Synergistic interaction and mode of action of Citrus hystrix essential oil against bacteria causing periodontal diseases

Abstract

Context: Citrus hystrix de Candolle (Rutaceae), an edible plant regularly used as a food ingredient, possesses antibacterial activity, but there is no current data on the activity against bacteria causing periodontal diseases.

Objective: C. hystrix essential oil from leaves and peel were investigated for antibiofilm formation and mode of action against bacteria causing periodontal diseases.

Materials and methods: In vitro antibacterial and antibiofilm formation activities were determined by broth microdilution and time kill assay. Mode of action of essential oil was observed by SEM and the active component was identified by bioautography and GC/MS.

Results and discussion: C. hystrix leaves oil exhibited antibacterial activity at the MICs of 1.06 mg/mL for P. gingivalis and S. mutans and 2.12 mg/mL for S. sanguinis. Leaf oil at 4.25 mg/mL showed antibiofilm formation activity with 99% inhibition. The lethal effects on P. gingivalis were observed within 2 and 4 h after treated with 4 × MIC and 2 × MIC, respectively. S. sanguinis and S. mutans were completely killed within 4 and 8 h after exposed to 4 × MIC and 2 × MIC of oil. MICs of tested strains showed 4 times reduction suggesting synergistic interaction of oil and chlorhexidine. Bacterial outer membrane was disrupted after treatment with leaves oil. Additionally, citronellal was identified as the major active compound of C. hystrix oil.

Conclusions: C. hystrix leaf oil could be used as a natural active compound or in combination with chlorhexidine in mouthwash preparations to prevent the growth of bacteria associated with periodontal diseases and biofilm formation.

• Biofilm
• citronellal
• kaffir lime
• Porphyromonas gingivalis
• Streptococcus mutans
• Streptococcus sanguinis

Introduction

Periodontal diseases are infectious diseases caused by more than 300 bacterial species in the oral cavity (Mayrand & Greniner, 1998). Bacterial species associated with development of periodontal diseases are members of the genera Porphyromonas, Bacteroides, Fusobacterium, Wolinella, Actinobacillus, Capnocytophaga, and Eikenella, while members of genera Actinomyces and Streptococcus may not be directly involved in the progression (Stanley & Thomas, 1991). The results from epidemiologic studies during the past decade indicated that untreated periodontal diseases could be a risk factor of low birth weight infants, diabetes, coronary heart diseases, and cerebrovascular accidents (Loesohe, 1999). Moreover, the incidences of bacterial resistance to antibiotics have been reported because of their imprudent use (Robert & Moellering, 1998). Many medicinal plants and their constituents have been extensively studied for their biological activities and applications, including raw and processed potential as natural agents for food preservatives, pharmaceuticals, and alternative medicines (Chomnawang et al., 2005; Cos et al., 2002; Panossian et al., 2010; Rocha et al., 2005; Rota et al., 2008). Thai edible plants have demonstrated various biological activities such as cancer prevention, antidiabetic, and antimicrobial activities (Eidi et al., 2006; Lantz et al., 2005; Manosroi, 2005; Sindhu et al., 2011; Siripongvutikorn et al., 2005; Wannissorn et al., 2005). In this study, Citrus hystrix de Candolle (Rutaceae) (kaffir lime), with a long history Thai folk medicine, was extensively studied. Kaffir lime fresh juice has been used for antiscurvy, expectorants, and antidandruff. Extracts from fruit peel showed physiological action as carminatives and stomachache alleviator (Saralamp et al., 1996). Ethyl acetate extract of C. hystrix fruit peel exhibited antibacterial activity against the growth of Staphylococcus aureus, Bacillus cereus, and Listeria monocytogenes (Chanthaphon et al., 2008). Essential oil of C. hystrix exhibited fungicidal activity against Aspergillus flavus, A. fumigatus, A. parasiticus, and Saccharomyces cerevisiae (Chanthaphon et al., 2008; Rammanee & Hongpattarakere, 2011; Thanaboripat et al., 2006). This study examined antibacterial activity of leaves and fruit peel of C. hystrix essential oils against three oral pathogens, Porphyromonas gingivalis, Streptococcus mutans, and Streptococcus sanguinis. Although several essential oils have been reported to have antibacterial activity against oral pathogens, there are no present data on the essential oil of C. hystrix (Alviano et al., 2005; Chaudhari et al., 2012; Hammer et al., 2003; Nalina & Rahim, 2006). Additionally, the active compound of the C. hystrix essential oil and the mode of action were also investigated.

Materials and methods

Microorganisms and culture condition

Streptococcus mutans ATCC 25175 and Streptococcus sanguinis ATCC 10556 were obtained from the Department of Medical Science, Ministry of Public Health, Thailand. These two strains of streptococci were cultured at 37 °C in 5% CO₂ on brain heart infusion agar (Difco Laboratories, Dickinson and Company, Franklin Lakes, NJ) supplemented with 5% v/v sheep blood. Porphyromonas gingivalis ATCC 33277 was purchased from the American Type Culture Collection which was grown anaerobically at 37 °C on brain heart infusion agar containing with 5% v/v sheep blood.

Plant essential oils and chemicals

Essential oils from C. hystrix leaves and peel were purchased from Thai-China Flavours and Fragrances Industry Company, Limited. Chlorhexidine and terpenes (linalool, pinene, and citronellal) were purchased from Sigma-Aldrich, Inc. (St. Louis, MO).

Antimicrobial susceptibility test of plant essential oils

Antimicrobial susceptibility test was determined by broth microdilution assay on 96-well polystyrene microtiter plates according to Chomnawang et al. (2005) with some modifications. Tween 80 at 2 and 3% ethanol were added into BHI broth to enhance oil solubility. Then, two-fold serial dilutions of each oil was performed in 96-well plates. Prepared inoculum was adjusted to an optical density to 0.2 at 600 nm (approximately 10⁷cfu/mL). An aliquot of tested microorganisms was added equally into each well. The plate was incubated under anaerobic conditions or 5% CO₂ atmosphere at 37 °C for 18 h and the minimum inhibition concentration (MIC) was observed by recording the lowest concentration which could inhibit the growth of tested bacteria. The minimal bactericidal concentration (MBC) was determined by transferring diluted broth from each well to a BHI agar plate. The MBC value was defined as the lowest concentration of substance that prevented the growth of cultures. Chlorhexidine was used as the standard compound (positive control).

Effect of plant essential oil on biofilm formation

The effect of C. hystrix leaves oil on biofilm formation of S. mutans was determined by the crystal violet staining method (Peeters et al., 2008). The culture and plant essential oil were prepared as previously described. Briefly, bacterial suspension in the exponential growth phase (optical density 0.2 at 600 nm) was incubated in 96-well polystyrene plates composed of 1% sucrose in brain heart infusion and various concentrations of C. hystrix leaf oil. After a designated time, the culture medium containing planktonic cells was removed, and wells were washed three times with 0.9% normal saline. The adherent biofilm was stained with 0.1% crystal violet. Then, samples were rinsed with distilled water twice and the dye bound to the biofilm was solubilized by adding 95% ethanol. The extracted dye was quantified by measuring the absorbance at 540 nm. The activity of antibiofilm formation was calculated and demonstrated in the term of percent reduction according to the equation as described below: B was defined as the average absorbance of blank, U was defined as the average absorbance of untreated cells (control), T was defined as the average absorbance of treated cells.

The biofilm removal property of C. hystrix leaves oil was also determined. Culture in the exponential phase was incubated under 5% CO₂ conditions at 37 °C for 8 h to allow biofilm formation. The biofilm was treated with various concentrations of essential oil until a designated time and the remaining biofilm was quantified by crystal violet staining method as described above.

Time-kill assay

Three oral pathogens in exponential growth phase were tested for time kill assay, which was performed according to the procedure described by Koo et al. (2002). Cultures were incubated with C. hystrix leaf oil at concentrations of 0.25 × MIC, 0.5 × MIC, 1 × MIC, 2 × MIC, and 4 × MIC. After designated times (0, 2, 4, 8, 12, 18, and 24 h), samples were removed for viable cell count on BHI agar. Killing curves were constructed by plotting the number of viable cells (cfu/mL) on a semi-log scale versus time over 24 h compared with the control.

Checkerboard microdilution assay

Synergistic effect between the plant essential oil and chlorhexidine was determined by checkerboard assays as previously described by Botelho (2000). The prepared inoculums of test strains were at exponential growth phase and the final concentration of test strains were approximately 10⁶cfu/mL. The mixed concentrations between essential oil and chlorhexidine were 1/16 × MIC to 4 × MIC. The fractional inhibitory concentration (FIC) index was derived from the lowest concentration of chlorhexidine and essential oil combination with no visible growth of the test organisms. FIC index was calculated using the formula:

Synergistic effect was defined as an FIC index of 0.5 or less and indifferent as a FIC index between >0.5 and 4.0. The antagonistic effect was defined as FIC index >4.0.

Scanning electron microscope assay

Scanning electron microscope (SEM) was used to study the effect of C. hystrix leaves oil at 4 times the MIC on P. gingivalis morphological changes. The method of SEM was modified from Kockro et al. (2000). The untreated and essential oil treated bacterial cells were fixed with 4% glutaraldehyde solution and 1% osmic acid solution. The fixed specimens were washed with 0.1 M buffer solution (pH 7.2). Furthermore, the samples were sequentially dehydrated for 10 min in cold ethanol series of 30, 50, 70, 90, and 95% and then twice for 20 min in absolute ethanol. After dehydration, the specimens were dried with CO₂. Finally, the effect of essential oil on morphological changes of bacterial cells was observed under scanning electron microscope.

Biological guided separation of active compound

The active components of C. hystrix leaf oil were classified by bioautographic assay. Two sets (A, B) of thin layer chromatography plates (TLC) were used in this assay. After developing with toluene and ethyl acetate (93:7), the plate (set A) was sprayed with 25% sulfuric acid and heated at 110 °C to visualize the chromatogram. Another set was subjected to the bioautographic assay according to Chomnawang et al. (2005). Developed TLC plates were carefully dried for complete solvent removal. The inoculum of each tested strains approximately 10⁶cfu/mL in the molten agar was distributed over the plate before incubation at 37 °C for 18 h. The active spots were identified by comparison with the first chromatogram. To isolate the active component, the active essential oil was separated by preparative TLC using the same condition as above. The active bands were scraped and eluted with dichloromethane, then the extract was analyzed by GC/MS-QP2010 gas chromatography mass spectrometer (Shimudsu) using a DB-5 ms bonded phase fused capillary column (30 m × 0.25 mm, film thickness 0.25 µm; J&W scientific, Folsom, CA). Helium was used as carrier gas at a constant flow rate of 0.67 mL/min. The oven temperature was initially 70 °C (isothermal, 2 min) and was increased to 300 °C at 4 °C/min. The injector temperature was 250 °C. Identification of components was made by Wiley & NIST 147 spectral library. After that, antimicrobial activity of active component was confirmed by broth microdilution method.

Results

Antibacterial and antibiofilm formation activity

Antibacterial activities of C. hystrix leaf and fruit peel oil to the growth of three oral pathogens were investigated. The results indicated that essential oil from C. hystrix leaves gave the highest efficacy against all three tested bacterial strains with MICs in the range of 1.06–2.12 mg/mL and MBCs in the range of 1.06–4.25 mg/mL (Table 1). The MICs and MBCs of chlorhexidine, the active ingredient of commercial mouthwashes, were found in the range of 0.001–0.004 mg/mL. However, essential oil of C. hystrix peel did not exhibit antibacterial activity. The effect of C. hystrix leaves oil on biofilm development which produced by S. mutans was investigated and the result indicated that the inhibitory effect on the development of the oral biofilm depended on the amount of essential oil. At the concentrations of 1.06 mg/mL and 2.12 mg/mL, the antibiofilm formation efficacy of oil were at about 92.77 and 98.77%, respectively (Figure 1). Moreover, more than 99% biofilm reduction was detected at the concentrations of 4.25 mg/mL of C. hystrix leaves oil while C. hystrix peel oil did not exhibit antibiofilm formation activity.

Figure 1. Effect of C. hystrix leaf oil on S. mutans biofilm formation. The antibiofilm formation efficacy of the oil were about 92.77% at MIC and 98.77% at MBC. Moreover, more than 99% biofilm reduction was detected at 4 × MIC and 8 × MIC of C. hystrix leaf oil.

Table 1. Antimicrobial activity of essential oils and major components.

	P. gingivalis		S. sanguinis		S. mutans
Plant essential oil	MIC (mg/mL)	MBC (mg/mL)	MIC (mg/mL)	MBC (mg/mL)	MIC (mg/mL)	MBC (mg/mL)
C. hystrix leaf oil	1.06	1.06	2.12	4.25	1.06	2.12
C. hystrix peel oil	>4.62	>4.62	>4.62	>4.62	>4.62	>4.62
citronellal	0.64	2.57	0.64	2.57	0.15	0.39
linalool	4.35	4.35	4.35	>4.35	2.17	2.17
pinene	>4.36	>4.36	>4.36	>4.36	>4.36	>4.36
chlorhexidine	0.002	0.004	0.001	0.002	0.001	0.004

Time-kill assay

In the present study, time kill assay was designed to determine the killing rate of C. hystrix leaf oil on tested bacteria. C. hystrix leaf oil was exposed to P. gingivalis, S. sanguinis, and S. mutans and the viable cells were determined at different incubation intervals (Figure 2). The results demonstrated that essential oil of C. hystrix leaves at the concentrations of 2 and 4 times of MIC rapidly reduced the growth of P. gingivalis within 2 h and 4 h, respectively. Moreover, essential oil at both concentrations had lethal effect on S. sanguinis and S. mutans within 4 h and 8 h, respectively. At the MICs, C. hystrix leaf oil could inhibit the growth of all three oral pathogens up to 24 h.

Figure 2. Time-kill plots of C. hystrix leaf oil against oral pathogens. At the concentrations of 2 × MIC and 4 × MIC, the essential oil of C. hystrix leaves significantly inhibit the growth of P. gingivalis (A) within 2 h and 4 h, respectively. The essential oil at both concentrations had lethal effect on S. sanguinis (B) and S. mutans (C) within 4 h and 8 h, respectively.

Scanning electron microscope assay

The effect of C. hystrix leaf oil on P. gingivalis was further determined by SEM. The result showed that after exposed to C. hystrix leaf oil at four times the MIC for 4 h, bacterial outer membrane was disrupted and showed abnormality in shape when compared with untreated cells (Figure 3). Moreover, the bacterial cell membrane was completely destroyed after treatment with four times of MIC for 8 h.

Figure 3. Effect of C. hystrix leaf oil on P. gingivalis cell membrane. After treated with C. hystrix leaf oil at 4 × MIC for 2 h (B), 4 h (C), and 8 h (D), bacterial outer membrane was disrupted and showed abnormality in shape when compared with untreated cells (A). Moreover, the bacterial cell membrane was completely destroyed after treated with four times of MIC for 8 h.

Checkerboard assay

Chlorhexidine has been widely used as an active ingredient in daily mouthwash products to prevent the growth of oral pathogens. However, long-term use of mouthwash products containing high concentration of chlorhexidine are not recommended due to its toxicity. For this reason, the checkerboard assay was chosen to determine the interaction between chlorhexidine and other active ingredients in order to decrease the amount of chlorhexidine in preparation. The result indicated that chlorhexidine showed synergistic interaction with C. hystrix leaf oil (Table 2). It was demonstrated that to combination of essential oil and chlorhexidine showed more sensitivity to all of tested strains than given alone. The combination MICs of chlorhexidine needed to inhibit the growth of all tested strains were reduced approximately 4 times from their MIC values. Moreover, the amount of C. hystrix leaf oil used for inhibiting growth of pathogenic bacteria was decreased to one-fourth of the single compound.

Table 2. Synergistic effect of chlorhexidine and C. hystrix leaves oil.

	Chlorhexidine (MIC mg/mL)			C. hystrix leaves oil (MIC mg/mL)
Test strain	Alone	Combined	FIClowest	Alone	Combined	FIClowest	FICI
P. gingivalis	0.002	0.0004	0.2	1.06	0.265	0.25	0.45
S. sanguinis	0.001	0.0002	0.2	2.12	0.531	0.25	0.45
S. mutans	0.001	0.0002	0.2	1.06	0.265	0.25	0.45

Biological guided separation of the active compound

The active component of C. hystrix leaf oil was determined by TLC bioautography assay and GC/MS technique. The TLC bioautography assay of C. hystrix leaves oil showed a clear spot (inhibition zone) at the same position with citronellal. The major component of essential oil was confirmed by GC/MS (Figure 4). As shown in Table 3, the components detected in the essential oil of C. hystrix leaves oil were β-citronellal (78.11%), citronellyl acetate (6.24%), β-citronellol (5.28%), 2,6-dimethyl-5-heptenal (3.10%), linalool (2.75%), isopulegol (2.08%), sabinene (1.3%), β-pinene (0.5%), (-)-caryophyllene oxide (0.37%), and d-nerolidol (0.27%). The results revealed that beta-citronellal was a major active compound of C. hystrix leaf oil. Additionally, terpene compounds were also evaluated for antibacterial activity. Citronellal exhibited the best inhibitory effect against the growth of P. gingivalis and S. sanguinis with MIC values of 0.64 mg/mL and at concentration of 0.15 mg/mL for S. mutans. The concentrations of citronellal which exhibited the bactericidal activity were 2.57 mg/mL for P. gingivalis and S. sanguinis and at 0.39 mg/mL for S. mutans.

Figure 4. GC/MS spectrum of C. hystrix leaves oil.

Table 3. Chemical composition of C. hystrix leaves oil.

Peak	Retention time (min)	Base peak (m/z)	Area %	Name
1	6.638	93	1.30	Sabinene
2	6.836	93	0.50	β-Pinene
3	9.659	82	3.10	2,6-Dimethyl-5-heptenal
4	12.067	71	2.75	Linalool
5	15.150	41	2.08	Isopulegol
6	15.444	41	78.11	β-Citronellal
7	22.186	69	5.28	β-Citronellol
8	41.513	81	6.24	Citronellyl acetate
9	64.719	69	0.27	d-Nerolidol
10	65.184	79	0.37	(−)-Caryophyllene oxide

Discussion

Nowadays, the development of daily healthcare products from natural sources is in the spotlight to reduce an amount of chemical composition in healthcare products and to lower the side effects of chemical ingredients after long term use. The oral care products, especially mouthwashes, normally consist of chlorhexidine as the major active ingredient. Although previous studies have been reported that mouthwashes containing 0.2% chlorhexidine could prevent the formation of dental plaque and gingivitis incidence, long term use at high concentration of chlorhexidine was able to promote extrinsic tooth stain, calculus formation, and taste aberrations (Eley, 1999; Flotra et al., 1972). For this reason, the present study was focused on searching for the active ingredients from Thai edible plants to be developed as the natural active compound to decrease the amount of chlorhexidine in mouthwash products. It was demonstrated that leaf oil from C. hystrix exhibited strong activity against cariogenic and periodontopathic bacteria with MIC and MBC in the range of 1.06–4.25 mg/mL. Previous studies on other essential oils such as cinnamon oil, manuka oil, tea tree oil, and eucalyptus oil have reported the ability to inhibit the growth of S. mutans which were considerably higher than the MICs of C. hystrix as determined in this study (Takarada et al., 2004; Hammer et al., 2003). Time kill study demonstrated that the complete lethal effect was observed at the four times of MIC after 2, 4, and 8 h for P. gingivalis, S. sanguinis, and S. mutans, respectively. The outer membrane of P. gingivalis was eliminated after exposure to C. hystrix leaf oil at a concentration of 4 × MIC within 4 h. The action of C. hystrix oil could be explained by its composition which was lipophilic, therefore, it could diffuse and penetrate the into bacterial outer membrane causing loss of membrane permeability and eventually cell death (Filoche et al., 2005; Helander et al., 1998). Synergistic interaction of C. hystrix leaf oil and chlorhexidine was first demonstrated in this study. An approximate four-fold reduction in the MICs of C. hystrix essential oil and chlorhexidine was observed. The action of chlorhexidine is by increasing the membrane permeability and precipitation of cytoplasmic molecules (Marrie & Costerton, 1981; Richards & Cavill, 1979). The synergistic activity of chlorhexidine and essential oil may be due to their actions on the same target at bacterial cell membrane.

Dental plaque is caused by biofilm formation of microorganisms on the tooth surface which exhibits several properties including reduced susceptibility to antibacterial agents (Loesohe, 1986). S. mutans was classified as the primary bacteria causing dental plaque so that prevention of biofilm formation by S. mutans could reduce the incidence of tooth decay and periodontal diseases (Coenye et al., 2007). The data demonstrated that essential oil from C. hystrix leaves at a concentration of 4.25 mg/mL was able to reduce the formation of bacterial biofilm higher than 99% when compared with untreated strain. However, we could not observe the biofilm removal property from C. hystrix leaf oil. From the result of this study, antibiofilm formation property of C. hystrix leaf oil could be possibly by inhibiting the growth of S. mutans. It was possible that the essential oil could not eliminate or remove bacteria because the biofilm acted as the barrier, so that the compound might not be able to completely permeate through the biofilm (Jefferson et al., 2005).

Conclusions

C. hystrix leaves oil demonstrated potential antimicrobial activity against growth of P. gingivalis, S. sanguinis, and S. mutans, which were a group of bacteria highly related with periodontal diseases. This oil might be developed as mouthwash products used as plaque protection agents. Further investigation with in vivo studies should be performed.

Declaration of interest

The authors have declared no conflict of interest. The authors alone are responsible for the content and writing of this article. This study was supported by the Coordinating Center for Research and Development to increase the value of Plants Indigenous to Thailand, Mahidol University and the Thailand Research Fund.

Acknowledgements

The authors wish to thank the staff in the Department of Microbiology and Miss Pattamapan Lomarat for their help and suggestion on this work.