µl) diluted to 1 × 106 cfu/ml was added into each well of a microtiter plate with 100µl of antimicrobial agent. The optical density of the plate was taken using an ELISA plate reader at 595 nm at 0 and 18 hours post-
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inoculation. The assay was carried out in triplicate and repeated thrice. The mean percentage inhibition was calculated as - % inhibition = (1-(At18– At0)/Acontrol18-Acontrol0) * 100 where At18 and At0 are absorbance readings of test compounds and Acontrol18 and Acontrol0 are absorbance readings of growth control at 18 and 0 hours, respectively. The MIC was taken as minimal value of the compound showing ≥ 95% inhibition. Broth containing phytochemical without bacterial inoculum was considered for the background colour of the substance. The antimicrobial activity of 0.25% DMSO was kept as control (Quave et al., 2008). 2.5 The antimicrobial activity of cinnamaldehyde in combination with thymol, thymoquinone and eugenol against S. epidermidis in suspension The antimicrobial activity of cinnamaldehyde in combination with thymol, thymoquinone and eugenol was determined by the checkerboard method (Wayne, 2009). In brief, serial dilutions of the antimicrobial agents were prepared [cinnamaldehyde (3.12 mg/l to 200 mg/l), thymol (3.12mg/l to 1000 mg/l), thymoquinone (3.12 mg/l to 1000 mg/l) and eugenol (6.25 mg/l to 1000 mg/l)]. Each dilution of cinnamaldehyde was mixed with various concentrations of other compounds in a 96-well microtitre plate (50 µl each) in a checkerboard manner and incubated with a S. epidermidis suspension containing 1 × 106 cfu/ml (100 µl) at 37°C for 18 hours. To assess the synergistic, antagonistic or additive interaction of antimicrobial compounds, the fractional inhibitory concentration index (FICI) was determined using the following formula: FICA = MICA+B/MICA, FICB = MICB+A/MICB, FICI = FICA +FICB. Where, MICA+B is the MIC of drug A in combination with B and MICB+A is vice versa. The results were interpreted as synergistic if FICI ≤ 0.5; additive if FICI is between 0.5–4 and antagonistic if FICI > 4 (Orhan et al., 2005).
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2.6 Determination of minimum biofilm inhibitory concentration (MBIC) of essential oil components against S. epidermidis biofilm The ability of essential oil constituents to inhibit the biofilm formation was determined by checkerboard assay. Bacterial suspension equivalent to 1 × 106 cfu/ml and an equal volume of different concentrations of plant compounds (final volume 200 µl) in a 96-well plate were incubated at 37°C for 48 hours. The culture supernatant was discarded after the incubation and the plate was washed thoroughly with phosphate buffer saline (PBS). Subsequently, the plate wells were stained with 100 µl of 0.2% solution of crystal violet (CV) and incubated at 37°C for 10-15 minutes. The plate was rinsed with distilled water and dried. The absorbed CV was solubilised with 100 µl of 90% ethanol. Biofilm disruption was evaluated by taking the OD595nm of a 96-well microtiter plate in an ELISA reader (Biorad, California, USA). MBIC was calculated as the lowest concentration showing ≥ 95% biofilm inhibition (Bhaduri et al., 1987). 2.7 The antibiofilm activity of cinnamaldehyde in combination with thymol, thymoquinone and eugenol against S. epidermidis The antibiofilm activity of cinnamaldehyde in combination with thymol, thymoquinone and eugenol was assessed by the CV method (Bhaduri et al., 1987). The essential oil components in combination, as discussed in the above section, were plated along with S. epidermidis and after 48 hours, the CV assay was carried out and FICI was calculated. 2.8 Permeabilization of the inner membrane of E. coli ML-35p
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E. coli ML-35p, is a mutant strain which constitutively expresses cytoplasmic β-galactosidase but lacks lac permease. It was grown overnight at 200 rpm at 37°C and was washed thrice in phosphate buffer, pH 7.4 (10 mM phosphate buffer, 0.1 M NaCl). The bacterial culture (1 × 106 cfu/ml), diluted in incubation buffer (10 mM phosphate buffer, pH 7.4, 0.3 g/L TSB) was added to each well in a 96-well microplate, along with test compound and 2.5 mM ortho-nitrophenyl-β-galactoside (ONPG). 0.1% SDS was taken as positive control. Absorbance was recorded at 420 nm for 60 min at intervals of 2 min in a spectrophotometer (Shimadzu, Japan) (Epand et al., 2010).
2.9 Cytotoxicity assay The toxicity of the phytochemicals on the green monkey kidney vero cell line was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyl-tetrazolium bromide (MTT; Sigma-Aldrich) cytotoxicity assay. Vero cells were cultured in Dulbecco's modified Eagle's medium (Sigma) supplemented with 10% foetal bovine serum (Hi Media) and plated in triplicate wells at 1 × 105 cells per well, respectively. The various concentrations of essential oil components were added to the cells and incubated at 37°C in 5% CO2 atmosphere for 24 hours. MTT (0.5 mg/ml) was added and cells were further incubated for 4 hours at 37°C. Solubilisation of formazan, the product of the metabolic reduction of MTT, was carried out in DMSO. The plate was read at 570 nm in an ELISA plate reader. 3. RESULT AND DISCUSSION
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3.1 Antibacterial and anti-biofilm activity
S. epidermidis is a common commensal bacterium that is responsible for more than 50% of all nosocomial infections (Otto, 2014). Essential oils are combinations of various heterogeneous compounds, and the actual components responsible for their antimicrobial activity and the mode of their activity are not well understood. S. epidermidis was challenged with different concentrations of cinnamaldehyde, thymol, thymoquinone and eugenol and the MIC was calculated by broth micro-dilution assay. All the essential oil constituents exhibited antibacterial activities against S. epidermidis in a dose-dependent manner (data not shown) with MIC values ranging from 125 to 400 mg/l (Table 1). The antibiofilm property of the selected essential oil components was determined using the CV assay in a microtiter plate and they exhibited a significant inhibitory effect on biofilm formation of S. epidermidis in a dosedependent manner and MBIC was determined (Table 1). The results demonstrated that cinnamaldehyde, thymol, thymoquinone and eugenol showed antimicrobial activity against S. epidermidis when grown both in suspension and as a biofilm and S. epidermidis was found to be the most sensitive to cinnamaldehyde (125 mg/l). The concentrations required to inhibit the growth both in planktonic and biofilm were the same, although the biofilm is considered to be more resistant to antimicrobial agents than planktonic cells (Spoering and Lewis, 2001). Cinnamaldehyde/thymol was found to be synergistically effective; however, cinnamaldehyde/thymoquinone and cinnamaldehyde/eugenol had an additive effect against S. epidermidis for both planktonic culture (Table 2) and biofilm formation (Table 3).
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The synergistic combination of cinnamaldehyde and curcumin has been reported earlier by our group to inhibit the growth of S. epidermidis both in planktonic and biofilm with FIC values of 15.6mg/l and 3.12mg/l, respectively (Sharma et al., 2014). Cinnamaldeyde has also been reported to prevent the E. coli biofilm on plates and catheters at 400mg/l (Pei et al., 2009). Thymoquinone has been shown to be effective against the S. aureus ATCC 25923 and S. epidermidis with MIC and MBC (minimum bactericidal concentration) values ranging from 8-16 µg/ml and was able to prevent 90% of S. epidermidis biofilm formation (Chaieb et al., 2011). Thymol has been reported to inhibit the growth of S. epidermidis in both suspension and in a biofilm with an MIC value of 0.5-4g/l (Karpanen et al., 2008). Recently, cinnamaldehyde in combination with streptomycin was shown to exert synergistic effects against planktonic and biofilm formed by food-borne pathogens Listeria monocytogenes and Salmonella typhimurium (Liu et al., 2015). One of the major advantages of potential synergy among polyphenolic compounds is to improve the antimicrobial potential due to their multi-targeting activity at lower concentrations (Tomas-menor et al., 2015).
3.2 Permeabilization of inner membrane of E. coli ML-35p
E. coli ML-35p was used to study the kinetics for permeabilization of the selected essential oil components alone and in combination. E. coli ML-35p has been altered to lack lac permease and hence will not be able to convert ONPG to orthonitrophenol (ONP) by the constitutive β-galactosidase unless the cellular membrane is damaged. As shown in Fig. 1, cinnamaldehyde, thymol, thymoquinone and eugenol showed an increase in conversion of ONPG to ONP by β-galactosidase, indicating a time-dependent effect on the inner membrane permeability. The β-galactosidase activity increased considerably when E. coli cells were treated with a mix of 13
cinnamaldehyde and other essential oil components in contrast to cinnamaldehyde alone, which indicates that the E. coli ML-35p permeability was more pronounced with the combination. It has been reported in the literature that cinnamaldehyde interacts with the membrane and allows the leakage of small ions (Gill & Richard, 2004). In certain studies, the result of electron microscopy indicates that the essential oil components in combination affect the integrity of the cell membrane, which results in release of the cell constituents (Lv et al., 2011).
3.3 Cytotoxicity assay
The WHO established the temporary acceptable daily intake for cinnamaldehyde for humans as 0-0.7 mg/kg body weight. According to the WHO, the various animal studies have indicated that cinnamaldehyde intake can result in "slight hepatic cell swelling" and a "slight hyperkeratosis of squamous portion" of the stomach and can also result in histological changes in the kidney cells (Gowder et al., 2008). Dose-dependent effects of the combination exhibiting a synergistic outcome (cinnamaldehyde and thymol) against S. epidermidis were tested for in vitro cytotoxicity on the vero cell line by MTT assay. Vero is an established cell line from the normal kidney of an African green monkey and has been extensively used as a cellular model to evaluate cytotoxic effects (Fernández et al., 2009; Sharma et al., 2012).
Cinnamaldehyde and thymol in combination displayed improved cell viability (78%) on the vero cell line suggesting that these two compounds in combination were less toxic than the cinnamaldehyde alone (Fig. 2).
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4. CONCLUSION This study was undertaken to analyse the possible synergistic effect of cinnamaldehyde and other essential oil components, eugenol, thymol and thymoquinone, against S. epidermidis. The cinnamaldehyde and thymol combination was found to be synergistic as an antimicrobial agent against S. epidermidis as well as its biofilm development. The combinations of cinnamaldehyde with thymoquinone and eugenol were additive against planktonic and biofilm cultures of S. epidermidis. These phytochemicals have previously been shown to mediate the killing of the organism by altering the membrane permeability. The synergistic combinations were also found to enhance the membrane permeability as determined by a β-galactosidase assay on E. coli ML-35p cells. The cytotoxicity data on mammalian cell line indicate that cinnamaldehyde and thymol together were less toxic than cinnamaldehyde alone. 5. ACKNOWLEDGEMENT We thank the Department of Biotechnology, Jaypee Institute of Information Technology, Noida, UP, India, for providing the infrastructural facility to carry out the research work. 6. CONFLICT OF INTEREST Authors
declare
no
conflict
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of
interest.
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Legends Figures Figure 1. Time-dependent inner membrane permeabilization of E. coli ML35p by (a) cinnamaldehyde 125 mg/l (♦), eugenol 250 mg/l (■), cinnamaldehyde 62.5 mg/l and eugenol 15.62 mg/l in combination (▲); (b) cinnamaldehyde 125 mg/l (♦), thymol 400 mg/l (■), cinnamaldehyde 15.62 mg/l and thymol 100 mg/l in combination (▲); (c) - cinnamaldehyde 125 mg/l (♦), thymoquinone 200 mg/l (■), cinnamaldehyde 15.62 mg/l and thynoquinone 200 mg/l in combination (▲), 0.1% SDS(ж) was taken as positive control and cells only (×) as negative control. Hydrolysis of ONPG was monitored at 420 nm. The bar represents mean ± standard error. Figure 2. MTT assay for cytotoxicity analysis of cinnamadehyde (a), thymol (b) and combination of cinnamaldehyde and thymol (c) on vero cell line. Each bar indicates mean ± standard error. The experiment was repeated minimum thrice and this data is the representative result from triplicate experiments.
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Table 1. Antibacterial activity of plant essential oil components against S. epidermidis planktonic cells and biofilm, as determined by checkerboard microtiter dilution assay. MIC and MBIC were calculated from at least three independent experiments.
Essential oil
MIC against S.
MBIC against S.
components
epidermidis (mg/l)
epidermidis (mg/l)
Cinnamaldehyde
125
125
Thymol
400
400
Thymoquinone
200
200
Eugenol
250
250
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Table 2. Antibacterial activity of cinnamaldehyde alone and in combination with thymol, thymoquinone and eugenol against S. epidermidis.
Combination
MIC of compound alone/
FIC
FICI
Result
0.375
Synergy
1.125
Additive
0.562
Additive
combination (mg/l) Cinnamaldehyde
125 /15.625
0.125
Thymol
400/100
0.25
Cinnamaldehyde
125 /15.625
0.125
Thymoquinone
200/200
1
Cinnamaldehyde
125/62.5
0.5
Eugenol
250/15.62
0.062
23
Table 3. Antimicrobial activity of cinnamaldehyde and in combination with thymol, thymoquinone and eugenol against S. epidermidis biofilm. Combination
MBIC of compound alone/
FIC
FICI
Result
0.375
Synergy
1.125
Additive
1
Additive
combination (mg/l) Cinnamaldehyde
125 /15.625
0.125
Thymol
400/100
0.25
Cinnamaldehyde
125 /15.625
0.125
Thymoquinone
200/200
1
Cinnamaldehyde
125/62.5
0.5
Eugenol
250/125
0.5
24
25
26
S2210-8033(16)30094-X http://dx.doi.org/doi:10.1016/j.hermed.2016.11.001 HERMED 159
To appear in: Received date: Revised date: Accepted date:
19-8-2015 29-2-2016 21-11-2016
Please cite this article as: Sharma, Garima, Raturi, Kiran, Dang, Shweta, Gupta, Sanjay, Gabrani, Reema, “Inhibitory effect of cinnamaldehyde alone and in combination with thymol, eugenol and thymoquinone against Staphylococcus epidermidis”.Journal of Herbal Medicine http://dx.doi.org/10.1016/j.hermed.2016.11.001 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Title: “Inhibitory effect of cinnamaldehyde alone and in combination with thymol, eugenol and thymoquinone against Staphylococcus epidermidis” Garima Sharma#, Kiran Raturi#, Shweta Dang, Sanjay Gupta, Reema Gabrani* Centre for Emerging Diseases, Department of Biotechnology, Jaypee Institute of Information Technology, A-10, Sector-62, NOIDA, India
Garima Sharma: Tel: +91-120-2594536; e-mail: [email protected] Kiran Raturi: Tel: +91-9910677541; e-mail: [email protected] Shweta Dang:Tel: +91-120-2594207; e-mail: [email protected] Sanjay Gupta: Tel: +91-120-2594204; e-mail: [email protected] #
Both authors have contributed equally.
*
Corresponding author. Tel: +91-120-2594210; Email: [email protected]
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Title:
Inhibitory effect of cinnamaldehyde alone and in combination with thymol, eugenol and thymoquinone against Staphylococcus epidermidis
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ABSTRACT Staphylococcus epidermidis has emerged as an important cause of nosocomial infections. These infections are very difficult to cure due to formation of persistent and recurrent biofilm, which decreases the susceptibility of bacteria to antimicrobial agents. Phytochemicals, especially in combination, can lower the dose of antimicrobial agent needed, reduce the toxicity and diminish the probability of developing resistance to microorganisms. The present investigation was undertaken to study the effect of cinnamaldehyde with other essential oil components (thymol, thymoquinone and eugenol) on the growth of S. epidermidis planktonic culture and its biofilm. All compounds used in the current study were found to be effective against the planktonic culture and biofilm of S. epidermidis. Thymol and cinnamaldehyde showed synergistic antimicrobial activity which considerably reduced the dose needed compared with cinnamaldehyde alone. Cinnamaldehyde in combination with other compounds showed time- and concentrationdependent enhanced bacterial membrane permeabilization as determined by β-galactosidase activity on Escherichia coli ML35p strain. Moreover, the synergistic combination was less cytotoxic on the vero cell line compared with cinnamaldehyde alone. The results of the current study indicate that cinnamaldehyde and thymol in combination can be effective against S. epidermidis planktonic culture and biofilm development. KEY WORDS: Antibiofilm, Cytotoxicity, Eugenol, Phytochemical, Thymol, Thymoquinone
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1. INTRODUCTION S. epidermidis, Gram-positive bacteria, is generally found on human skin as a commensal but can be the major cause of severe nosocomial infection in certain patients (Decker et al., 2015). It has been shown to develop resistance to antibiotics, highlighting the necessity for urgent development of novel therapeutics and antimicrobial strategies (Perazzi et al., 2006). Over the past few years, medicinal plants have gained a lot of interest and the attention of the scientific community and have demonstrated the promising potential of antimicrobial plant-derived substances (Mustaffa et al., 2011; Wise et al., 2011). A recent study indicates that essential oil components may target the bacterial cell by disrupting its membrane and interacting with various biochemical and molecular targets (Lopez-Romero et al., 2015). Essential oil components like cinnamaldehyde, thymol, thymoquinone and eugenol have been reported to be active on Gram-positive and Gram-negative bacteria (Hyldgaard et al., 2012; Nazzaro et al., 2013). Trans-cinnamaldehyde is a major constituent of cinnamon essential oil obtained from the bark extract of Cinnamomum zeylanicum. Its antimicrobial activity has been evaluated against various bacteria including food-borne pathogens (Siddiqua et al., 2015). Thymol is a major component of oregano oil, which is retrieved from Origanum glandulosum. Thymol and its derivative have been shown to be effective against various bacteria including Salmonella enterica serovar Typhimurium and Escherichia coli K88 (Levent et al., 2016). Eugenol is an active constituent of clove (Eugenia caryophillis) and is widely reported for its use in dental care due to its analgesic and antibacterial effects (Freires et al., 2015). Thymoquinone, found in the volatile oil of Nigella sativa, has been recently reported for potential antimethicillin-resistant Staphylococcus aureus activity (Hariharan et al., 2016). Considering the interest of researchers and an 5
increase in consumer demand for effective, safe, natural products, there is a need for compiling and generating quantitative data on phytochemical activity and ingenious screening programs. One of the main causes of resistance to antibiotics is the formation of biofilm. Biofilm is a microbial community in which the cells are irreversibly attached to a substratum or to each other embedded in a matrix of self-produced extra polymeric substance (EPS) exhibiting an altered phenotype with respect to growth rate, stress tolerance and gene transcription (Piette et al., 2009; Trulzsch et al., 2007). It has been argued that targeting the organism at multiple sites can facilitate its killing in a synergistic manner and reduces its chance of developing drug resistance. Interaction of components could have synergistic, additive or antagonistic effects on the growth of microorganisms (Van Zyla et al., 2010). Essential oil constituents are divided into terpenes, terpenoids, phenylpropanoids and others. The phenylpropanoids and terpenoid phenols in combination can cooperate in a synergistic manner to enhance the biological activity, probably due to the occurrence of different functional groups (Bassolé et al., 2012). Different components of essential oils often interact in a complex way which leads to varied pharmacological effects and therapeutic outcomes. Hence cinnamaldehyde, an aromatic aldehyde and a phenylpropanoid, was tested for possible synergistic activity with another phenylpropanoid with an alcoholic functional group, eugenol, and the phenolic terpenoids thymol and thymoquinone. Cinnamaldehyde has shown synergistic effects with curcumin against S. epidermidis (Sharma et al., 2014), eugenol against Laetiporus sulphureus (Yen and Chang, 2008), and fluconazole against Aspergillus fumigatus and Trichophyton rubrum (Khan and Ahmad, 2011). Trans-cinnamaldehyde was found to be effective in preventing the growth of E. coli biofilm, which is an uropathogen on plates
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and catheters without any cytotoxic effects on human bladder epithelial cells (Amalaradjou et al., 2010). Cinnamon oil has been reported for antibacterial activity against clinical S. epidermidis strains on both planktonic and biofilm cultures and decrease in biofilm formation was directly correlated to reduction in ica expression (Nuryastuti et al., 2009). In this study, the antimicrobial activities of cinnamaldehyde, thymol, thymoquinone and eugenol, as well as cinnamaldehyde in combination with other essential oil components were evaluated against S. epidermidis planktonic culture and biofilm genesis. The mechanism of action was also explored by assessing their effect on the inner membrane permeability of E. coli by β-galactosidase assay. The cytotoxicity of the synergistic combination was tested on a mammalian cell line.
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2. MATERIALS AND METHODS 2.1 Materials E. coli ML35p was generously gifted by Dr. Dinkar Sahal from ICGEB, New Delhi. Trans-cinnamaldehyde and eugenol were purchased from Sigma-Aldrich (Dorset, UK) and thymol and thymoquinone were purchased from HiMedia, India. 2.2 Preparation of essential oil compounds Cinnamaldehyde (1g/ml), eugenol (1g/ml), thymol (50 mg/ml) and thymoquinone (50 mg/ml) were initially diluted (1:10) in Luria Bertani (LB) broth containing 0.25% DMSO and were further diluted in LB. These compounds were filter sterilized prior to use. 2.3 Preparation of inoculum S. epidermidis (MTCC no.435) was procured from MTCC, Chandigarh, India. S. epidermidis, the indicator organism in the current study was grown overnight at 37°C with shaking at 200 rpm. The inoculum from the secondary culture equivalent to 1 × 106 cfu/ml was used to set up the antimicrobial assay (Wiegand et al., 2008). 2.4 Determination of minimum inhibitory concentration (MIC) of essential oil components against S. epidermidis The MICs of cinnamaldehyde, thymol, thymoquinone and eugenol were determined using a microbroth dilution assay using CLSI guidelines (2009). S. epidermidis suspension (100 µl) diluted to 1 × 106 cfu/ml was added into each well of a microtiter plate with 100µl of antimicrobial agent. The optical density of the plate was taken using an ELISA plate reader at 595 nm at 0 and 18 hours post-
8
inoculation. The assay was carried out in triplicate and repeated thrice. The mean percentage inhibition was calculated as - % inhibition = (1-(At18– At0)/Acontrol18-Acontrol0) * 100 where At18 and At0 are absorbance readings of test compounds and Acontrol18 and Acontrol0 are absorbance readings of growth control at 18 and 0 hours, respectively. The MIC was taken as minimal value of the compound showing ≥ 95% inhibition. Broth containing phytochemical without bacterial inoculum was considered for the background colour of the substance. The antimicrobial activity of 0.25% DMSO was kept as control (Quave et al., 2008). 2.5 The antimicrobial activity of cinnamaldehyde in combination with thymol, thymoquinone and eugenol against S. epidermidis in suspension The antimicrobial activity of cinnamaldehyde in combination with thymol, thymoquinone and eugenol was determined by the checkerboard method (Wayne, 2009). In brief, serial dilutions of the antimicrobial agents were prepared [cinnamaldehyde (3.12 mg/l to 200 mg/l), thymol (3.12mg/l to 1000 mg/l), thymoquinone (3.12 mg/l to 1000 mg/l) and eugenol (6.25 mg/l to 1000 mg/l)]. Each dilution of cinnamaldehyde was mixed with various concentrations of other compounds in a 96-well microtitre plate (50 µl each) in a checkerboard manner and incubated with a S. epidermidis suspension containing 1 × 106 cfu/ml (100 µl) at 37°C for 18 hours. To assess the synergistic, antagonistic or additive interaction of antimicrobial compounds, the fractional inhibitory concentration index (FICI) was determined using the following formula: FICA = MICA+B/MICA, FICB = MICB+A/MICB, FICI = FICA +FICB. Where, MICA+B is the MIC of drug A in combination with B and MICB+A is vice versa. The results were interpreted as synergistic if FICI ≤ 0.5; additive if FICI is between 0.5–4 and antagonistic if FICI > 4 (Orhan et al., 2005).
9
2.6 Determination of minimum biofilm inhibitory concentration (MBIC) of essential oil components against S. epidermidis biofilm The ability of essential oil constituents to inhibit the biofilm formation was determined by checkerboard assay. Bacterial suspension equivalent to 1 × 106 cfu/ml and an equal volume of different concentrations of plant compounds (final volume 200 µl) in a 96-well plate were incubated at 37°C for 48 hours. The culture supernatant was discarded after the incubation and the plate was washed thoroughly with phosphate buffer saline (PBS). Subsequently, the plate wells were stained with 100 µl of 0.2% solution of crystal violet (CV) and incubated at 37°C for 10-15 minutes. The plate was rinsed with distilled water and dried. The absorbed CV was solubilised with 100 µl of 90% ethanol. Biofilm disruption was evaluated by taking the OD595nm of a 96-well microtiter plate in an ELISA reader (Biorad, California, USA). MBIC was calculated as the lowest concentration showing ≥ 95% biofilm inhibition (Bhaduri et al., 1987). 2.7 The antibiofilm activity of cinnamaldehyde in combination with thymol, thymoquinone and eugenol against S. epidermidis The antibiofilm activity of cinnamaldehyde in combination with thymol, thymoquinone and eugenol was assessed by the CV method (Bhaduri et al., 1987). The essential oil components in combination, as discussed in the above section, were plated along with S. epidermidis and after 48 hours, the CV assay was carried out and FICI was calculated. 2.8 Permeabilization of the inner membrane of E. coli ML-35p
10
E. coli ML-35p, is a mutant strain which constitutively expresses cytoplasmic β-galactosidase but lacks lac permease. It was grown overnight at 200 rpm at 37°C and was washed thrice in phosphate buffer, pH 7.4 (10 mM phosphate buffer, 0.1 M NaCl). The bacterial culture (1 × 106 cfu/ml), diluted in incubation buffer (10 mM phosphate buffer, pH 7.4, 0.3 g/L TSB) was added to each well in a 96-well microplate, along with test compound and 2.5 mM ortho-nitrophenyl-β-galactoside (ONPG). 0.1% SDS was taken as positive control. Absorbance was recorded at 420 nm for 60 min at intervals of 2 min in a spectrophotometer (Shimadzu, Japan) (Epand et al., 2010).
2.9 Cytotoxicity assay The toxicity of the phytochemicals on the green monkey kidney vero cell line was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyl-tetrazolium bromide (MTT; Sigma-Aldrich) cytotoxicity assay. Vero cells were cultured in Dulbecco's modified Eagle's medium (Sigma) supplemented with 10% foetal bovine serum (Hi Media) and plated in triplicate wells at 1 × 105 cells per well, respectively. The various concentrations of essential oil components were added to the cells and incubated at 37°C in 5% CO2 atmosphere for 24 hours. MTT (0.5 mg/ml) was added and cells were further incubated for 4 hours at 37°C. Solubilisation of formazan, the product of the metabolic reduction of MTT, was carried out in DMSO. The plate was read at 570 nm in an ELISA plate reader. 3. RESULT AND DISCUSSION
11
3.1 Antibacterial and anti-biofilm activity
S. epidermidis is a common commensal bacterium that is responsible for more than 50% of all nosocomial infections (Otto, 2014). Essential oils are combinations of various heterogeneous compounds, and the actual components responsible for their antimicrobial activity and the mode of their activity are not well understood. S. epidermidis was challenged with different concentrations of cinnamaldehyde, thymol, thymoquinone and eugenol and the MIC was calculated by broth micro-dilution assay. All the essential oil constituents exhibited antibacterial activities against S. epidermidis in a dose-dependent manner (data not shown) with MIC values ranging from 125 to 400 mg/l (Table 1). The antibiofilm property of the selected essential oil components was determined using the CV assay in a microtiter plate and they exhibited a significant inhibitory effect on biofilm formation of S. epidermidis in a dosedependent manner and MBIC was determined (Table 1). The results demonstrated that cinnamaldehyde, thymol, thymoquinone and eugenol showed antimicrobial activity against S. epidermidis when grown both in suspension and as a biofilm and S. epidermidis was found to be the most sensitive to cinnamaldehyde (125 mg/l). The concentrations required to inhibit the growth both in planktonic and biofilm were the same, although the biofilm is considered to be more resistant to antimicrobial agents than planktonic cells (Spoering and Lewis, 2001). Cinnamaldehyde/thymol was found to be synergistically effective; however, cinnamaldehyde/thymoquinone and cinnamaldehyde/eugenol had an additive effect against S. epidermidis for both planktonic culture (Table 2) and biofilm formation (Table 3).
12
The synergistic combination of cinnamaldehyde and curcumin has been reported earlier by our group to inhibit the growth of S. epidermidis both in planktonic and biofilm with FIC values of 15.6mg/l and 3.12mg/l, respectively (Sharma et al., 2014). Cinnamaldeyde has also been reported to prevent the E. coli biofilm on plates and catheters at 400mg/l (Pei et al., 2009). Thymoquinone has been shown to be effective against the S. aureus ATCC 25923 and S. epidermidis with MIC and MBC (minimum bactericidal concentration) values ranging from 8-16 µg/ml and was able to prevent 90% of S. epidermidis biofilm formation (Chaieb et al., 2011). Thymol has been reported to inhibit the growth of S. epidermidis in both suspension and in a biofilm with an MIC value of 0.5-4g/l (Karpanen et al., 2008). Recently, cinnamaldehyde in combination with streptomycin was shown to exert synergistic effects against planktonic and biofilm formed by food-borne pathogens Listeria monocytogenes and Salmonella typhimurium (Liu et al., 2015). One of the major advantages of potential synergy among polyphenolic compounds is to improve the antimicrobial potential due to their multi-targeting activity at lower concentrations (Tomas-menor et al., 2015).
3.2 Permeabilization of inner membrane of E. coli ML-35p
E. coli ML-35p was used to study the kinetics for permeabilization of the selected essential oil components alone and in combination. E. coli ML-35p has been altered to lack lac permease and hence will not be able to convert ONPG to orthonitrophenol (ONP) by the constitutive β-galactosidase unless the cellular membrane is damaged. As shown in Fig. 1, cinnamaldehyde, thymol, thymoquinone and eugenol showed an increase in conversion of ONPG to ONP by β-galactosidase, indicating a time-dependent effect on the inner membrane permeability. The β-galactosidase activity increased considerably when E. coli cells were treated with a mix of 13
cinnamaldehyde and other essential oil components in contrast to cinnamaldehyde alone, which indicates that the E. coli ML-35p permeability was more pronounced with the combination. It has been reported in the literature that cinnamaldehyde interacts with the membrane and allows the leakage of small ions (Gill & Richard, 2004). In certain studies, the result of electron microscopy indicates that the essential oil components in combination affect the integrity of the cell membrane, which results in release of the cell constituents (Lv et al., 2011).
3.3 Cytotoxicity assay
The WHO established the temporary acceptable daily intake for cinnamaldehyde for humans as 0-0.7 mg/kg body weight. According to the WHO, the various animal studies have indicated that cinnamaldehyde intake can result in "slight hepatic cell swelling" and a "slight hyperkeratosis of squamous portion" of the stomach and can also result in histological changes in the kidney cells (Gowder et al., 2008). Dose-dependent effects of the combination exhibiting a synergistic outcome (cinnamaldehyde and thymol) against S. epidermidis were tested for in vitro cytotoxicity on the vero cell line by MTT assay. Vero is an established cell line from the normal kidney of an African green monkey and has been extensively used as a cellular model to evaluate cytotoxic effects (Fernández et al., 2009; Sharma et al., 2012).
Cinnamaldehyde and thymol in combination displayed improved cell viability (78%) on the vero cell line suggesting that these two compounds in combination were less toxic than the cinnamaldehyde alone (Fig. 2).
14
4. CONCLUSION This study was undertaken to analyse the possible synergistic effect of cinnamaldehyde and other essential oil components, eugenol, thymol and thymoquinone, against S. epidermidis. The cinnamaldehyde and thymol combination was found to be synergistic as an antimicrobial agent against S. epidermidis as well as its biofilm development. The combinations of cinnamaldehyde with thymoquinone and eugenol were additive against planktonic and biofilm cultures of S. epidermidis. These phytochemicals have previously been shown to mediate the killing of the organism by altering the membrane permeability. The synergistic combinations were also found to enhance the membrane permeability as determined by a β-galactosidase assay on E. coli ML-35p cells. The cytotoxicity data on mammalian cell line indicate that cinnamaldehyde and thymol together were less toxic than cinnamaldehyde alone. 5. ACKNOWLEDGEMENT We thank the Department of Biotechnology, Jaypee Institute of Information Technology, Noida, UP, India, for providing the infrastructural facility to carry out the research work. 6. CONFLICT OF INTEREST Authors
declare
no
conflict
15
of
interest.
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Legends Figures Figure 1. Time-dependent inner membrane permeabilization of E. coli ML35p by (a) cinnamaldehyde 125 mg/l (♦), eugenol 250 mg/l (■), cinnamaldehyde 62.5 mg/l and eugenol 15.62 mg/l in combination (▲); (b) cinnamaldehyde 125 mg/l (♦), thymol 400 mg/l (■), cinnamaldehyde 15.62 mg/l and thymol 100 mg/l in combination (▲); (c) - cinnamaldehyde 125 mg/l (♦), thymoquinone 200 mg/l (■), cinnamaldehyde 15.62 mg/l and thynoquinone 200 mg/l in combination (▲), 0.1% SDS(ж) was taken as positive control and cells only (×) as negative control. Hydrolysis of ONPG was monitored at 420 nm. The bar represents mean ± standard error. Figure 2. MTT assay for cytotoxicity analysis of cinnamadehyde (a), thymol (b) and combination of cinnamaldehyde and thymol (c) on vero cell line. Each bar indicates mean ± standard error. The experiment was repeated minimum thrice and this data is the representative result from triplicate experiments.
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Table 1. Antibacterial activity of plant essential oil components against S. epidermidis planktonic cells and biofilm, as determined by checkerboard microtiter dilution assay. MIC and MBIC were calculated from at least three independent experiments.
Essential oil
MIC against S.
MBIC against S.
components
epidermidis (mg/l)
epidermidis (mg/l)
Cinnamaldehyde
125
125
Thymol
400
400
Thymoquinone
200
200
Eugenol
250
250
22
Table 2. Antibacterial activity of cinnamaldehyde alone and in combination with thymol, thymoquinone and eugenol against S. epidermidis.
Combination
MIC of compound alone/
FIC
FICI
Result
0.375
Synergy
1.125
Additive
0.562
Additive
combination (mg/l) Cinnamaldehyde
125 /15.625
0.125
Thymol
400/100
0.25
Cinnamaldehyde
125 /15.625
0.125
Thymoquinone
200/200
1
Cinnamaldehyde
125/62.5
0.5
Eugenol
250/15.62
0.062
23
Table 3. Antimicrobial activity of cinnamaldehyde and in combination with thymol, thymoquinone and eugenol against S. epidermidis biofilm. Combination
MBIC of compound alone/
FIC
FICI
Result
0.375
Synergy
1.125
Additive
1
Additive
combination (mg/l) Cinnamaldehyde
125 /15.625
0.125
Thymol
400/100
0.25
Cinnamaldehyde
125 /15.625
0.125
Thymoquinone
200/200
1
Cinnamaldehyde
125/62.5
0.5
Eugenol
250/125
0.5
24
25
26