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Staphylococcus epidermidis is notorious for its biofilm formation on medical devices, and novel approaches to prevent and kill S. epidermidis biofilms are desired. In this study, the effect of cinnamon oil on planktonic and biofilm cultures of clinical S. epidermidis isolates was evaluated. Initially, susceptibility to cinnamon oil in planktonic cultures was compared to the commonly used antimicrobial agents chlorhexidine, triclosan, and gentamicin. The MIC of cinnamon oil, defined as the lowest concentration able to inhibit visible microbial growth, and the minimal bactericidal concentration, the lowest concentration required to kill 99.9% of the bacteria, were determined using the broth microdilution method and plating on agar. A checkerboard assay was used to evaluate the possible synergy between cinnamon oil and the other antimicrobial agents. The effect of cinnamon oil on biofilm growth was studied in 96-well plates and with confocal laser-scanning microscopy (CLSM). Biofilm susceptibility was determined using a metabolic 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Real-time PCR analysis was performed to determine the effect of sub-MIC concentrations of cinnamon oil on expression of the biofilm-related gene, icaA. Cinnamon oil showed antimicrobial activity against both planktonic and biofilm cultures of clinical S. epidermidis strains. There was only a small difference between planktonic and biofilm MICs, ranging from 0.5 to 1% and 1 to 2%, respectively. CLSM images indicated that cinnamon oil is able to detach and kill existing biofilms. Thus, cinnamon oil is an effective antimicrobial agent to combat S. epidermidis biofilms.
Staphylococcus epidermidis is a gram-positive bacterium and an important agent of nosocomial infections worldwide. Treatment of these infections is increasingly problematic because of the resistance of clinical isolates to an increasing number of antimicrobial agents and, more importantly, due to its ability to grow as a biofilm. Biofilm formation by S. epidermidis (35) can be governed in part by the production of polysaccharide intercellular adhesin. Polysaccharide intercellular adhesin is produced by enzymes encoded by the ica operon which comprises four intercellular adhesion genes: icaA, icaB, icaC, and icaD. The expression of the ica operon and biofilm formation are tightly regulated by icaR under in vitro conditions (15). Biofilm formation can be influenced by changing environmental conditions, such as the presence of subinhibitory concentrations of antimicrobials like tetracycline and quinopristin-dalfopristin, as well as high temperatures, anaerobiosis, ethanol stress, and osmolarity (8, 9, 26, 37).
Previous studies have demonstrated that microorganisms within biofilms are less susceptible to antimicrobial treatment than their planktonic counterparts (4), probably due to a combination of poor antimicrobial penetration, nutrient limitation, adaptive stress responses, induction of phenotypic variability, and persister cell formation (28). For this reason, current research has been focused on identifying new compounds that have antimicrobial activity against microorganisms, both in planktonic and biofilm modes of growth. Plant essential oils have been used in food preservation, pharmaceutical therapies, alternative medicine, and natural therapies for many thousands of years (23, 36).
Cinnamon oil is one of the essential oils commonly used in the food industry because of its special aroma (6). Cinnamomum is a genus in the family Lauraceae, many species of which are used for spices. One of the species is Cinnamomum burmannii from Indonesia, also called Indonesian cassia (the commercial name is “cinnamon stick”). Several publications have demonstrated the antibacterial activity of cinnamon oil isolated from the bark of this species (12, 18, 22, 39). Cinnamon oil was also shown to be effective against biofilm cultures of Streptococcus mutans and Lactobacillus plantarum (14). In addition, essential oil derived from the leaves of another closely related species within this plant family, Cinnamomum osmophloeum (endemic to Taiwan), had an excellent inhibitory effect on planktonic cultures of nine gram-positive and gram-negative bacteria, including methicillin-resistant Staphylococcus aureus and S. epidermidis (6). Previous studies reported that the predominant active compound found in cinnamon oil was cinnamaldehyde (36, 39). Cinnamaldehyde causes inhibition of the proton motive force, respiratory chain, electron transfer, and substrate oxidation, resulting in uncoupling of oxidative phosphorylation, inhibition of active transport, loss of pool metabolites, and disruption of synthesis of DNA, RNA, proteins, lipids, and polysaccharides (11, 13, 33). In addition, an important characteristic of volatile oils and their components is their hydrophobicity, which enables them to partition into and disturb the lipid bilayer of the cell membrane, rendering them more permeable to protons. Extensive leakage from bacterial cells or the exit of critical molecules and ions ultimately leads to bacterial cell death (36).
The susceptibility of S. epidermidis to cinnamon oil derived from the bark of Cinnamomum burmannii, however, has never been published, neither for planktonic organisms nor for staphylococci in a biofilm mode of growth. Hence, the current study was undertaken to establish the efficacy of this oil as an antimicrobial agent against clinical S. epidermidis isolates in planktonic and biofilm cultures. Chlorhexidine, triclosan, and gentamicin were used as positive controls in addition to examination of possible synergistic effects by combining cinnamon oil with any of these clinically used antimicrobials.
Sixteen clinical isolates of S. epidermidis (Table (Table1)1) were collected from Sardjito Hospital, Yogyakarta, Indonesia, and identified as reported previously in the Microbiology Department, Gadjah Mada University, Yogyakarta, Indonesia (32). Isolates were obtained from blood, cerebrospinal fluid, pus, and urine. S. epidermidis strains RP62A (ATCC 35984) and ATCC 12228 were included as ica-positive and ica-negative reference strains, respectively. All strains were cultured at 37°C in tryptic soy broth (TSB; Oxoid) with or without agar.
Clinical isolates of S. epidermidis were screened for the presence of icaA by PCR using the primers listed in Table Table22 (19, 32, 40). Briefly, S. epidermidis strains were grown overnight at 37°C on a TSB agar plate. A colony of each isolate was taken and resuspended in 20 μl sterile demineralized water (dH2O). Samples were heated to 100°C for 5 min, and the bacterial debris and unlysed organisms were removed by centrifugation (21,000 × g for 10 min). Five microliters of the supernatants was used as template DNA in a PCR analysis using gyrB as a control for the presence of DNA.
Cinnamon stick (Cinnamomum burmannii), originally produced in Indonesia, was obtained from a local market in Tawangmangu in the center of Java, Indonesia, and was authenticated by botanical experts. Cinnamon oil was extracted by steam distillation to obtain a volatile oil (38). Stock solutions of 16% cinnamon oil in 5% propylene glycol (PG) and 128 mg/liter triclosan (Flochea) in 5% PG were made to enhance their solubility in suspension (42) and used the following dilution. Equal amounts (final concentrations) of PG were included in cultures in order to determine the effect of PG in the absence of cinnamon. Chlorhexidine (Sigma-Aldrich) and gentamicin (Sigma-Aldrich) were diluted with sterile dH2O to obtain fourfold stock solutions of 64 mg/liter and 128 mg/liter, respectively, as positive controls.
The MIC and minimal bactericidal concentrations (MBCs) for cinnamon oil, chlorhexidine, triclosan, and gentamicin of planktonic S. epidermidis cultures were determined in TSB using the broth microdilution method (1). Suspensions of S. epidermidis were prepared by resuspending one colony of an overnight culture from TSB agar in TSB broth. The bacterial density was adjusted to 1 × 108 bacteria/ml in 0.9% NaCl, using optical densities at 625 nm of 0.08 to 0.10 as a reference, corresponding to 0.5 McFarland units. The bacteria were further diluted with TSB to obtain inocula containing 1 × 106 bacteria/ml. Each well of a tissue culture polystyrene microtiter plate (water contact angle, 56 degrees; Falcon; Becton Dickinson Labware, Franklin Lakes, NJ) containing 100 μl of the antimicrobial agent at different concentrations or PG solution was inoculated with 100 μl of the bacterial suspension. Following a 24-h incubation at 37°C, the wells were visually inspected for growth. The MIC was defined as the lowest concentration that did not show growth. Controls containing antimicrobial agents in broth without bacterial inocula were included. Following the MIC assay, MBCs were determined by plating 10 μl of each of the clear wells onto TSB agar plates. The MBC was defined as the lowest concentration yielding no growth following incubation at 37°C for 24 h. Data from at least three biological replicates were evaluated, and averages were calculated.
To determine possible synergistic effects of cinnamon oil in combination with chlorhexidine, triclosan, and gentamicin, a checkerboard assay was performed as described previously (3, 14, 34). Briefly, each well of a 96-well microtiter plate was filled with 100 μl of TSB, and 100 μl of cinnamon oil was added to the first row in a twofold-decreased concentration, while 100 μl of the other antimicrobial agent was added to the right column in decreasing concentrations. Thus, serial twofold dilutions of the antimicrobial compounds were made in each row/column (final concentrations were 2 to 0.01% [vol/vol] for cinnamon oil, 32 to 0.2 mg/liter for gentamicin and triclosan, and 8 to 0.05 mg/liter for chlorhexidine). The wells were then inoculated with 100 μl of the bacterial suspensions containing 1 × 106 bacteria/ml. Controls containing inocula in TSB alone and antimicrobial compounds without inoculum were included. The microtiter plates were incubated at 37°C for 24 h, and the MIC of the combination of both antimicrobial compounds was determined by visual inspection.
To determine the synergistic or antagonistic activity of antimicrobial combinations, the fractional inhibitory concentration (FIC) and FIC index (FICI) were determined as described by Odds (34). Briefly, the FIC of cinnamon oil plus another antimicrobial agent was calculated as the MIC of each agent when used in combination with the other agent divided by the MIC when used alone (FIC = MIC in combination/MIC alone). Accordingly, each antimicrobial combination produced two FIC values, which were summed to produce the FICI (FICI = FIC of cinnamon oil + FIC of another antimicrobial agent). Synergy was defined as a FICI value of ≤0.5, no interaction was defined as a FICI value of >0.5 to 4.0, and antagonism was defined as a FICI value of >4 (10, 34).
Biofilms were grown as described previously (7, 32). Briefly, wells of a 96-well tissue culture polystyrene microtiter plate (Falcon; Becton Dickinson Labware, Franklin Lakes, NJ) were filled with 100 μl of TSB containing cinnamon oil (twofold final concentrations) and subsequently inoculated with a 1:100 dilution of an overnight culture. Final concentrations of cinnamon oil were 2 to 0.01% (vol/vol). After incubation for 24 h at 37°C, the plates were gently washed twice with phosphate-buffered saline (PBS; 10 mM potassium phosphate, 0.15 M NaCl, pH 7.0) and stained with 1% (wt/vol) crystal violet solution for 30 min at room temperature in order to determine the biofilm mass. The excess stain was washed off with dH2O. Subsequently, the biofilms were resuspended in acid-isopropanol (5% [vol/vol] 1 M HCl in isopropanol) and, finally, the A575 was measured in a Fluostar Optima microplate reader (BMG Labtech).
Total RNA was isolated from 24-h biofilm cultures grown with and without 0.01% cinnamon oil in 12-well tissue culture polystyrene plates (water contact angle, 59 degrees; Costar, Corning, NY) as described previously (32). Briefly, after resuspending the biofilms by pipetting, bacteria were pelleted by centrifugation and frozen at −80°C. Samples were thawed slowly on ice and resuspended in 100 μl diethylpyrocarbonate-treated water, after which the bacterial suspension was frozen in liquid nitrogen. Frozen bacteria were ground using a mortar and pestle. Total mRNA was isolated using the Invisorb Spin Cell RNA mini kit (Invitek, Freiburg, Germany) according to the manufacturer's instructions. DNA was removed using the DNA-free kit from Ambion, and the absence of genomic DNA was verified by real-time PCR prior to reverse transcription. For all samples, 35 cycles of PCR using the gyrB primer set (Table (Table2)2) did not result in any detectable signal. One μg of total RNA was used for cDNA synthesis (IScript; Bio-Rad) according to the manufacturer's instructions. Real-time PCR was performed as described previously (32). Reaction mixtures were prepared in duplicate using the CAS-1200 pipetting robot (Corbett Life Science, Sydney, Australia). Normalized expression levels of icaA (see primers in Table Table2)2) were calculated using the threshold cycle method (2−ΔΔCT) (27) with untreated biofilms as controls and gyrB as the reference.
Biofilms were grown as described above but without cinnamon oil. After a 24-h incubation at 37°C, the biofilms were washed three times with sterile PBS, after which the biofilms were exposed to 200 μl of cinnamon oil, with oil concentrations ranging from 2 to 0.01% (vol/vol). The plates were incubated for 1, 3, and 24 h at 37°C, after which the cinnamon oil was removed by washing twice with 200 μl PBS. Bacterial viability was analyzed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT; Sigma) as described previously (5). Briefly, 100 μl prewarmed MTT solution (0.5 mg/ml) in PBS containing 0.1% glucose and 10 μl of 10 μM menadion was added to each well. The plates were incubated at 37°C for 30 min, and the MTT solution was removed. Bacteria were washed once with PBS and resuspended in acid isopropanol (5% [vol/vol] 1 M HCl in isopropanol). Finally, the absorbance was measured (A560). The biofilm MIC was defined as the concentration of cinnamon oil showing A560 values equal to or lower than those of the control, i.e., the biofilm-negative strain, S. epidermidis strain ATCC 12228.
Confocal laser-scanning microscopy (CLSM) was used to visualize biofilms treated with 2% cinnamon oil for 1, 3, and 24 h. Biofilms grown in 12-well tissue culture polystyrene plates were washed with PBS and stained with the bacterial live/dead stain BacLight (Molecular Probes, Leiden, The Netherlands) for 30 min in the dark. Excess stain was removed, and the biofilms were submerged in 2 ml PBS. CLSM images were collected using a Leica TCS SP2 CLSM with a ×40 water objective using 488 nm excitation and 500 to 523 nm (green, alive) and 622 to 722 nm (red, dead) emission filter settings.
The susceptibility of clinical isolates of S. epidermidis to cinnamon oil, chlorhexidine, triclosan, and gentamicin is summarized in Table Table1.1. All clinical isolates included were susceptible to cinnamon oil, with a planktonic MIC ranging from 0.5 to 1%, except for strain 1239 which showed a higher MIC of 2%. Ten out of 18 S. epidermidis strains used in this study showed resistance to triclosan (MIC higher than 32 mg/liter) (2, 16, 21, 41). Gentamicin resistance was observed in 13 out of 18 strains (MIC higher than 32 mg/liter) (24). The planktonic MIC of clinical S. epidermidis strains for chlorhexidine, one of the most widely used skin antiseptics, was 1 to 4 mg/liter, and no resistant strains were observed (25). The planktonic MBCs of cinnamon oil were twofold higher (P < 0.001) than their MICs for the same strain. Chlorhexidine demonstrated a fourfold-higher (P < 0.001) MBC than MIC on average, while triclosan showed no bactericidal activity in the concentration range evaluated here (MBC > 32 mg/liter), except for strain 724.
Synergy between cinnamon oil and each of the other three antimicrobials was evaluated for three icaA-positive clinical isolates (strains 46, 64, and 236) and the two reference strains. Strain 46 is resistant to both triclosan and gentamicin, while strains 64 and 236 are resistant only to triclosan. Strains RP62A and ATCC 12228 are resistant only to gentamicin. A combination of cinnamon oil with gentamicin showed synergy for strains 64 and 236 (Table (Table3),3), while a combination of cinnamon oil with chlorhexidine showed synergy for all strains tested. A combination of cinnamon oil with triclosan showed only synergy for the two reference strains, RP62A and ATCC 12228 (Table (Table33).
The effects of cinnamon oil on biofilm formation by the high-biofilm-producing strains (46 and 64), intermediate biofilm formers (236 and RP62A), and the negative control (ATCC 12228) were determined. The negative control did not form a biofilm in the presence or absence of cinnamon oil. The growth of S. epidermidis strains 46 and 64, a high-biofilm-producing strain, and RP62A could be inhibited by a cinnamon oil concentration of 0.5%, while the intermediate strain 236 required significantly (P < 0.01) less cinnamon oil to prevent biofilm growth (0.25%). Interestingly, for S. epidermidis strains RP62A and 236, there was a twofold (P < 0.05) induction of biofilm formation by cinnamon oil at a concentration of 0.01 to 0.05% (Fig. (Fig.11).
Since the effect of 0.01% cinnamon oil seemed to stimulate biofilm formation, it was analyzed in more detail by following icaA expression using real-time PCR. Relative to the unexposed control, icaA was overexpressed in all strains when exposed to 0.01% cinnamon oil (Fig. (Fig.2).2). Normalized fold expression of icaA was between two and four times that of strains 46, 64, and 236 when exposed to 0.01% cinnamon. Interestingly, strain RP62A exposed to 0.01% cinnamon demonstrated 37 times the overexpression of icaA compared to the untreated control and a 10-fold-stronger overexpression of icaA compared to the other strains.
The susceptibility of S. epidermidis biofilms to cinnamon oil was studied by exposing 24-h-old biofilms to cinnamon oil, ranging from 0.01 to 2% (vol/vol) for 1, 3, and 24 h. The two high-biofilm-producing strains had a biofilm MIC of 2% (strains 46 and 64), whereas the intermediate biofilm-producing strains (236 and RP 62A) showed a biofilm MIC at 1% after 24 h. Interestingly, treatment of the intermediate biofilm-producing strains with 1% cinnamon oil for 3 h and 24 h resulted in the complete loss of metabolic activity, while a 1-h exposure resulted in a residual metabolic activity of 14% relative to that of the untreated control (Fig. (Fig.3A).3A). For the high-biofilm-producing strains, 24-h exposure to 1% cinnamon oil did not result in the complete loss of metabolic activity but showed 20 to 30% residual metabolic activity (Fig. (Fig.3A).3A). Exposure to 2% cinnamon oil for 24 h resulted in the complete disappearance of metabolic activity (Fig. (Fig.3B3B).
The effect of cinnamon oil on preexisting biofilms was also studied by using CLSM. For the high-biofilm-producing strains, treatment with 2% cinnamon oil for 1 and 3 h reduced the number of bacteria present in the biofilm (compare Fig. 4B and C with A), but the remaining bacteria were viable (Fig. 4B and C). After treatment for 24 h, cinnamon oil efficiently removed the majority of the bacteria from the biofilm, and in addition, the remaining bacteria were dead (Fig. (Fig.4D).4D). For an intermediate biofilm-producing strain exposed to 2% cinnamon oil, biofilm detachment was observed after 1 h of treatment (Fig. 4E and F), and remaining bacteria were viable, while 3 h and 24 h of treatment resulted in both bacterial detachment and death (Fig. 4G and H, respectively).
Several investigations have studied the antimicrobial effects of cinnamon oil (6, 14, 31, 36, 39). However, there is very limited information about its effect on S. epidermidis, either in planktonic or biofilm cultures. In the present study, it is shown that cinnamon oil has antimicrobial activity against both planktonic and biofilm cultures of clinical S. epidermidis strains. This is in line with other reports showing that cinnamon oil had the most potent bactericidal properties compared to 20 other essential oils against different important pathogens (36, 39). Remarkably, many of the clinical S. epidermidis strains used in this study showed resistance to triclosan (10 out of 18 strains), an antimicrobial agent widely used in medical practice. In addition, resistance to gentamicin, the most commonly used antibiotic in bone cement with its wide antibacterial spectrum, was also common (13 out of 18 strains) (30). These large proportions of resistant strains are probably due to the Indonesian origin of the strains, since antibiotic use is relatively widespread there compared to, e.g., in The Netherlands (20). Strains showing resistance to gentamicin, triclosan, or both were still susceptible to cinnamon oil. Interestingly, the planktonic MIC and MBC to cinnamon oil were similar to the biofilm MIC and MBC of the same strain, ranging from 0.5 to 1% and 1 to 2% (vol/vol), respectively. This suggests that cinnamon oil has similar antimicrobial activity against planktonic bacteria and bacteria in biofilms. In addition, another advantage of the use of essentials oil over antibiotics may be that bacteria do not develop resistance to essential oils (29). The present study showed that cinnamon oil has synergistic activity with chlorhexidine, triclosan, and gentamicin except for strains that are resistant against triclosan or gentamicin. Importantly, no antagonism between cinnamon oil and any of the generally used disinfectants or antimicrobials included was observed. In combination with cinnamon oil, the amount of chlorhexidine, gentamicin, and triclosan required to achieve growth inhibition was reduced significantly (10-, 8-, and 50-fold, respectively) (data not shown). For chlorhexidine, this finding is in line with results of a previous study showing that in combination with cinnamon oil, a 10-fold-lower chlorhexidine concentration was needed for equivalent inhibition of biofilm cultures of S. mutans and L. plantarum (14). The synergistic activity between an essential oil and an antimicrobial agent may be due to their action on either different (17) or similar targets of bacterial cells, i.e., cell membranes (14, 25). This is supported by results presented here. Cinnamon oil and one of its main components, cinnamaldehyde, act on the plasma membranes, similarly to chlorhexidine (inhibition of the same target), while gentamicin inhibits protein synthesis and triclosan inhibits a specific metabolic pathway required for fatty acid synthesis in bacteria (inhibition of different targets) (14, 25). The synergistic activity of cinnamon oil with other antimicrobial agents could be beneficial in clinical settings, for example, to improve skin antisepsis and to eliminate antimicrobial-resistant S. epidermidis strains (25). Using combinations of relatively cheap cinnamon oil with relatively expensive antimicrobials can lower the cost of therapy significantly.
Our results clearly indicated that the expression of icaA is strongly enhanced by the presence of sub-MIC concentrations of cinnamon oil. To our knowledge, this is the first report that cinnamon oil could act as an inducer of biofilm formation in clinical S. epidermidis strains. Biofilm formation can be induced by conditions that are potentially toxic for bacterial cells, such as high levels of osmolarity, detergents, urea, ethanol, oxidative stress, and the presence of sub-MICs of some antibiotics (9, 26, 37).
Interestingly, CLSM imaging of cinnamon-treated biofilms shows not only that biofilm bacteria are effectively killed by cinnamon oil but that cinnamon oil is also able to detach biofilms. This shows that cinnamon oil has a dual mode of action against S. epidermidis biofilms; it is able to detach adhering bacteria from a substratum surface and it can kill bacteria. Furthermore, from the CLSM analysis it appears that detachment of biofilm bacteria is a more rapid process than the actual killing (compare the decrease in biomass with the absence of dead bacteria after a 1-h incubation with cinnamon oil in Fig. Fig.4).4). This also illustrates that the reduction in metabolic activity upon exposure of the biofilm to cinnamon oil, as observed here, is predominantly caused by the detachment rather than killing of biofilm bacteria.
In conclusion, this study demonstrated that cinnamon oil has excellent antibacterial activity, either alone or in combination with triclosan, gentamicin, or chlorhexidine, against clinical S. epidermidis isolates. This essential oil was able to inhibit biofilm formation, detach existing biofilms, and kill bacteria in biofilms of clinical S. epidermidis strains. Importantly, biofilms were equally as sensitive to cinnamon oil as their planktonic counterparts, probably due to the dual activity of cinnamon oil on existing biofilms. Further study is warranted to elucidate the complex mode of action of cinnamon and its components against biofilms of S. epidermidis and other clinically relevant microbes.
This study has been funded by the University Medical Center Groningen and the University of Groningen, Groningen, The Netherlands.
Published ahead of print on 11 September 2009.