Antimicrobial susceptibility of selected Essential Oils and their compounds against Streptococcus suis

Background: Streptococcus suis is an emerging zoonotic pathogen causing different diseases, in both humans and pigs. Generally, the control of diseases caused by this pathogen is based on antimicrobial therapy, but the development of bacterial resistance has led to look for new options. In this sense, the Essential Oils (EOs) constitute an alternative to the use of conventional antimicrobials. The activity of oregano, cinnamon, common thyme and red thyme EOs and their main active compounds (carvacrol, cinnamaldehyde and thymol) against 56 S. suis isolates from pigs (n=50) and human (n=6) was determined by broth microdilution. MIC50-90, MBC50-90 and the bactericidal index (MBC/MIC) were calculated. Also, the time-kill curve of each product against the S. suis P1/7 European reference strain was determined. Results: No differences in the MIC or MBC values were observed between all the tested products, which suggest a homogenous behaviour of S. suis, independently of their origin, organ of isolation or resistance profile. All the products showed a concentration-dependent and time-dependent killing activity and achieved the virtual eradication of S. suis (reduction of ≥ 4 log10) at suprainhibitory (2x-4x MIC) concentrations within the first 5 minutes of exposure, except cinnamaldehyde, that showed only bacteriostatic effect (reduction of < 3 log10). Conclusions: While all the tested products demonstrated an antimicrobial activity, red thyme and cinnamon followed by thymol showed the best results. It suggests that these products would be recommended as antimicrobials in veterinary medicine for the control of this zoonotic pathogen, although more pharmacology, toxicology, tolerability and formulation studies are necessary.

The control of these diseases should focus on the correct use of antimicrobials and sanitary measures implemented on the affected farms (5). However, the increasing cases of antimicrobial resistance in S. suis to many classes of antimicrobial agents, such as lincosamides, macrolides, sulphonamides and tetracycline, have been a global problem in recent years (6,7). It has been suggested that S. suis may be responsible for the spread of resistance genes to important human pathogens like Streptococcus pyogenes, S. pneumoniae and S. agalactiae (5,8).
Nowadays, an important pressure to reduce the use of antimicrobials in pig farming worldwide, to avoid the development of bacterial resistance, does exit (9). Different studies have proposed the use of Essential Oils (EOs) as natural antimicrobial agents alone or in combination with conventional antimicrobials (10,11). EOs are natural bioactive compounds derived from plants obtained by steam distillation and composed by terpenes, aldehydes and alcohols (12). The EOs activity is the result of the effects of all components and their interactions. Nevertheless, just a limited number of compounds account for up to 85 per cent of the total mixture compared with the minors (10,(13)(14)(15) and their mechanisms of action include enzymatic systems inactivation, membrane proteins alteration and increased membrane permeability (16).
Previous studies have shown the in vitro antibacterial activity of cinnamon, oregano and common and red thyme oils against Streptococcus spp. (10,11,17). However, the effect of their main compounds (cinnamaldehyde, carvacrol and thymol) has not been studied yet in S. suis. For this, the antimicrobial activity of these essential oils and their main active components against S. suis isolates obtained from human and pigs was analysed in this study. Furthermore, the time required to get a bactericidal effect of each product against the S. suis P1/7 European reference strain was determined.

Bacterial strains
A total of 56 S. suis isolates belonging to pigs (n = 49) and humans (n = 6) were analysed. The European reference S. suis strain P1/7 was also included (

Essential oils and their main components
Cinnamon, oregano, common thyme and red thyme EOs (purity ≥ 95%) were purchased from Aromium™ (Barcelona, Spain) and analysed by Gas Chromatography/Quadrupole Mass Spectroscopy to chemotype (data provided by manufacturer). Cinnamaldehyde, carvacrol and thymol were supplied by Sigma-Aldrich, Inc. (Madrid, Spain) with a purity of ≥ 95% ( Table 2). All the products were stored following the manufacturer instructions. Table 2 Botanical classification and chemotype of the studied essential oils. In vitro susceptibility test The broth microdilution method (18) was carried out with slight modifications. Mueller-Hinton broth was replaced with Brain-Heart Infusion (BHI) and supplemented with 0.15% agar (Oxoid Ltd., Cheshire, UK) to improve the dilution of the products (17,19). Double serial dilutions of natural products (ranging from 39.0625 µg/ml to 5000 µg/ml) were prepared and mixed with an equal volume  Regarding to the MIC 90 values, carvacrol showed the highest inhibitory activity (156.25 µg/mL), followed by thymol, cinnamaldehyde, oregano, common and red thyme (312.5 µg/mL) and cinnamon (1250 µg/mL). All tested products presented the same values for MICs and MBCs (Table 3), except for red thyme, cinnamaldehyde and carvacrol for MBC 90 . Apparently, all the tested S. suis isolates showed a homogenous behaviour against to all the products, independently of their origin, organ of isolation or resistance profile.
The BI showed the bactericidal or bacteriostatic character of all the tested products (BI < 4) ( Table 3).
The four products that showed the highest BI (BI = 1) were cinnamon, oregano, common thyme and thymol.

Time-kill assay
This assay was carried out to determine the bactericidal power of different concentrations of EOs and their main compounds throughout time. In general, concentration-dependent and time-dependent killing activities were observed in all the products tested against the European reference strain P1/7 ( Figs. 1a-1c).
Cinnamon and cinnamaldehyde showed marked differences in their bactericidal activities (Fig. 1a). At Oregano and carvacrol showed similar kinetics for all tested concentrations (Fig. 1b). Both products were bacteriostatic at 1x MIC, but the effectiveness of oregano decreased significantly at the end of evaluated period, while the carvacrol reached values close to the bactericidal limit. At supra-inhibitory concentrations, both products achieved the virtual eradication of the bacteria within 5 minutes of exposure.

Discussion
The extensive use of antimicrobials in veterinary medicine is considered one of the main causes of the emergence and diffusion of resistant microorganisms (5,25). Recent studies about S. suis report a remarkable decrease in the susceptibility of this zoonotic pathogen to antimicrobials commonly used in livestock, such as lincosamides, macrolides, sulphonamides and tetracyclines (25,26).
Furthermore, this microorganism has been identified as reservoir for antibiotic resistance genes which can be transferred horizontally to streptococcal human pathogens such as S. pyogenes, S.
Essential oils are natural products with hydrophobic character and a complex chemical composition (13), which could be used for the treatment of diseases caused by resistant bacteria (27)(28)(29). In previous studies, we showed that cinnamon, oregano, thyme and their main compounds (cinnamaladehyde, carvacrol and thymol) could be used in combination with gentamicin and tetracycline for the control of resistant S. suis isolates from swine (19). However, the efficacy of these products alone against porcine and human S. suis isolates is poorly known. This study includes not only an considerable number of S. suis strains (n = 56), but also human isolates.
According to our results, values of MIC 90 of these EOs against all the S. suis isolates analysed in this study ranged from 1250 µg/ml (cinnamon) to 312.5 µg/ml (oregano and common and red thyme), like those previously obtained against 20 porcine isolates (11). Regarding the main compounds, the MIC 90 ranged from 321,5 µg/ml (cinnamaldehyde and thymol) to 156,25 µg/ml (carvacrol). Moreover, in this work, all the products showed a bactericidal character (BI < 4).
Time-kill curves that monitor bacterial growth and death have been frequently used to evaluate the effect of antimicrobials over time (21,30). In our work, these assays showed a time-dependent and concentration-dependent activity for all the tested products. In general, all of them have shown an antimicrobial potential against S. suis as described for other Streptococcus species (15,17), although a huge variability was observed regarding concentration and time among them. EOs showed better results than their main compounds, showing stronger antimicrobial activity at the same concentration (1x MIC) at relatively earlier times. It has already been suggested that the antimicrobial activity of the EOs is better, which can be attributed to the wide variety of components, which may act in synergy against different targets (19,(31)(32)(33).
The best results were obtained for red thyme and cinnamon, that reached the virtual eradication after 1 minute and 30 minutes of exposure, respectively, coinciding with previous results which demonstrated the effectiveness of both products against S. suis isolated from pigs (21,34,35).
Regarding the main compounds, thymol demonstrated a bactericidal activity after 2 hours of exposure against the porcine and human S. suis isolates, which are in accordance with previous studies with Streptococcus species (15,36). Nevertheless, the seven products achieved the virtual eradication at 2x MIC and 4x MIC within 1-5 minutes, except the cinnamaldehyde that reached this effect at 4 h.
Previous studies with S. pyogenes ATCC 19615 and S. aureus ATCC 25923 have also highlighted the strong and quick bactericide effect of oregano (5-10 min) at doses equal to or higher than the MIC (21,34). However, our study showed slightly different results, decreasing its effectiveness at the end of the evaluated period. Variations in the EOs composition (extraction methods, geographic region, plant part, botanical species) and the susceptibility of studied strains would explain these differences.
A slight regrowth of the bacteria was observed with some of the products (cinnamon, oregano, red thyme and thymol) at concentrations 1x MIC, which could be associated with the persistence phenomena described for other concentration-dependent antimicrobials such as fluoroquinolones (30).
One of the main controversies is the cytotoxic effect of EOs when used at high doses. Some studies have shown a cytotoxic effect of these EOs at concentrations like our MIC 90 and the MBC 90 results (34.55-500 µg/ml) (36)(37)(38)(39)(40). However, recent studies on pig tracheal epithelial cell lines showed that cinnamon or thyme had a minimal effect on cell viability following a 2 h exposure at concentrations effective against this pathogen (35). Moreover, in a study carried out on vero cell lines, the thyme showed a marked inhibitory activity against many bacteria, despite a low cytotoxicity (38).
Furthermore, the toxicity tests carried out in rats, showed a low oral toxicity with the oregano used in this work (LD50 < 2000 mg/kg and NOAEL < 200 mg/kg/day) (39,41).
The knowledge of EOs is scarce, therefore, research efforts must be intensified to clarify the role that these products play as antimicrobials. In this context, time-kill assays and other dynamics studies together with MIC or MBC, are of interest to monitor the behavior of different bacterial species against the EOs.
The lack of the standardized method, the differences on the EOs composition (geographical origin, growth climatic and environmental conditions, part of the plant and the extraction method) and the variability of the botanical names, difficult the comparison of the results between different studies (29,32,35,42). In our study, EOs showed better antimicrobial activity than their main compounds, results that can be explained if consider that the molecules contained in very low proportion can also play an important role (32). However, the use of these main compounds of the EOs represents an interesting research line, since the standardisation is easier and, thus, the comparison of the results and their subsequent application in the diseases control (43,44).
We found a homogenous behaviour of all the S. suis isolates against all the tested products, independently of their origin, organ of isolation or resistance profile, which encourage further studies to favor the use of these EOs in S. suis infections control. To do so, a complete assessment (e.g. in vitro cell and in vivo animal model) on the toxicity and safety of these products is strongly advisable.

Conclusion
Cinnamon, oregano and Thyme (red and common thyme) and their main compounds showed an antimicrobial activity against strains of S. suis but thyme, its main compound (thymol) and cinnamon were revealed with the best results. These findings suggest that these products can be an alternative to conventional antimicrobials to reduce outbreaks of diseases caused by this zoonotic pathogen, although further studies are recommended.

Declarations
Ethics approval and consent to participate Not applicable

Consent to publish
Not applicable Availability of data and materials The dataset analyzed during the current study is available from the corresponding author on reasonable request.

Competing interests
None of the authors of this manuscript has a financial or personal relationship with other people or organizations that could inappropriately influence the content of this work.