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 AiM  Vol.11 No.2 , February 2021
In Vitro Efficacy of Clove Oil and Eugenol against Staphylococcus spp and Streptococcus mutans on Hydrophobicity, Hemolysin Production and Biofilms and their Synergy with Antibiotics
Abstract: The present study aimed to evaluate Syzygium aromaticum (clove) plant extract, clove oil and eugenol for their antibacterial activity and their potential to eradicate bacterial biofilms alone and in combination with antibiotics. Anti-bacterial efficacy of S. aromaticum extract, clove oil and eugenol was evaluated as minimum inhibitory concentration (MIC) and subsequently sub-MICs was selected for inhibition of virulence factors against test bacterial strains. Biofilm cultivation and eradication was assayed using XTT reduction in 96-well microtiter plate. Checkerboard method was used to study the interaction between essential oils and antibiotics. Staphylococcus aureus MTCC3160, Staphylococcus epidermidis MTCC435, Staphylococcus sciuri (SC-01), Staphylococcus auricularis (SU-01) and Streptococcus mutans MTCC497 were found strong biofilm former among all the test bacterial strains. The potency of test agents was found in the order of eugenol > clove oil > S. aromaticum methanolic extract. Sub-MIC (0.5 × MIC) of clove oil and eugenol showed a significant reduction in cell surface hydrophobicity (p < 0.05) and hemolysin production in the test bacterial strains. Eugenol showed no increase in sessile MIC (SMIC) against S. auricularis (SU-01), S. epidermidis MTCC435 and S. mutans MTCC497 compared to planktonic MIC (PMIC). Antibiotics (vancomycin and azithromycin) exhibited upto 1000-folds increased in SMIC compared to PMIC against all the test bacterial strains. Synergy was observed between eugenol and antibiotics (vancomycin/azithromycin) against all the test bacterial strains in both planktonic and sessile mode. Highest synergy was exhibited between eugenol and azithromycin in planktonic mode (FICI value 0.141). Further, microscopy also confirmed the spectacular effect of combination treatment on pre-formed S. aureus MTCC3160 and S. mutans MTCC497 biofilms. These findings highlighted the promising role of clove oil and eugenol alone and in combination on pathogenic bacterial biofilms.

1. Introduction

Infectious diseases are still a major global cause of mortality and morbidity in humans mainly in developing countries. Many opportunistic and drug resistant pathogenic bacterial species that are widely studied include Staphylococcus and Streptococcus species [1]. The tendency of these bacteria to cause infections is often related to the expression of several virulence factors, such as cell surface hydrophobicity, hemolysin production and biofilm formation. These factors aid bacteria to invade the host tissue and escape immune defence system [2]. Generally, Staphylococcus aureus is frequently responsible to cause bacteremia, pneumonia, deep seated and device related infections. S. aureus bacteremia is responsible for significant burden on health care system and accounts about 20 to 30% mortality and morbidity rates [3]. Certainly, the importance of coagulase negative Staphylococcus species (Staphylococcus epidermidis, Staphylococcus auricularis and Staphylococcus sciuri) has also increased over the past decades. S. epidermidis is opportunistic pathogen as it causes infections in immune-compromised patients. This pathogen is liable to cause infection in healthy person only when it penetrates into the host tissue [4]. The clinical significance of other coagulase negative Staphylococci (S. auricularis and S. sciuri) has been increasing since it can colonize and form biofilms on the host tissues as well as on the medical devices [5] [6] [7]. S. auricularis are inhabitants of human skin and mucous membranes. This bacterium has also been linked with various types of infections in immmuno-compromised patients and catheter related blood streams infections, osteomyelitis, bone and joint infections, prosthetic valve endocarditis and wound infections [8]. S. sciuri has also been isolated from various infection sites in humans, such as wound infections, soft tissue infections, abscesses, boils, periodontitis and endocarditis [5]. The involvement of Streptococcus mutans in oral biofilm associated infections is well known. As long as these bacterial species adhere and form multilayered biofilms on host tissues and different surfaces, it persists and prolongs the infections [9]. Several studies have revealed that the microorganisms in biofilm mode of growth are more tolerant to antibiotics than the planktonic mode [10]. Once biofilm gets established, it becomes difficult to eradicate through common antimicrobial drug therapy. Therefore, new antimicrobials or strategies effective against pathogenic bacterial biofilms are of practical significance in chemotherapy. There are several strategies that can be used to combat bacterial biofilm infections depending upon nature and site of infection. However, use of antipathogenic and antibiofilm agents which act through modulation of quorum sensing, inhibition of biofilm formation, biofilm degrading enzymes, application of specific bacteriophages, broad spectrum activity of natural products, and nanoparticles are considered as promising strategies [11]. To achieve broad spectrum and enhanced efficacy, combinational therapy may be more effective promising approach to combat pathogenic bacterial biofilm infections. Several advantages may be expected through this approach, such as less chance of emergence of resistance, improved efficacy of single drug in combination and dose related toxicity [12] [13].

Natural products of plant origin such as plant extracts, essential oils and phytochemicals provide infinite opportunities for the discovery of new drugs due to the abundance of chemical diversity [14] [15]. The crude extracts of different parts of the medicinal plants such as root, stem, flower, fruits and twigs have been commonly used for the treatment of microbial infections in traditional system of medicine and modern phytomedicine. Syzygium aromaticum extracts and its active compounds are well known for antimicrobial properties against gram positive and negative bacteria [16]. Antimicrobial activity of clove essential oils and eugenol is attributed by disturbing the cell wall and membrane, cell lysis, leakage of cellular contents and inhibition of proton motive force [17] [18] [19]. It is reported that they kill bacteria without promoting the development of resistance [17] [20]. However little is known about anti-infective and antibiofilm activity of S. aromaticum and its active compound against pathogenic biofilms and synergy with antibacterial drugs. Therefore, the present study investigated antibiofilm activity of clove essential oil and eugenol alone and in combination with the commonly used antibiotics against the selected strong biofilm forming bacterial strains including S. aureus, S. epidermidis, S. auricularis, S. sciuri and S. mutans under in vitro settings.

2. Material and Methods

2.1. Bacterial Strains Used Under Study

Nine clinical strains of Staphylococcus species including S. aureus (SAJ-01 to 04), S. epidermidis (SEJ-01 to 04) and S. auricularis (SU-01) were isolated and or kindly provided by the division of Microbiology, Jawaharlal Nehru Medical College and Hospital, Aligarh Muslim University, Aligarh. S. sciuri (SC-01) was isolated from the skin of healthy person in our laboratory by swab culture on nutrient agar (NA) plate. Reference strains of Staphylococcus species viz S. aureus MTCC3160, S. epidermidis MTCC435 and S. mutans MTCC497 were procured from Microbial Type Culture Collection, CSIR, IMTECH, Chandigarh, India. The details of above strains are shown in TableS1. All the test bacterial strains were identified and characterized by morphological and biochemical tests like gram staining, mannitol salt agar, coagulase test, novobiocin test and nitrate reduction test. The test bacterial strains were maintained on nutrient agar (NA) slants at 4˚C.

Characterization of bacterial isolates based on 16S rRNA gene sequence analysis:

The partial gene sequencing of 16S rRNA of the S. sciuri (SC-01) and S. auricularis (SU-01) strains was done commercially at the Macrogen, Inc., Seoul, Korea. The amplified and purified PCR products were sequenced by using 2 primers 785F 5' (GGA TTA GAT ACC CTG GTA) 3' and 907R 5' (CCG TCA ATT CMT TTR AGT TT) 3'. Sequencing was performed by using Big Dye terminator cycle sequencing kit v.3.1 (Applied BioSystems, USA). Sequencing products were resolved on an Applied Biosystems model 3730XL automated DNA sequencing system (Applied BioSystems, USA). These sequences were submitted to Gen-Bank sequence database after verification and editing by using Bioedit 7.2.0 software. Next, phylogenetic tree was constructed by using software programme (Neighbor-joining method of Molecular Evolutionary Genetics Analysis (MEGAv6.0)). The evolutionary distance was calculated by using Maximum Composite Likelihood program [21] [22].

2.2. Test Agents Used: S. aromaticum Extract, Essential Oil, Eugenol and Antibiotic

A 100 g of air-dried S. aromaticum (bud) was collected from local authorized shop and identified by the Department of Botany, Aligarh Muslim University, Aligarh and deposited in the Department of Agricultural Microbiology with No. SA-C/2016/Agric 02. Sample was powdered and soaked in 500 ml of 97% methanol for 5 days with intermittent shaking and filtered out through Whatman filter paper No 1 (Whatman Ltd., UK). Further, clear filtrate was obtained by centrifugation 10,000 rpm for 15 min. The filtrate was concentrated in rotary evaporator at 40˚C and stored at 4˚C until use. The dried extract was further dissolved in 1% DMSO to obtain the required concentration of extract. S. aromaticum essential oil (Clove oil) and pure compound (eugenol 99% purity) were purchased from Dabur India Ltd, Delhi, India and Hi-Media Laboratory, Mumbai, India respectively. Essential oil and its active compound (eugenol) were diluted ten times in 1% DMSO. Aqueous stock solution of vancomycin (Cipla, Mumbai, India) and azithromycin (Cipla, Mumbai, India) were prepared at a concentration of 25 mg/ml.

2.3. Gas Chromatography and High Resolution Gas Chromatography-Mass Spectrometry (GC-MS) Analysis of Plant Extract

The composition of S. aromaticum methanolic extract was identified by GC-MS analysis. GC-MS analysis was conducted on Shimadzu QP-2010 Plus with Thermal Desorption System TD 20 and separation was obtained in column of 30 m × 0.22 mm × 0.25 µm (Thermo Scientific) at Jawaharlal Nehru University, New Delhi, India. Helium gas was used as a carrier gas while the flow rate of mobile phase was adjusted at 1.21 ml/min. The sample was infused into the column with a split ratio of 1:10. The linear temperature was attained from 60˚C to 300˚C. Hold time was set at 60˚C for 2 min. The peaks of the sample compounds were identified by using the above system with the reference database NIST libraries.

2.4. Determination of Planktonic Minimum Inhibitory Concentration (PMIC) of S. aromaticum Extract, Essential Oil, Eugenol and Antibiotics

The MIC of test agents (S. aromaticum plant extract, essential oil, eugenol and vancomycin) for bacterial strains was determined by the broth microdilution technique followed by [23] with some modification. Briefly, overnight grown culture prepared in Brain Heart Infusion Broth (BHIB). 0.1 ml of two-fold serial dilutions of test agents (2 × final concentration) was made in respective medium in the wells of microtiter plates. 0.1 ml of inoculum medium (1 × 106 CFU/ml) was added to each well and incubated at 37˚C for 48 h. Agent free wells were included as a control. Lowest concentration of test extract/essential oil/eugenol/antibiotic inhibiting the visible growth of the bacteria is defined as MIC. The experiment was repeated at least two times with three replicates.

2.5. Biofilm Forming Ability of the Test Bacterial Strains

Standard method of XTT reduction assay using 96-well microtiter plates was adopted as described earlier [24]. Briefly, the strains of Staphylococcus and S. mutans were grown in NA (glucose 0.25% w/v) and BHIB (sucrose 5% w/v) at 37˚C for 24 h respectively. 0.1 ml of the standardized cell suspension (approx. 1 × 108 of CFU/ml) and equal volume of respective medium was added into the wells of microtiter plates. The plates were incubated at 37˚C for 48 h. Thereafter, non-adherent cells were washed off with sterile PBS. XTT was added in each well to observe oxidation. XTT was used to assay biofilm formation by taking the absorbance at 490 nm using Lab system Multiskan Ex MTP reader. The experiment was repeated at least two times with three replicates. The mean absorbance value was calculated to measure the biofilms quantitatively.

2.6. Assay for Cell Surface Hydrophobicity

The microbial adhesion to hydrocarbons assay was performed to determine the cell surface hydrophobicity (CSH) using the method as adopted by [25] with little modifications. In brief, the bacterial strains were grown in 10 ml of BHIB (pH 7.0) to a shaking incubator at 37˚C for 18 h. The bacterial cells were obtained by centrifugation at 13,000 rpm for 15 min. The pellet were washed three times in PBS and finally suspended in PBS to attain an OD600 of 0.5. Two ml of bacterial suspension was mixed with 0.4 ml of xylene and shaken vigorously for 1 min. The absorbance of aqueous phase was determined at 600 nm using UV-visible spectrophotometer (LMSP-V325, India) and the percentage of adherence was calculated as: %CSH = {(1 − A)/Ao) × 100}. Where A and Ao were the absorbance values of aqueous phase at 600 nm before and after exposure to the organic phase.

2.7. Hemolysin Production

Quantitative hemolysin production was determined by using the modified method of [26]. Bacterial strains were grown in 10 ml TSB for 48 h at 37˚C on shaking incubator (200 rpm) until reaching the optical density of 2.5 at 600 nm. The cell supernatant was obtained by centrifugation at 10,000 rpm for 10 min. 0.1 ml of culture supernatant was mixed with equal volume of 2.5% defibrinated sheep blood in PBS buffer (pH 7.0) and incubated at 37˚C for 15 min. The sheep blood was collected from local slaughter shop. The blood cells (unlysed) were peletted by centrifugation at 5500 rpm for 1 min to obtain supernatant. The absorbance of supernatants was read at 543 nm. RBCs treated with Triton X-100 and saline water were served as a positive (100% lysis) and negative control respectively. Percentage of hemolysis was calculated as: Absorbance of the sample-Absorbance of the negative control/Absorbance of the positive control-Absorbance of the negative control.

2.8. Inhibition of Cell Surface Hydrophobicity in the Bacterial Strains

The inhibition of microbial adhesion was determined by using the method as adopted by [25] with little modifications as described previously. Briefly, the cultures of bacterial strains were grown at 0.25 and 0.5 × MIC of essential oils, compounds and drugs. The bacterial strains without any treatment considered as untreated control. Percent reduction in cell surface hydrophobicity of treated strains was calculated over untreated strains. The experiment was repeated at least two times with three replicates and data was recorded as mean values for calculation of percent CSH.

2.9. Inhibition of Hemolysin Production in the Bacterial Strains

The reduction in hemolysin production in the presence of test agents were determined by using the modified method of [26] as described earlier. The bacterial cells were grown in the presence of essential oil, compounds and drugs at sub-MICs (0.25 and 0.5 × MIC) for 48 h at 37˚C on shaking incubator (200 rpm) until reaching the optical density of 2.5 at 600 nm. The absorbance of supernatants was read at 543 nm. The untreated culture supernatant considered as the 100% hemolysis control, and relative percentage of hemolysis was calculated by comparison to untreated control.

2.10. Determination of Eradication of Biofilms by Clove Oil, Eugenol and Antimicrobial Drugs

The sessile MIC (SMIC) of antimicrobial agents for bacterial strains was determined to assess the biofilm eradication by the broth microdilution technique followed by [27]. Briefly, biofilms were allowed to form in microtiter plates as described in previous section. The pre-formed biofilm cells were treated with different concentrations of test agents (clove oil, eugenol, vancomycin (for Staphylococcus species) and azithromycin (for S. mutans)) prepared in BHIB medium and incubated at 37˚C for 48 h. Agent free wells were included as control. Sessile minimum inhibitory concentration (SMIC) was calculated as the reduction in the mean absorbance of the test clove oil/eugenol treated biofilm to the untreated control and expressed as the concentration eradicating 80% of pre-formed biofilms.

2.11. Assay for Synergistic Interaction between Clove Oil, Eugenol and Antibiotics in Planktonic Mode of Bacterial Growth

The synergy between test agents was determined against the test strains of bacterial species using previously described method [28]. In brief, 0.05 ml of prepared dilution of essential oil was added to the 96-well microtiter plates in the vertical direction and same amount of antibacterial drug was added in horizontal direction to obtain the various test combinations. Further, 0.1 ml of inoculum suspension (1 × 106 CFU/ml) of bacterial species was added to each well followed by incubation at 37˚C for 48 h. The interaction was determined as fractional inhibitory concentrations (FICI). The FICI result was interpreted as synergistic: FICI ≤ 0.5, no interaction > 0.5 - 4.0, and antagonistic > 4.0.

2.12. Assay for Synergistic Interaction between Clove Oil, Eugenol and Antimicrobial Drugs in Sessile Mode of Bacterial Growth

The synergistic effect of essential oils and antibiotics against the bacterial strains was determined by using the checkerboard microtiter assay. Briefly, bacterial biofilms were cultivated in 96-well plates. A two-fold dilution of essential oils and antibiotics were prepared in BHIB. 0.05 ml prepared dilution of essential oils in vertical direction and 0.05 ml of antibiotics (vancomycin for Staphylococcus species and azithromycin for S. mutans) in horizontal direction was added in the wells. The microtiter plates were observed after 48 h of incubation at 37˚C. The type of interaction was determined by calculating the FICI value as described in the above section.

2.13. Scanning Electron Microscopy (SEM)

The pre-formed bacterial biofilms treatment with combinations of eugenol and antibiotics (vancomycin and azithromycin) were prepared on glass coverslips using the method as described by [24] and processed for scanning electron microscopy (JSM 6510, LV, JEOL, JAPAN). Briefly, treated biofilm cells were washed with PBS and fixed with 5% glutaraldehyde in cocodylate buffer in a graded concentration of ethanol (25%, 50%, 75%, 95% and 100%), immersed in hexamethyldisilazane. The glass disc were dried and mounted on aluminium stubs with silver paint, sputter coated with gold and observed under scanning electron microscope.

2.14. Statistical Analysis

The data was analyzed statistically by one way ANOVA using Duncan’s method (IBM SPSS statistics, version 20). The significance was determined using P value less 0.05.

3. Results

3.1. Characteristics of Bacterial Strains and Plant Extract

The test bacterial strains previously obtained and characterized as indicated in the supplementary data (TableS1, TableS2 and FigureS1 and FigureS2). Staphylococcus isolates SC-01 and SU-01 were characterized in this study by 16S rRNA gene sequence analysis. The accession numbers of Staphylococcus scuiri (SC-01) and S. auricularis (SU-01) are KY027071 and KX953265 respectively. The data showed 99% sequence homology of SC-01 with S. sciuri DSM20345 and SU-01 with S. auricularis ATCC33753 as indicated in supplementary file (FigureS1 and FigureS2). Further, the biofilm forming ability of these bacterial strains was determined on the basis of optical density by XTT reduction assay (Table1). Biofilm forming ability of the test strains varied from moderate to strong. S. aureus MTCC3160, S. epidermidis MTCC435, SC-01, SU-01, SAJ-01 and S. mutans MTCC497 formed strongest biofilms (OD490 ≥ 1). Among 13 bacterial strains, hemolysin production was detected in eight strains. Similarly, ten (10) strains demonstrated the varying level of cell surface hydrophobicity (CSH). Significant amount of hemolysin production and CSH were noticed in the strains of S. epidermidis MTCC435 and S. sciuri (SC-01) respectively.

S. aromaticum methanolic crude extract was subjected to GC-MS analysis. It is observed that crude extract of S. aromaticum showed the presence of 12 phytocompounds where eugenol, eugenolacetate and caryophyllene were more

Table 1. Production of biofilms and virulence factors in the bacterial strains.

ND: Not detected.SA: S. aureus; SE: S. epidermidis.

predominant. Other detected compounds include eugenol acetate, caryophyllene, gamma.-Sitosterol, olean-12-en-28-oic acid, 2.beta., 3.beta., 23-trihydroxy, caryophyllene oxide, n. Hexadecanoic acid, alpha-Farnesene, benzyl benzoate, vitamin E, octyl cyclodecane and methyl stearate (Table 2 and Figure 1).

3.2. Effect of Test Agents on Bacterial Growth and Virulence Factors

Methanolic extract of clove bud, clove oil and eugenol were assessed to observe the antibacterial potency in term of MIC against the test bacterial strains. The results are presented in Table 3. MIC was ranged from 25 to >6400 µg/ml. MIC

Table 2. List of compounds of S. aromaticum methanolic extract identified by GC-MS analysis.

Table 3. Minimum inhibitory concentration (MIC) of S. aromaticum extract, clove oil and eugenol against bacterial species.

Figure 1. Chromatogram of S. aromaticum methanolic extract.

data clearly indicated that eugenol as the most effective in antibacterial potency compared to methanolic clove extract and oil. The clove oil and eugenol were further evaluated for their effects on bacterial virulence factors at sub-MIC values. Cell surface hydrophobicity of test bacterial strains in the presence of sub-MICs of eugenol and clove oil is shown in Table 4. Both clove oil and eugenol exhibited anti hydrophobicity potential being higher for eugenol. Eugenol showed similar inhibitory activity on CSH against SC-01 and SU-01. On the other hand, eugenol significantly reduced CSH in S. mutans MTCC497 from 68.06 to 8.28% (p < 0.05). Antibiotic (control) was found least effective against the test bacterial strains. Similarly, the reduction in hemolysin production was also recorded at sub-MICs of eugenol and clove oil in the test bacterial strains. Significant reduction in hemolysin production was exhibited in the presence of eugenol in S. sciuri SC-01 (78%) and S. epidermidis MTCC435 (69%) (Table 5).

Table 4. Effect of sub-inhibitory concentration of clove oil, eugenol and antibacterial drug on cell surface hydrophobicity of the test bacterial strains.

Where a, b, c letters indicate meanvalues which are significantly different according to the Duncan’s multiple range tests (p ≤ 0.05). Antibiotics: Vancomycin used for Staphylococcus species and azithromycin for S. mutans.

Table 5. Effect of sub-inhibitory concentrations of clove oil, eugenol and antibacterial drug on production of hemolysin in the test bacterial strains.

Antibiotics: Vancomycin used for Staphylococcus species and azithromycin for S. mutans.

3.3. Biofilm Eradication by Test Agents

The concentration of test agents eradicating more than 80% of pre-formed biofilms was considered as SMIC. Clove oil and eugenol showed no increase or only 2-folds increase in SMIC compared to PMIC. No increase in SMIC of eugenol compared to PMIC was observed against SU-01, S. epidermidis MTCC435 and S. mutans MTCC497. Similarly, SMIC of clove oil recorded equivalent to PMIC against SU-01. However, antibacterial drug (vancomycin and azithromycin) exhibited 4 to 1000-folds increased in SMIC compared to PMIC against all the test bacterial strains (Table 6).

3.4. Synergistic Interaction between Clove Oil/Eugenol and Antibiotics

Synergy between clove oil/eugenol and antibiotics were tested against the bacterial strains both in planktonic and sessile mode of growth. Various test combinations of essential oils and antibiotics (vancomycin/azithromycin) were tested against the bacterial strains in planktonic mode (Table 7 and Table 8). The combination of clove oil and vancomycin showed indifferent interaction (FICI

Table 6. PMIC and SMIC of essential oils and drugs against bacterial strains.

Antibiotics: Vancomycin used for Staphylococcus species and azithromycin for S. mutans MTCC497.

Table 7. In vitro interaction of essential oils with antibiotics against Staphylococcus strains in planktonic and sessile mode of growth.

Where A: Alone, C: Combination, IT: Type of interaction, Nature of interaction, I: Indifferent, S: Synergy, FICI: Fractional Inhibitory Concentration Index, CLV: Clove, EUG: Eugenol, VAN: vancomycin.

Table 8. In vitro interaction of essential oils with azithromycin against S. mutans in planktonic and sessile mode.

Where A: Alone, C: Combination, IT: Type of interaction; Nature of interaction, I: Indifferent, S: Synergy, FICI: Fractional Inhibitory Concentration Index, CLV: Clove, EUG: Eugenol, AZI: Azithromycin.

values 0.75) against S. aureus MTCC3160 strains. In contrast, other test staphylococcal strains (S. epidermidis MTCC435, SU-01 and SC-01) showed synergy between clove oil and vancomycin with FICI value of 0.312 to 0.5. However, synergistic interaction was exhibited between eugenol and vancomycin against all the test staphylococcal strain in planktonic mode and FICI value was found in the range of 0.25 to 0.375. Eugenol showed maximum synergy with azithromycin against S. mutans MTCC497 (FICI = 0.141) in planktonic mode (Table 8).

Similarly, interaction studies on eugenol with antibacterial drug (vancomycin/azithromycin) against pre-formed bacterial biofilms are depicted in Table 7 and Table 8. There was varied extent of synergy between eugenol and vancomycin with FICI index ranged from 0.25 to 0.375 against Staphylococcus pre-formed biofilms. No test combinations of oils and drugs exhibited antagonistic effect. Eugenol also showed synergy with azithromycin against S. mutans MTCC497 pre-formed biofilms [29].

Furthermore, SEM images indicated the prominent effect of the combine treatment of eugenol and antibiotics on pre-formed bacterial biofilms compared to the untreated control (Figure 2).

4. Discussions

Plants and plants products have significant antimicrobial properties against variety of pathogens. Problems such as microbial resistance to currently used antibiotics and lack of development of new antibiotics created an increased interest in the anti-infective herbal medicines. Natural products comprising of plant extracts and phytochemicals have been used to treat various infectious diseases since several decades [30].

The pathogenic bacteria included in the present study were characterized and identified based on cultural, morphological and biochemical characteristics. Among the 13 bacterial strains, 10 clinical and 3 reference strains were identified and characterized by the biochemical, cultural and morphological basis. The strains included in this study were S. aureus, S. epidermidis, S. sciuri, S. auricularis and S. mutans. S. sciuri (SC-01) and S. auricularis (SU-01) were identified by 16S rRNA gene sequence analysis as they were showing varied biochemical tests. Production of common virulence attributes such as cell surface hydrophobicity and hemolysin production were studied in the test bacterial strains. These

Figure 2. SEM images of pre-formed bacterial biofilm cells treated with combination of eugenol and antibiotics. (A) Untreated S. aureus MTCC3160 cells; (B, B1) Eradication of S. aureus MTCC3160 biofilm cells by the treatment of combination of eugenol (12.5 µg/ml) and vancomycin (256 µg/ml); (C) Untreated S. mutans MTCC497 biofilm cells; (D, D1) Eradication of S. mutans MTCC497 biofilm cells by the treatment of combination of eugenol (12.5 µg/ml) and azithromycin (64 µg/ml).

attribute aids in the disruption of the host tissues and also inactivate host antimicrobial mechanisms [31] [32]. Bacterial hemolysin mediate damage to the RBC of the host to meet and satisfy the iron requirement which may further enhance the virulent behavior in staphylococcal and S. mutans strains [33] [34]. Hemolysis is an important virulence factor which causes penetration and lysis of the host cells by the bacterial pathogens [34]. The bacterial strains included in this study such as S. aureus, S. auricularis (SU-01), S. epidermidis, S. sciuri (SC-01) and S. mutans produced above studied virulence factors. Production of cell surface hydrophobicity and hemolysin were detected in Staphylococci and Streptococci strains. These observations on Staphylococci are similar to the reports of other workers [32] [35] [36]. Similarly, production of various virulence factors in S. mutans is also reported by many workers [37] [38]. We were concerned with the strains that are pathogenic and exhibit significant virulence traits. Indeed, virulence traits are directly linked with adherence and biofilm development. Interestingly, association of Staphylococcus species such as S. sciuri and S. auricularis from the skin surface were reported in this study which are potentially pathogenic in nature. The pathogenecity of S. sciuri and S. auricularis were also documented by several other researchers [5] [6] [39].

Biofilm forming ability of the bacteria was evaluated under in vitro by XTT reduction assay in 96-well tissue culture plate. About 85% of the test strains developed strong biofilms which has clearly indicated that majority of clinical bacterial pathogens may involve biofilms mode of growth in the disease development in their respective host. Therefore, in vitro screening provides an initial idea on intrinsic ability of the strains to form biofilms. In the recent past, many workers have reported the biofilm development by S. aureus [40] [41], S. epidermidis [42] [43] and S. mutans [44] [45], S. sciuri [7] [46] and S. auricularis [6] [47].

In this study, S. aromaticum plant extract, clove essential oil and eugenol were screened for their efficacy against the bacterial pathogens. GC-MS analysis of S. aromaticum methanolic plant extract revealed that eugenol was the major compound. Other compounds such as caryophyllene, caryophyllene oxide, n. Hexadecanoic acid and alpha-Farneseneare also reported by other workers [48] [49]. The efficacy of test agents was determined in terms of minimum inhibitory concentration (MIC) so that a range of sub-MICs and above MICs values of active extract/essential oil/eugenol may be carefully selected for their anti-virulence and antibiofilm activities. The methanolic plant extract exhibited moderate to low activity against the test bacterial pathogens. Clove oil and eugenol showed significant antibacterial and antivirulence activity in the staphylococcoal and S. mutans strains. It is evident that pure compounds namely eugenol was more effective than essential oil against bacterial pathogens. Antimicrobial activity of S. aromaticum plant extract, clove oil and eugenol has been well documented [17] [50] [51] [52].

Since, targeting virulence factors and pathogenicity is considered as promising approach to combat localized and systemic infections [53]. Nowadays, new alternatives have been attempted to attenuate microbial virulence and pathogenicity rather than killing the pathogenic microorganisms. It is believed that targeting virulence and pathogenicity will not result in development of resistance to such agents compared to agents that are used to kill the pathogens [54]. Therefore, agents affecting the production of virulence factors could be used in controlling bacterial infections. In this study, the essential oil of S. aromaticum and eugenol showed significant anti-infective activity at sub-MICs. The antimicrobial activity of these oils was further evaluated for their ability to inhibit the production of CSH and hemolysin production in bacterial strains. It is reported that hydrophobic cells have the ability to adhere and escape phagocytosis and these attributes enhance the virulent behavior of these pathogens. Our data clearly showed that eugenol apparently suppress the CSH of test microbial strains. However, inhibition of CSH by the essential oils is not widely studied against the bacterial strains. A report on the inhibition of CSH at sub-MICs of geraniol against S. aureus strain has been documented [55]. This is the first report on the inhibition of CSH and hemolysin production in S. epidermidis, S. sciuri, S. auricularis and S. mutans strains by clove oil and eugenol.

Furthermore, damage to the host cells is mediated by the production of bacterial hemolysin which is an important virulence factor of these pathogens. Hemolysin aids in the release of iron from the hemoglobin which is used as a nutrient source during the bacterial infections. Our findings suggested the varying level of inhibition of hemolysin production in bacterial strains (S. aureus, S. epidermidis, S. sciuri, S. auricularis and S. mutans) at sub-MICs of the test essential oils (eugenol and S. aromaticum). Eugenol significantly reduced the hemolysin production (p < 0.05) at 0.5 and 0.25 × MIC in the test bacterial strains. Similarly, inhibition of hemolysin production in the presence of eugenol and thymol in S. aureus strains has also been reported [56]. Thus, anti-hemolysin activity of the test oils may further contribute to the anti-pathogenic action against the bacterial strains. However, anti-hemolysin activity of eugenol was not widely studied in other Staphylococcus species and S. mutans.

The essential oils are bactericidal at higher concentration, but at lower concentration (sub-MICs) suppression in virulence activity of the microbial pathogens is evident. We found that viability of bacterial strains at sub-MICs of the test oils were not significantly reduced compared to the untreated control (data not shown). This has clearly indicated the inhibition of the virulence factors without significantly affecting the growth of the microbial cells. However, further investigation is needed to know the mechanism of action of the essential oils in the virulence inhibition. In this study essential oil inhibited more than one virulence factors which may further aid in lowering the pathogenicity of the organisms without affecting the growth of the organisms. In this way, chances of development of resistance might be minimized by the treatment of the clove oil and eugenol.

In our study, clove oil and eugenol eradicated the Staphylococcus species and S. mutans biofilm cells. Sessile MIC of clove oil and eugenol was only increased 2-folds against the bacterial strains. Interestingly, no increase in SMIC of eugenol was observed against SU-01, S. epidermidis MTCC435 and S. mutans MTCC497. However, SMICs of antibiotics (vancomycin/azithromycin) increased upto 1000-folds against bacterial strains. The incidence of increased resistance to vancomycin against Staphylococcus species under biofilm mode of growth is also reported by other workers [57] [58] [59]. Similarly, the clinical strains of S. mutans under biofilm mode have also showed resistance to various antibiotics [60] [61]. Similarly, eradication of Staphylococcus pre-formed biofilms in the presence of essential oils of Cymbopogon flexuosus, Citrus paradisi, Thymus vulgaris, Cinnamomum verum, Rosmarinus officinalisis reported by other researchers [62] [63] [64]. [65] reported the eradication of S. aureus biofilm forming strains (S. aureus ATCC29213 and five isolated methicillin resistant strains) in the presence of thymol. [66] investigated the eradication of S. epidermidis biofilm by eucalyptus oil. Very few reports are available on the effect of essential oils on pre-formed S. epidermidis biofilms. So far no investigation is conducted on the eradication of pre-formed S. auricularis and S. sciuri biofilms by the essential oils.

Interestingly, essential oils/phytocompounds displayed enhanced bactericidal activity against the bacterial strains in biofilm mode compared to existing antimicrobial drugs. Thus, the cidal mode of action of test agents is quite effective in eradicating pre-formed biofilms. Thus, research on the selection of a new effective treatment in combating bacterial infections; necessitate a search for an agent with different mode of action. Moreover, the agents should not only possess direct antimicrobial activity but also should have synergistic activity with antimicrobial drugs, involve inhibition of expression of virulence factors such as production of cell surface hydrophobicity, hemolysin, adhesion, and aggregation that may further aid in combating microbial infections. As our test oils satisfy all the above characteristics, therefore an attempt has conducted to study the interaction of clove oil/eugenol with antibiotics in planktonic and sessile mode.

Combinational antimicrobial therapy is known for its therapeutic efficacy in controlling various infectious diseases including infection caused by the drug resistant pathogens [67]. It is expected that synergy between plant derived products/phytocompounds and antimicrobial drugs against bacterial pathogens could provide a new formulation for disease treatment [68]. The present study highlights the synergistic interaction between the essential oils with antibiotics against the test bacterial strains under planktonic and biofilm mode. The essential oils of S. aromaticum and eugenol have promising antibacterial activity alone against the test bacterial pathogens. Thus, above essential oils were investigated in combination with antimicrobial drugs against the test pathogens. Combination of essential oils (S. aromaticum and eugenol) and vancomycin revealed synergistic interaction against coagulase positive and coagulase negative Staphylococci. However, clove oil showed indifferent interaction with vancomycin against S. aureus MTCC3160. The MIC of vancomycin was reduced upto 8 and 16-folds against Staphylococcus species in planktonic and sessile mode. The combination of eugenol with vancomycin exhibited equal synergy against S. aureus MTCC3160, S. sciuri and S. auricularis in planktonic and sessile mode. Very few reports are available on interaction of essential oils with antibiotics against S. aureus in planktonic and sessile mode. However, this may be the first report on the interaction of clove oil/eugenol with vancomycin against Staphylococcus species such as S. epidermidis, S. sciuri and S. auricularis in planktonic and sessile mode of growth. The interaction between eugenol and vancomycin was reported earlier against S. aureus MTCC3160 pre-formed biofilms [69]. Vancomycin alters the cell membrane permeability, inhibits the cell wall biosynthesis and also inhibits the synthesis of ribonucleic acid [70] [71]. The antibacterial activity of eugenol depends on its ability to permeabilize the cell membrane and interact with cellular proteins. Non-specific permeabilization of the cytoplasmic membrane is linked with the leakage of K+ and ATP from the bacterial cell in the presence of eugenol [72]. Thus, these two antibacterial agents target the different cellular structures and functions which ultimately lead to the lethal effect on the bacterial pathogens. Synergistic interaction between Zataria multiflora Boiss essential oil and vancomycin against S. aureus is also reported by [73]. Similarly, synergistic interaction between the citral and norfloxacin against methicilin resistant S. aureus was also reported by [74]. Other reports on Carum copticum essential oil with vancomycin against S. aureus [75], essential oil of Melaleuca armillaris with cloxacillin against S. aureus [76] were also documented. The essential oils of clove and eugenol also showed synergy with azithromycin against S. mutans MTCC497 in planktonic mode. However, eugenol showed highest synergy with azithromycin. The MIC of the drug was reduced upto 16-folds against S. mutans MTCC497. Azithromycin targets various metabolic processes including protein synthesis, quorum sensing and also biofilm formation [77]. A possible of mode of action of combination of eugenol and antibiotics on Staphylococcus and S. mutans biofilms seems to have multiple targets on bacterial cells structure including altered cell structure, damaged cell wall, changes in cell membrane permeability and inhibition of protein and ribonucleic synthesis. Additionally, the combination treatment reduces the dose of the antibiotics and thus it helps to overcome the drug toxicity issue. Further, the eradication of biofilms was also confirmed by the scanning electron microscopy by the treatment of combination of eugenol and antibiotics (vancomycin/azithromycin). Microscopy revealed the eradication of S. aureus MTCC3160 and S. mutans MTCC497 biofilm cells compared to the untreated control at 12.5 µg/ml concentration of eugenol in combination treatment compared to the untreated control. Altered cell morphology and shrinkage of bacterial cells is quite evident from SEM, indicating the possible mode of action of combination of eugenol and antibiotics. Synergy with antibiotics reduced the dose related toxicity issue and also restores the available drug to a state reduced resistance [12]. This clearly suggested that natural products could be used to potentiate the efficacy of antibiotics in appropriate combinations. Moreover, lowering the dose of drugs in combination treatment may improve the overall efficacy of the treatment.

5. Conclusions

Our findings clearly established that clove oil and its active compounds like eugenol exhibited broad spectrum effect on growth, virulence and biofilms of bacterial strains. Synergy between essential oils and antibiotics is of great practical significance as these combinations can be used to surmount pathogenic biofilms associated infections. However, therapeutic application of combined treatment warrants in vivo investigation for its efficacy and safety. Further, molecular understanding and in vivo efficacy and safety of such combination needs to be evaluated carefully for future application in therapy.

Acknowledgements

Thanks are due to University grant commission for providing non-net fellowship to H.J. We are also thankful to University Sophisticated Instrumentation Facility (USIF), Aligarh Muslim University, Aligarh, India for SEM analysis. Advanced Instrumentation Research Facility (AIRF), Jawaharlal Nehru University, New Delhi, India is duly acknowledged for GC-MS analysis.

Supplementary File

Table S1. Designation, clinical condition and source of isolation of bacterial strains.

Table S2. Biochemical characterization of the SC-01 strain.

+: indicates positive reaction, −: indicates negative reaction.

Figure S1. Phylogenetic tree of SU-01 strain (S. auriuclaris J5) based on 16S rRNA gene sequence based homology.

Figure S2. Phylogenetic tree of SC-01 strain (S. sciuri JA1) based on 16S rRNA gene sequence based homology.

Cite this paper: Jafri, H. and Ahmad, I. (2021) In Vitro Efficacy of Clove Oil and Eugenol against Staphylococcus spp and Streptococcus mutans on Hydrophobicity, Hemolysin Production and Biofilms and their Synergy with Antibiotics. Advances in Microbiology, 11, 117-143. doi: 10.4236/aim.2021.112009.
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