Bactericidal Effect of Clove Oil against Multidrug-Resistant Streptococcus suis Isolated from Human Patients and Slaughtered Pigs

Streptococcus suis is a zoonotic pathogen that is currently considered an emerging multidrug-resistant (MDR). Increasing antibiotic resistance can lead to the unsuccessful treatment of S. suis infection. Recently, many investigations of medicinal plants were conducted for the treatment of infection as a result of the increase of antibiotic-resistant bacteria. The aims of this study were to determine the chemical composition of essential oil from Syzygium aromaticum (L.) Merr. & L.M. Perry and the antibacterial activities of clove oil on MDR S. suis. Using gas chromatography coupled to a mass spectrometer, eugenol (97.76%) was found to be the major active ingredient of clove oil. In vitro antibacterial activities of clove oil against MDR S. suis were evaluated. Using the agar disc diffusion test, the clove oil showed a maximum zone of inhibition at 15% (v/v) oil concentration. In a broth microdilution method, the minimum bactericidal concentration of clove oil against all MDR S. suis isolates was 0.1% (v/v). A time-kill analysis was performed, and the killing kinetics of clove oil showed that MDR S. suis was completely reduced after 15 min of exposure to clove oil. In addition, clove oil exhibited a strong antibacterial activity at all pH values applied following incubation of MDR S. suis in pH-adjusted media with clove oil. Moreover, scanning electron microscopy revealed the nonviable S. suis isolates clearly showed atypical form and cell membrane lysis after incubation with clove oil. This study confirms the efficacy of clove oil as a natural antimicrobial against MDR S. suis and suggests the possibility of employing it as a promising alternative product for control of infectious diseases caused by S. suis in animal and human patients.


Introduction
Streptococcus suis is an emerging zoonotic pathogen that causes severe systemic infection in humans who come in close contact with pigs or pork-derived products. It also causes economic losses in the intensive swine industry. S. suis infections have been reported worldwide and a high human morbidity rate has been observed in Thailand, with the majority of infections caused by the consumption of raw pork [1].

Antibacterial Activity of Clove Oil
Clove oil was found effective against all of the tested MDR S. suis isolates, as summarized in Table 2. The diameter of inhibition zones depended on the concentration of the oil. Maximum inhibition was found at 15% concentration, and the minimum effect was observed at 5% concentration. The inhibition zones were 5.07 ± 0.12 to 13.07 ± 0.12 mm, 5.03 ± 0.05 to 14.03 ± 0.05 mm, and 8.00 ± 0.00 to 15.07 ± 0.05 mm on discs impregnated with 5%, 10%, and 15% (v/v) oil, respectively. At 1% (v/v), the oil could not inhibit any the tested isolates. As a positive control, high sensitivity to penicillin (10 µg) was observed for all MDR S. suis strains. No inhibition zone was observed in the discs with methanol. This indicates that the methanol used to prepare the clove oil solution had no effect on bacterial growth.

Determination of MIC and MBC
The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) results are summarized in Table 2 and the distribution of MICs and MBCs for 15 isolates is shown in Table 3. The results showed that the MDR S. suis isolates were inhibited by clove oil at a very low concentration, with the MIC50 value at 0.05% and the MIC90 value at 0.1%. The clove oil showed bactericidal effect against all tested MDR S. suis with the MBC values were 0.1 ± 0.00 %. In addition, as a vehicle control, methanol was checked for its antibacterial capability at the various concentrations applied for preparing the essential oil in the experiment. We found that methanol at each concentration did not affect the growth of the tested isolates (data not shown).

Antibacterial Activity of Clove Oil
Clove oil was found effective against all of the tested MDR S. suis isolates, as summarized in Table 2. The diameter of inhibition zones depended on the concentration of the oil. Maximum inhibition was found at 15% concentration, and the minimum effect was observed at 5% concentration. The inhibition zones were 5.07 ± 0.12 to 13.07 ± 0.12 mm, 5.03 ± 0.05 to 14.03 ± 0.05 mm, and 8.00 ± 0.00 to 15.07 ± 0.05 mm on discs impregnated with 5%, 10%, and 15% (v/v) oil, respectively. At 1% (v/v), the oil could not inhibit any the tested isolates. As a positive control, high sensitivity to penicillin (10 µg) was observed for all MDR S. suis strains. No inhibition zone was observed in the discs with methanol. This indicates that the methanol used to prepare the clove oil solution had no effect on bacterial growth.

Time-Kill Study
To determine the killing kinetics of clove oil against MDR S. suis, survival of the MNCM06 isolate was evaluated over a 24 h period in the presence of clove oil at MBC. The data on CFU showed that treatment with 0.1% (v/v) essential oil exhibited a strong bactericidal effect on the MNCM06 isolate in a rapid and time-dependent manner. The cell population was reduced by approximately 2-log CFU/mL after only 5 min of incubation. Within 15 min, the bacterial population was completely inactivated ( Figure 2, Table S1). Loss of viability was apparent for the MNCM06 isolate after prolonged exposure to the clove oil, while the growth rate was not decreased for the control without clove oil.

Effect of pH on Bactericidal Activity
The highest bactericidal activity of clove oil was observed at pH 4. However, the bactericidal effect at the same time of treatment were not significantly different (p > 0.05) among pH values applied ( Figure 3). The results show that at an MBC of 0.1%, clove oil completely inhibited the tested isolate within 15 min of incubation, with a strong activity at pH 4 through pH 8. The growth of the tested isolate in the control tube without clove oil was unaffected by changes in pH value.

Effect of Clove Oil on Cell Surface of S. suis
The morphological changes of the treated bacteria were compared with the untreated control, as shown in Figure 4. The cell surface of the untreated MNCM06 isolate was found to have typical morphology, which is an intact spherical shape ( Figure 4a). In contrast, the treatment of cells with clove oil for 1 h induced important morphological damage (Figure 4b

Effect of pH on Bactericidal Activity
The highest bactericidal activity of clove oil was observed at pH 4. However, the bactericidal effect at the same time of treatment were not significantly different (p > 0.05) among pH values applied ( Figure 3, Table S2). The results show that at an MBC of 0.1%, clove oil completely inhibited the tested isolate within 15 min of incubation, with a strong activity at pH 4 through pH 8. The growth of the tested isolate in the control tube without clove oil was unaffected by changes in pH value.

Effect of pH on Bactericidal Activity
The highest bactericidal activity of clove oil was observed at pH 4. However, the bactericidal effect at the same time of treatment were not significantly different (p > 0.05) among pH values applied ( Figure 3). The results show that at an MBC of 0.1%, clove oil completely inhibited the tested isolate within 15 min of incubation, with a strong activity at pH 4 through pH 8. The growth of the tested isolate in the control tube without clove oil was unaffected by changes in pH value.

Effect of Clove Oil on Cell Surface of S. suis
The morphological changes of the treated bacteria were compared with the untreated control, as shown in Figure 4. The cell surface of the untreated MNCM06 isolate was found to have typical morphology, which is an intact spherical shape (Figure 4a). In contrast, the treatment of cells with clove oil for 1 h induced important morphological damage (Figure 4b-d). At the MIC and MBC of

Effect of Clove Oil on Cell Surface of S. suis
The morphological changes of the treated bacteria were compared with the untreated control, as shown in Figure 4. The cell surface of the untreated MNCM06 isolate was found to have typical morphology, which is an intact spherical shape (Figure 4a). In contrast, the treatment of cells with clove oil for 1 h induced important morphological damage (Figure 4b-d). At the MIC and MBC of clove oil, it was found that cells became deformed, with cell wall ruptures and cell lysis in which leakage of cytoplasmic contents were apparent. clove oil, it was found that cells became deformed, with cell wall ruptures and cell lysis in which leakage of cytoplasmic contents were apparent.

Discussion
Four phytoconstituents were identified in essential oil from clove buds in this study, and eugenol (97.76%) was the major constituent. This is in agreement with previous studies that reported eugenol was the main component of clove [22,26,27]. However, the percentage of eugenol in those studies was different, ranging from 50.3% to 76.8%. Our study obtained a higher eugenol content and found different components in the clove oil. The differences in the contents and components of the essential oil from clove buds can be explained as influences of the clove species, geographic region, environmental factors (temperature, humidity, and light) [28], and the difference in extraction methods used [27][28][29]. More importantly, the high concentration of eugenol in clove oil gives it potent bactericidal properties [30]. Moreover, the synergistic effects of phytochemicals are well-documented [31]. Interestingly, previous studies showed that the MIC of the purified eugenol is higher than the total clove oil [32]. Therefore, the antibacterial activity of clove is due to phytocomplexes of eugenol and the minor constituents.
The present study was performed on S. suis isolates, which are resistant to many antibiotics that are widely used to treat bacterial infections and used as feed additive in pigs to prevent infections in Thailand. The results from the agar disc diffusion method revealed that clove oil had inhibitory effects against all test MDR S. suis isolates. This is consistent with previous studies [33][34][35][36]. Incidentally, regarding the inhibition zones obtained from the essential oil, it is notable that differences can occur among studies due to the methods used in solubilizing the oils to obtain hydrophilic molecules [33], antimicrobial assays, and tested organisms. Even though the antibacterial properties of essential oil from clove bud against S. suis have already been reported [37], its effect against MDR S. suis has not been previously reported. Our findings suggest that the MDR S. suis isolated from both human patients and slaughtered pigs is susceptible to the essential oil of S. aromaticum (L) Merr. & L.M.Perry.

Discussion
Four phytoconstituents were identified in essential oil from clove buds in this study, and eugenol (97.76%) was the major constituent. This is in agreement with previous studies that reported eugenol was the main component of clove [22,26,27]. However, the percentage of eugenol in those studies was different, ranging from 50.3% to 76.8%. Our study obtained a higher eugenol content and found different components in the clove oil. The differences in the contents and components of the essential oil from clove buds can be explained as influences of the clove species, geographic region, environmental factors (temperature, humidity, and light) [28], and the difference in extraction methods used [27][28][29]. More importantly, the high concentration of eugenol in clove oil gives it potent bactericidal properties [30]. Moreover, the synergistic effects of phytochemicals are well-documented [31]. Interestingly, previous studies showed that the MIC of the purified eugenol is higher than the total clove oil [32]. Therefore, the antibacterial activity of clove is due to phytocomplexes of eugenol and the minor constituents.
The present study was performed on S. suis isolates, which are resistant to many antibiotics that are widely used to treat bacterial infections and used as feed additive in pigs to prevent infections in Thailand. The results from the agar disc diffusion method revealed that clove oil had inhibitory effects against all test MDR S. suis isolates. This is consistent with previous studies [33][34][35][36]. Incidentally, regarding the inhibition zones obtained from the essential oil, it is notable that differences can occur among studies due to the methods used in solubilizing the oils to obtain hydrophilic molecules [33], antimicrobial assays, and tested organisms. Even though the antibacterial properties of essential oil from clove bud against S. suis have already been reported [37], its effect against MDR S. suis has not been previously reported. Our findings suggest that the MDR S. suis isolated from both human patients and slaughtered pigs is susceptible to the essential oil of S. aromaticum lower than in a previous study by Perugini Biasi-Garbin et al. [38], which reported 0.125 to 0.5% (v/v), while Baskaran et al. [39] reported 0.4/0.8% (v/v) on mastitis pathogen S. agalactiae. However, it should be noted that the initial bacterial load, culture medium used, incubation time, and temperature are important variables that can affect MIC determination of clove oil [40][41][42][43].
The time-kill study demonstrated rapid inhibition of bacterial growth within only 5 min of treatment with clove oil at MBC concentration (0.1%). It was found that the clove oil reached maximal bactericidal effect within 15 min of incubation, when a complete loss of the viability of the MDR S. suis was observed. Our results are in accordance with a previous finding that the maximum kill rate of S. pneumoniae was observed during the first 15 min of eugenol exposure at MBC (0.12%) concentration [44] .However, it seems that S. suis was completely killed more rapidly than S. pneumoniae, which was completely killed within 60 min of exposure. This result suggests that clove oil has strong bactericidal activity on MDR S. suis and also indicates that MDR S. suis is very susceptible to clove oil.
With regard to the action of clove oil on MDR S. suis, the SEM images showed irregular morphology of cells treated with clove oil compared to untreated cells. Our results showed that cell breakdown is the principal phenomenon for MDR S. suis treated with clove oil. This corresponds to the result of the time-kill study, which demonstrated a progressive reduction in the number of viable S. suis cells in medium with clove oil and eventually led to complete death of the cells within 15 min. Our findings on the action of clove oil on bacterial morphology are consistent with other studies [22,23,38,[43][44][45][46]. It is already known from many research reports that the antibacterial activity of clove oil is due to its major component, eugenol. Eugenol is a phenolic structure that is highly active against various microorganisms [45]. It can readily pass through gram-positive bacterial cell walls, causing degradation of the cell wall [44], and then damage the cytoplasmic membrane, causing destruction of membrane proteins, impairment of bacterial enzyme systems, increased permeability leading to leakage of the cell contents, and eventually cell lysis [22,23].
Moreover, we described the results for the effect of pH on the bactericidal activity of clove oil against MDR S. suis, which showed strong antibacterial activity at all pH values applied, with the highest antibacterial activity observed at pH 4. Our result suggested that the bactericidal activity of clove oil against MDR S. suis is sustainable through neutral, acidic, and alkaline conditions. However, the variation of pH effect on the bactericidal activity of clove oil was reported in a previous study that found the highest activity of eugenol against Salmonella typhi at pH 9.0 [23], while Hoque et al. [47] reported the highest activity of clove oil against L. monocytogenes at pH 7.0, and Knight et al. [48] showed strong inhibition of the growth of E. coli O157: H7 at neutral (7.2) and acidic (4.5) pH values. The difference among these results could be due to factors such as test organisms, sample preparation, and the method used in pH assays. However, it is notable that the bactericidal effect of clove oil against MDR S. suis was not markedly inactivated by changes in pH, which makes it valuable and possible to apply as a treatment or preventive therapy for S. suis infection.
In conclusion, this study found that clove oil exhibits a strong potential bactericidal effect on MDR S. suis, which suggests the possibility of employing it in a promising alternative product for control of infectious diseases caused by S. suis. Further research with in vivo studies and clinical trials need to evaluate the potential of clove oil in medical applications.

Materials and Methods
This study was performed at the Department of Veterinary Biosciences and Public Health, Faculty of Veterinary Medicine, Chiang Mai University, Thailand. The ethical approval by the Ethics Committee of the Faculty of Veterinary Medicine, Chiang Mai University, Thailand was waived since this study did not involve the study of human or animal subjects, data, or tissues.

Bacteria and Culture Conditions
The MDR S. suis isolates from storage at −20 • C were used in the present study ( Table 4). All of them were resistant to tylosin, enrofloxacin, tetracycline, trimethoprim-sulfamethoxazole, Pathogens 2020, 9, 14 8 of 12 and oxytetracycline, which previously characterized antimicrobial resistance based on a breakpoint disc diffusion assay and a broth microdilution assay according to Clinical and Laboratory Standards Institute (CLSI) guidelines [49]. The bacterial strains were recovered from the bacterial storage medium by subculture on Columbia blood agar (Oxoid, Hampshire, U.K.) containing 5% (v/v) defibrinated sheep blood and incubated at 37 • C for 24 h. The colonies were confirmed as S. suis by the API 20 Strep kit (BioMérieux, Marcy l'Etoile, France). All of them presented the same resistance pattern as described above. -/-/+ human ** Serotypes were identified by multiplex-polymerase chain reaction (PCR) combined with serum agglutination [50]. † Sequence type (ST) were identified by Multilocus sequence typing (MLST) [50]. ‡ Virulence-associated gene profiles were identified by multiplex-PCR; mrp, muramidase-released protein; epf, extracellular factor; sly, suilysin; * the variant of epf [50]. § All strains were recovered from stock kept at −20 • C.

Essential Oil and Characterization
The essential oil of clove flower bud was purchased from Thai-China Flavours & Fragrances Industry Co. (Thailand). Its chemical characterizations were analyzed using a gas chromatography-mass spectrometry (GC-MS) method. The GC-MS analysis was performed using an Agilent 6890 gas chromatograph in positive ion electron impact (EI, 70 eV) mode coupled to an Agilent 5973 mass selective detector (Agilent Technologies Inc, USA) with an HP-5MS column (30 m × 250 µm i.d. × 0.25 µm film thickness). The individual compound was identified by retention times relative to those of authentic samples and by matching the spectral peaks available in the Wiley, NIST, and NBS mass spectral libraries.

Preparation of Clove Oil Concentration
The clove oil was prepared aseptically in methanol as a 30% (v/v) stock solution [23]. The concentrations used in subsequent experiments were prepared from the stock solution.

Determination of Antibacterial Activity
The antibacterial activity of clove oil was evaluated by disc diffusion method [23]. The MDR S. suis strains were inoculated into Bacto™ Todd-Hewitt broth (THB) medium (Becton Dickinson & Co., Sparks, MD, USA) and grown for 18 h at 37 • C. Bacterial cultures were adjusted to a concentration of 1.5 × 10 8 CFU/mL using a McFarland densitometer (Grant Instruments, Cambridgeshire, UK) and were swabbed on BBL™ Mueller-Hinton agar (MHA) (Becton Dickinson & Co., Sparks, MD, USA) plates containing 5% (v/v) defibrinated sheep blood. Sterile paper discs (Whatman no. 5, 6-mm diameter) were infused with 50 µL of different concentrations of clove oil (1%, 5%, 10%, and 15% [v/v]). The airdried discs were placed on MHA plates and incubated overnight at 37 • C. Penicillin (10 µg) was used as a positive control, and airdried discs of methanol were used as a vehicle control. The zones of inhibition were measured, and the determination was done in three independent experiments.

Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC)
MIC and MBC were measured by the broth microdilution method. The inoculums were prepared by suspending bacteria of each isolate in 3 mL of sterile 0.85% NaCl solution. Each suspension was adjusted to 0.5 using a McFarland densitometer and then diluted to 1:200 in BBL™ cation-adjusted Mueller-Hinton broth (CAMHB) (Becton Dickinson & Co., Sparks, MD, USA) supplemented with 5% (v/v) lysed horse blood. The cell density of the final inoculums was 5 × 10 6 CFU/mL. Equal volumes (100 µL) of bacterial suspension, and two-fold serial dilutions of clove oil in CAMHB supplemented with 5% (v/v) lysed horse blood (0.2-0.0125% final concentration) were mixed into the wells of 96-well plates. Control wells with no bacteria or no clove oil and methanol control were also prepared. After incubation for 24 h at 37 • C, MIC values were recorded as the lowest concentration of clove oil in which no visible growth occurred. To evaluate MBC values, 10 µL of the content from each well showing no visible growth were dropped on MHA plates containing 5% (v/v) defibrinated sheep blood and then incubated for 24 h at 37 • C. The MBCs of clove oil were recorded as the lowest concentration at which no colony formation occurred. The MIC and MBC values were determined in triplicate and in three independent experiments. The MIC and MBC against 50% (MIC50 and MBC50 values) and 90% (MIC90 and MBC90 values) of isolates were also calculated.

Time-Kill Study
The MDR S. suis MNCM06 isolate was randomly selected as a representative strain to determine the killing kinetics of clove oil. The overnight culture of bacteria was harvested by centrifugation and suspended in sterile phosphate-buffered saline (PBS; pH 7.2). The suspension was adjusted to a concentration of 1.5 × 10 8 CFU/mL and treated with clove oil at MBC. A tube without essential clove oil was assigned as the control. Samples were taken at 0, 5, 10, 15, 30, and 60 min, then serially diluted in PBS (pH 7.2) and dropped in triplicate on Columbia blood agar plates containing 5% (v/v) defibrinated sheep blood. Following 24 h of incubation at 37 • C, the colony-forming units (CFU) were calculated. The determination was done in three independent experiments.

Effect of pH on Bactericidal Activity
The effect of pH on the bactericidal activity of clove oil was determined as reported previously with slight modifications [51]. The pH was adjusted by adding 1 M of HCl or 1 M of NaOH to the THB to obtain various pH values (4, 5.5, 7, and 8), sterilized by membrane filtration, and used immediately. The overnight culture of MDR S. suis MNCM06 isolate (1.5 × 10 8 CFU/mL) was inoculated into 5 mL of pH-adjusted media with clove oil at MBC concentration, followed by incubation at 37 • C. Next, 100 µL of the sample was taken, serially diluted in PBS (pH 7.2), and dropped in triplicate on Columbia blood agar plates containing 5% (v/v) defibrinated sheep blood at regular time intervals (0, 15, 30, 60 min). Following 24 h of incubation at 37 • C, the CFU was calculated. All the determinations were done in triplicate.

Scanning Electron Microscopy (SEM)
The effect of clove oil on the morphology of the MNCM06 isolate of MDR S. suis was further observed by SEM. Overnight culture of the bacteria was harvested by centrifugation, suspended in PBS (pH 7.2), and then treated with clove oil at MIC and MBC for 1 h at 37 • C and processed as previously described [22]. A control sample without clove oil treatment was similarly prepared and examined.

Statistical Analysis
Data were analyzed and expressed as mean ± SD of experiments performed in triplicate. The data for the effect of pH on bactericidal activity were analyzed using analysis of variance. Pairwise comparisons were analyzed using the least squares means procedure with Tukey's adjustment (values of p < 0.05), and the statistical tests were performed using SAS 9.2 software.