Chemical composition and antibacterial activity of essential oils against pathogens often related to cattle endometritis

Introduction: Endometritis is a condition marked by inflammation of the endometrium that affects dairy cows from 21 days after parturition, causing damage to herd fertility and economic losses on farms. The use of active compounds obtained from plant sources has gained importance as disease treatment agents in farm animals due to the high resistance rates currently observed against traditional antibiotics commonly used. The study was carried out to examine the chemical composition and to investigate the antibacterial activity of rosemary, cinnamon, cloves, eucalyptus, lemon, oregano and thyme essential oils against the reference strain of Escherichia coli (ATCC 25922), Fusobacterium necrophorum (ATCC 25286), Trueperella pyogenes (ATCC 19411) and Staphylococcus aureus (ATCC 29213), considered as typical bacteria causing endometritis. Methodology: The chemical composition of the seven essential oils were analyzed by GC-MS and their antibacterial activity was evaluated by the disc diffusion method. Results: Thirty-six components were identified in total using GC-MS analyzes. The main compounds were cinnamaldehyde (86.5% for cinnamon essential oil), eugenol (85.7% for clove essential oil), 1,8-cineol (80% for eucalyptus and 47.8% rosemary essential oils), limonene (65.5% for lemon essential oil), carvacrol (72.1% for oregano essential oil) and thymol (48.8% for thyme essential oil). The disc diffusion assay revealed that cinnamon, clove, oregano, and thyme essential oils showed the best results compared to the other three essential oils, showing the largest zone of inhibition against all bacteria evaluated. Conclusions: These findings indicated that essential oils are a potential agent to be used as an alternative for bovine endometritis treatment.


Introduction
Uterine diseases cause profound economic losses in the dairy sector, mainly due to costs related to decreased milk production, increased use of medicines to treat diseases, discarding milk through antibiotics, and the damage caused by death or early culling of the cows [1,2]. Among uterine diseases, endometritis is one of the most important, being characterized by inflammation of the endometrium from 21 days after parturition [3][4][5], with purulent or mucopurulent uterine discharge [6].
The prevalence of endometritis reported in Brazil was 28.4% in 338 cows evaluated [6]. The bacteria most often described causing endometritis are Trueperella pyogenes, Escherichia coli and Fusobacterium necrophorum [1,7]. The use of antibiotics is the most used therapy against endometritis [1]. However, indiscriminate use of antibiotics may contribute to increased resistance of pathogenic bacteria, compromising the success of therapy, and may cause low efficacy of the drugs [8].
In this sense, the use of products of natural origin has become an alternative to reduce the use of antibiotics in dairy cows. Thus, essential oils are volatile substances naturally produced by plants as secondary metabolites, and are known for their antibacterial, antifungal, and antiviral properties, among others [9]. They can be extracted from various parts of plants, such as roots, leaves, bark, seeds, and fruits [10,11]. Their components include two classes of separate biological origin: the prime group consists of terpenes and terpenoids, and the second of aliphatic and aromatic components [12]. According to Pauli and Schilcher, [13], the antimicrobial activity of essential oils can be witnessed by in vitro tests, being the most three important ones are the agar diffusion, the agar or broth dilution and the vapor phase test.
The use of essential oils in cattle has increased in recent years, and the action in the treatment of diarrhea in calves [14] and mastitis [11] has been reported. However, there is little information on the use of essential oils as a therapy for endometritis. In this context, the aim of the present study were to characterize the chemical composition and to investigate the antibacterial properties of rosemary (Rosmarinus officinalis), cinnamon (Cinnamomum cassia), clove (Eugenia caryophyllus), eucalyptus (Eucalyptus globulus), lemon (Citrus limon), oregano (Origanum vulgare) and white thyme (Thymus vulgaris) essential oils against four bacteria' strains causing endometritis.

Gas chromatography/mass spectrometry (GC-MS) analysis
The essential oils chemical components were identified by gas chromatograph coupled to mass spectrometry (GC-MS). GC analyses were performed using a Shimadzu GC-2010 gas chromatograph, equipped with a GCMS-QP2010 Ultra mass spectrometer (Shimadzu, Suzhou, China). A split/splitless injector was used. Sample (1 μl) was injected into the injector with a split ratio of 1:10. Oven temperature was 40 o C for 3 min, then programmed heating from 40 to 280 o C at a rate of 8 o C/min. Injector temperature was 250 o C. Helium was used as carrier gas with 14 mL/minute flow rate. The volatile compounds were identified by comparison with mass spectra with those recorded in the National Institute of Standards and Technology database.

Disc diffusion assay
After incubation period the cultures were diluted in sterile saline solution and the turbidity adjusted to the 0.5 standard McFarland scale (~10 8 CFU/mL). With the use of a sterile cotton swab, surface of plates containing Mueller-Hinton agar (MHA; Difco) were inoculated with the bacterial suspension. To test T. pyogenes strain, the MHA was supplemented with 5% sheep blood and to test F. necrophorum strain, the medium used was Brucella agar (Acumedia, Lansing, MI, USA), supplemented with 5% sheep blood, hemine (Interlab Difco, Sao Paulo, Brazil) and vitamin K1 (Interlab Difco, São Paulo, Brazil).
Paper disks with 6 mm diameter (Whatman n o 3) soaked with 20 µL of each pure essential oil were laid on the surface of inoculated agar. Discs of ceftiofur (30 μg, Cefar Diagnósticos Ltda., São Paulo, Brazil) were used as positive control. A paper disc soaked with 20 μL of solution consisting of phosphate buffered saline (PBS, Sigma, São Paulo, Brazil) with 0.5% (v/v) polysorbate 80 (Tween 80) was used as negative control and loaded in each tested plate.
The plates were incubated at 37 °C for 24 hours (E. coli and S. aureus) or 48 hours (T. pyogenes) in aerobic conditions, or 37 °C for 48 hours in anaerobic condition (F. necrophorum). Anaerobic conditions were maintained by using an anaerobic jar with anaerobic atmosphere generator (Anaerobac, Probac, São Paulo, Brazil). After incubation, the inhibition zone diameter (IZD) was measured in accordance with the Clinical and Laboratory Standards Institute guidelines [15] and all experiments were carried out in three independent replicates.

Statistical analysis
Data were subjected to analysis of variance (ANOVA) and the differences between the means and standard error were tested by Tukey test. Statistical significance is considered as P ˂ 0.05.

Chemical composition of the essential oils
The volatile compounds for all studied essential oils are listed in Table 1. The major chemical constituent found in cinnamon essential oil was cinnamaldehyde (86.5%), and in clove essential oil was eugenol (85.7%). The eucalyptus essential oil was particularly rich in 1,8-cineol (80.0%), while the essential oil of lemon contained a high percentage of limonene (65.5%). In oregano essential oil the most abundant compound was carvacrol (72.1%), and in rosemary essential oil was 1,8-cineol (47.8%). Thymol (48.8%) and p-cymene (26.4%) were the main compounds identified in the thyme essential oil.

Antibacterial activity
The in vitro antibacterial activities of seven essential oils against several bacteria strains were qualitatively and quantitatively assessed by the measuring of IZD using the agar disc diffusion method as shown in Table 2.
The results obtained with ceftiofur against E. coli ATCC 25922 (27.86 mm of IZD) and S. aureus ATCC 25923 (29.00 mm of IZD) strains were within the expected values according to [15] ( Table 2). These results revealed that the cinnamon oil presented the greater IZD that varied from 29.67 to 38.33 mm. The larger IZD was observed in S. aureus (38.33 mm), and   For E. coli, essential oil of cinnamon and thyme had the larger (P ˂ 0.05) IZD compared to the other essential oils. For T. pyogenes and F. necrophorum, cinnamon essential oil had the larger (P ˂ 0.05) IZD compared to the other essential oils. Against the S. aureus, essential oil of cinnamon, oregano and thyme had the highest (P ˂ 0.05) IZD compared to the other essential oils and positive control.

Discussion
Goñi et al. [16] showed that the major components of the essential oil of cinnamon is cinnamaldehyde. The content of cinnamaldehyde (86.5%) identified for cinnamon essential oil in our results is similar to Li et al. [17] (66.2-81.9), higher than Lv et al. [18] (77.3%) and lower than Zhang et al. [19] (92.4%). Our results highlighted the significant higher activity (P ˂ 0.05) of cinnamon essential oil when compared to ceftiofur for S. aureus and T. pyogenes. T. pyogenes is considered one of the most important pathogens causing endometritis in dairy cows and cephalosporin-based drugs are most commonly used as treatments in cows with endometritis [1,20]. In addition, it is important to emphasize that cinnamon essential oil showed significantly higher antibacterial activity (P ˂ 0.05) than the other essential oils (clove, eucalyptus, lemon, oregano, rosemary and thyme) investigated in the present study against the pathogenic species of F. necrophorum and T. pyogenes. Studies regarding antibacterial activity of essential oils in relation to these two pathogenic species causing endometritis have not been found. This is the first study to show the in vitro activity of essential oils in potentially endometritiscausing bacteria. According to our results, it was demonstrated the promising potential of cinnamon essential oil as therapy in cows with endometritis.
Based on the results of the clove essential oil components, our results are in accordance with literature data, which show that eugenol (> 85%) is the major component identified [21,22]. 1,8-cineol (> 80%) was the main component identified in eucalyptus essential oil, corroborating the results of previous study [23]. According to the identification of the lemon essential oil components, the main constituents identified were limonene (65.5%) and β-pinene (15%), which is in agreement with the study by Hsouna et al. [24] (39.7% and 25.44, respectively). Carvacrol (>70%) was the major component of oregano essential oil, similar results were described by Ebani et al. [25] (65.90%) and Fratini et al. [26] (65.94%). The main constituent of the rosemary essential oil was 1,8-cineol (47%) and is similar to that described by Yang et al. [27] (46%). The main compound identified in thyme 32.00 ± 1.13 a 21.67 ± 1.13 b 6.00 ± 1.13 c 6.00 ± 1.13 c 22.00 ± 1.13 b 6.00 ± 1.13 c 24.67 ± 1.13 b 37.33 ± 1.13 a 6.00 ± 1.13 c Staphylococcus aureus ATCC 25923 38.33 ± 0.75 a 15.33 ± 0.75 c 8.00 ± 0.75 d 6.00 ± 0.75 e 36.00 ± 0.75 a 10.67 ± 0.75 b 36.00 ± 0.75 a 29.00 ± 0.75 b 6.00 ± 0.75 e a Inhibition zone diameter, values represent mean of three replicates ± standard error. Different letters in the same line represent statistical difference (P ˂ 0.05) in the size of inhibition zones including diameter of disc 6 mm formed under the paper disc by each essential oil. essential in oil in this study was thymol (48%), our results are in agreement with those described by Sokovic et al. [28] (48%) and Ebani et al. [25] (52%). Different growing environments such as altitude, hours of sunshine, temperature, rainfall, and parts of the plant extracted for the supply of essential oil may contribute to the difference between the percentages of identified active components [29,30]. The results of this study shown that the essential oils tested have different activity against the bacteria evaluated considering the IZD observed. To date, there have been no reports in the literature on the use of essential oils against strains of T. pyogenes and F. necrophorum strains.
Several authors have reported the antibacterial activity of Cinnamon cassia essential oil [19,[31][32][33][34]. Our results of IZD of cinnamon essential oil against E. coli (32 mm) are similar to those described by Nimje et al. [31] (32 mm), Melo et al. [32] (30 mm) and Zhu et al. [34] (30 mm), and larger than those described by Zhang et al. [19] (19 mm). Based on the results observed in the present study the IZD (38 mm) of cinnamon essential oil against S. aureus, our results are in agreement with those described by Melo et al. [32] (40 mm) and Cieslak et al. [33] (35 mm), and larger than those described by Zhu et al. [34] (29 mm), Zhang et al. [19] (28 mm) and Nimje et al. [31] (21 mm). The main component of Cinnamon cassia oil used in this study was cinnamaldehyde (86%). The antibacterial activity of Cinnamon cassia essential oil is mainly due to the cinnamaldehyde component, that have hydrophobic properties, and can react with bacterial cell membranes, contributing to damage the membrane, another action is the ability to inhibit bacterial peptide and protein synthesis, thus having gram-positive and gram-negative bacteria action [34,35].
The antibacterial effects of clove essential oil have been described in the literature [36]. Our results of IZD of clove essential oil against E. coli (9 mm) were smaller than those noted by Oulkheir et al. [37] (16 mm), Prabuseenivasan et al. [38] (17 mm) and Bartkiene et al. [39] (11 mm). The IZD of clove essential oil against S. aureus (15 mm) noted in this study were similar than those described by Prabuseenivasan et al. [38] (16 mm) and Bartkiene et al. [39] (16 mm). The main component of clove essential oil was eugenol (85%), this compound is responsible for the antibacterial effect of clove essential oil. The eugenol has the ability to denature proteins and react with cell membrane phospholipids, altering membrane permeability [36].
Our study showed least inhibitory activity of eucalyptus essential against E. coli (8 mm) and S. aureus (8 mm). Fratini et al. [26] also did not observe IZD results using eucalyptus essential oil against S. aureus and E. coli.
Hsouna et al. [24] using lemon essential oil noted IZD against the reference strain of E. coli (15 mm) and S. aureus (22 mm). However, in the present study no antibacterial activity was identified against the bacteria tested.
Previous studies showed the antibacterial activity of oregano essential oil [26,40]. Our results of IZD of essential oil of oregano against E. coli (20 mm) were smaller than those noted by Melo et al. [32] (38 mm), while against S. aureus, our results of IZD (36 mm) are larger than those described by Ebani et al. [25] (13 mm). The major constituent of oregano essential oil in this study was carvacrol (72%). The main mechanism of action of carvacrol against the bacterial cell is the collapse of the proton motor force, the depletion of the ATP pool, and may act on the phospholipid bilayer of the cell membrane, increasing the permeability and leakage of vital intracellular components, which can cause membrane disruption and contribute to cell death [25].
The antibacterial activity of rosemary essential oil has been previously reported [41]. Our results of IZD of rosemary oil against E. coli (8 mm) and S. aureus (10 mm) were smaller than those showed by Prabuseenivasan et al. [38] (17 mm and 12 mm, respectively). The differences might be related with distinct composition of the essential oils tested.
The high antimicrobial activity of thyme essential oil has been previously revealed [11]. Thyme essential oil showed a range of IZD of 24-36 mm in this study. These results are in agreement with those reported by Oulkheir et al. [37] that showed activity of thyme essential oil against E. coli (18 mm) and S. aureus (22 mm). Thymol (48%), the main compound of thyme essential oil, have been found to exhibit antimicrobial activity [42], acting on the membrane of bacteria, contributing to the release of lipopolysaccharides, increasing the permeability of the cell membrane, and increasing the loss of ATP and the leakage of vital intracellular constituents [25].

Conclusion
This study revealed that essential oils have antibacterial activity against the main bacteria tested causing endometritis. Therefore, essential oils have great potential as an alternative to be explored as endometritis therapy in dairy cows. Further in vivo studies are recommended to evaluate the use in clinical applications.