In Vitro Antimicrobial Activity of Thymus vulgaris, Origanum vulgare, Satureja montana and Their Mixture against Clinical Isolates Responsible for Canine Otitis Externa

Simple Summary Otitis externa of dogs is the inflammation of the external ear canal and may be acute or chronic, persistent or recurrent. Several bacterial species are involved in otitis externa and often Malassezia yeasts are simultaneously present. Otitis externa is often a severe problem in veterinary medicine because of the resistance of the involved pathogens to conventional drugs. Essential oils (EOs) could be promising products with which to treat these inflammations. EOs from Origanum vulgare, Satureja montana, and Thymus vulgaris seem to be active to the main bacterial species and M. pachydermatis cultured from the ears of dogs with otitis; moreover, a mixture of these three components seems to improve the antibacterial property. Abstract Otitis externa is a frequent inflammation among dogs, mainly caused by bacteria and yeasts that are often resistant to conventional drugs. The aim of the present study was to evaluate the in vitro antibacterial and antifungal activities of commercial essential oils (EOs) from Origanum vulgare, Satureja montana, and Thymus vulgaris, as well as a mixture of these three components, against 47 clinical bacterial strains (Staphylococcus sp., Streptococcus sp., Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumoniae, Serratia marcescens) and 5 Malassezia pachydermatis strains, previously cultured from the ears of dogs affected by otitis externa. The tested Gram-positive bacteria were sensible to the analysed EOs with MICs ranging from 1.25% (v/v) to <0.0195% (v/v); Gram-negative isolates, mainly P. aeruginosa, were less sensitive with MICs from >10% (v/v) to 0.039% (v/v). M. pachydermatis isolates were sensitive to all EOs with MICs from 4.25% (v/v) to 2% (v/v). However, the mixture was active against all bacterial (except one P. aeruginosa strain) and fungal tested isolates. The three EOs and their mixture seem to be an interesting alternative for treating canine otitis externa when conventional antimicrobials are not active.


Introduction
Otitis externa of dogs is the inflammation of the external ear canal, including the ear pinna, and may be acute or chronic, persistent or recurrent. Changes occur in the canine external ear canal with otitis such as glandular hyperplasia, glandular dilation, epithelial hyperplasia, and hyperkeratosis, which cause increased cerumen production. This condition, characterized by higher local humidity and pH, predisposes the ear to secondary infection [1]. The bacteria most commonly isolated from ear canals of dogs affected by otitis are Staphylococcus spp., but other bacterial species, including Streptococcus spp., Pseudomonas spp., Escherichia coli and other Enterobacteriaceae may be involved, too [1][2][3]. Malassezia yeast is another frequent agent encountered in canine otitis externa. The frequency of infection as sole causative agent is reported between 8% and 26% [4,5]. Some dogs appear to develop an allergic response to Malassezia spp., leading to significant discomfort and pruritus [1] and, when not properly treated the affection can evolve in otitis media, as well [6]. Therefore, an effective therapy, together with the correction of predisposing factors and concurrent diseases, is mandatory to prevent relapses [7].
Topical antimicrobial therapy is preferred to systemic treatment in case of otitis externa, but frequently it is not successful because of the resistance of bacteria and yeast agents to conventional drugs. Therefore, essential oils (EOs) are welcome as alternative therapies and studies about their effectiveness against bacteria and yeasts responsible for otitis externa are necessary.
EOs from oregano (Origanum vulgare L.), savory (Satureja montana L.) and thyme (Thymus vulgaris L.) showed in vitro antimicrobial properties when tested against bacterial and fungal species [8], but specific information about their effectiveness against clinical strains isolated from dogs affected by otitis are very scanty [3].
The present study focused on investigating the in vitro antibacterial and antifungal activities of three commercial EOs versus clinical strains previously isolated from the ears of dogs affected by otitis externa. In particular, EOs from O. vulgare, S. montana, and T. vulgaris, as well as a mixture of these three components, were tested against isolates of Staphylococcus sp., Streptococcus sp., Pseudomonas aeruginosa, E. coli, Klebsiella pneumoniae, Serratia marcescens, and Malassezia pachydermatis.

Essential Oils
Three commercial EOs, obtained from oregano (Origanum vulgare L. subsp. hirticum), savory (Satureja montana L.), and thyme (Thymus vulgaris L.), were employed in the study. The EOs were obtained from FLORA ® (Pisa, Italy). A mixture was prepared with equal parts of the three oils. All EOs and the mixture were kept at 4 • C in dark glass vials; before being used in the analyses, they were microbiologically tested for quality control.

Gas Chromatography-Mass Spectrometry Analysis
The selected EOs were diluted to 5% in HPLC-grade n-hexane before the injection. The Gas Chromatography-Mass Spectrometry (GC-MS) analyses were carried out following the protocol previously reported by Pieracci et al. [9]. Agilent 7890B gas chromatograph (Agilent Technologies Inc., Santa Clara, CA, USA) equipped with an Agilent HP-5MS capillary column (30 m × 0.25 mm; coating thickness 0.25 µm) and an Agilent 5977B single quadrupole mass detector was used. The analytical conditions were the following: oven temperature increasing from 60 to 240 • C at 3 • C/min; injector and transfer line temperatures set at 220 • C and 240 • C, respectively; carrier gas helium flow set at 1 mL/min. The injection volume was 1 µL, with a split ratio of 1:25. The acquisition parameters were: full scan; scan range: 30-300 m/z; scan time: 1.0 s. The identification of the constituents was based on a comparison of the retention times with those of the authentic samples, comparing their linear retention indices relative to the series of n-hydrocarbons. Computer matching was also used against commercial (NIST 14 and ADAMS 2007) and laboratory-developed mass spectra libraries built up from pure substances and components of commercial essential oils of known composition and MS literature data [10][11][12][13][14][15] All strains were previously cultured from ears of dogs affected by otitis externa. After isolation, the strains were typed using API Staph, API Strep, API 20E, and API 20NE systems (BioMerieux, Milan, Italy) in relation to microbiological and Gram staining characters. To discriminate between S. aureus and S. pseudointermedius, a multiplex-PCR assay described by Sasaki et al. [16] was employed.
The strains were stored at −80 • C in glycerol broth. Before being employed in the antibacterial activity analyses, each isolate was cultured in brain hearth infusion broth (BHIB, Oxoid Ltd., Basingstoke, Hampshire, UK) at 37 • C for 24 h. Cultures of 1-2 × 10 7 CFU/mL, corresponding to 0.5 McFarland standard, were used in the tests.
The test was carried out on Mueller-Hinton agar plates (Oxoid) incubated at 35 • C for 16-20 h; the results were interpreted on the basis of the breakpoints reported by EUCAST or CLSI (The Clinical and Laboratory Standards Institute) [17,18].

EOs and Mixture Minimum Inhibitory Concentration
In order to verify the sensitivity of the bacterial strains to the three EOs and their mixture, each bacterial isolate was tested to determine the minimum inhibitory concentration (MIC) through the broth microdilution method, according to the guidelines reported by CLSI [19] and the protocol reported by Ebani et al. [20]. The MIC value was the lowest concentration, expressed in percentage (10%, 5%, 2.5%, 1.25%, 0.625%, 0.312%, 0.156%, 0.078%, 0.039%, 0.0195% (v/v)), of each EO and mixture at which bacteria show no visible growth. The test was executed simultaneously to control bacterial growth (tested strain and media) and sterility (tested EO and media). All tests were executed in triplicate.

Antifungal Activity
Five M. pachydermatis clinical strains were analyzed to verify their in vitro antifungal sensitivity through microdilution method. The test was assessed using liquid m-Dixon medium for preparing yeast suspensions, as previously described [21]. The isolates were cultured from dogs affected by external otitis onto Mycobiotic agar (Thermo Fisher Scientific, Rodano, Italy). The fungal isolates were tested against ketoconazole by E-test (AB Biodisk, Solna, Sweden) to verify their susceptibility to this antimycotic drug, frequently used to treat Malassezia infections [22]. The fungal strains were analyzed in triplicate versus EOs and mixture diluted into the medium at concentrations of 10%, 5%, 4.5%, 4%, 3.75%, 3,5%, 3, 25%, 3%, 2.75; 2.5%, 2.25%, 2%, 1.5%, and 1%. MIC was determined as the lowest EO/mixture concentration where no fungal growth was observed.

Gas Chromatography-Mass Spectrometry Analysis
The complete compositions of the analyzed commercial EOs and their mixture are reported in Table 1. Overall, 43 compounds were identified, covering 99.1-100.0% of the whole compositions.   The mixture was characterized by oxygenated monoterpenes as the main chemical class of compounds, accounting for 75.0%, mainly represented by carvacrol (50.8%) and thymol (17.1%). Carvacrol was found in very considerable percentages in the EOs of O. vulgare and S. montanta, where it constituted 78.0 and 60.0% of the whole compositions, respectively. The EO of T. vulgaris, instead, showed a much lower content of this component (5.5%), but a higher amount of thymol (43.6%), responsible for the great content of this molecule in the mixture.
Monoterpene hydrocarbons were the second most represented class in the mixture sample, reaching 14.0%. p-Cymene, with 9.2%, was the most abundant compound belonging to this class, and probably derived from the EO of T. vulgaris, in which it covered 18.7% of the composition. However, it was found in not negligible percentages also in O. vulgare (3.3%) and S. montana (5.7%) EOs.
Finally, sesquiterpenes in either their hydrocarbon or oxygenated forms were also detected in the mixture, as well as in the individual EOs. β-Caryophyllene and its oxide were the most representative molecules belonging to these classes, and their content in the mixture and in the singular EOs was very similar.
As regards bacteria of Enterobacteriaceae family, seven different resistance profiles were determined (Table S2). Analyzed bacterial strains were resistant from three to ten of the tested antimicrobials. The most effective antimicrobials were aminoglycosides and enrofloxacin, whereas 50.00% of tested isolates were resistant to doxycycline, tetracycline, and trimethoprim/sulfamethoxazole. More than 50% of tested E. coli were resistant to ampicillin. All Enterobacteriaceae were intrinsically resistant to clindamycin and rifampicin, S. marcescens is intrinsically resistant to ampicillin and amoxicillin/clavulanic acid, and K. pneumoniae is intrinsically resistant to ampicillin; obtained data are in line with these statements.

EOs and
Considering the bacterial species, one strain of E. coli (856A1) was resistant to T. vulgaris, and it was inhibited by O. vulgaris with 1.25% MIC value, but lowest MICs (0.039%) were obtained with S. montana and the mixture. The remaining five E. coli isolates were sensible to the three EOs, even though they showed the lowest MICs when assayed with the mixture. Both K. pneumoniae and S. marcescens were sensible to the three EOS and lower MICs were observed with the mixture (<0.0195% and 0.039%, respectively).
Different results were obtained when testing P. aeruginosa isolates in relation to EOs and bacterial strain. One isolate was resistant to O. vulgare, three to S. montana and ten to T. vulgaris. Except for isolate 535A, all strains were inhibited by the mixture.

Antifungal Activity
All isolates were sensible to ketoconazole. Satureja montana EO appeared as the more active compound, with MIC values of 2% in four out of five fungal isolates, followed by the other EOs with similar results. Interestingly, MICs of the mixture appeared slightly higher than those showed by S. montana, suggesting a possible antagonistic effect of the three oils, although the mixture was more effective with respect to O. vulgare and T. vulgaris alone (Table 4). Table 4. MIC values of the tested EOs and mixture expressed in percentage (v/v) against the selected Malassezia pachydermatis isolates.

Discussion
The findings of the present study showed antimicrobial properties of commercial EOs from O. vulgare, S. montana, and T. vulgaris against Gram-positive and Gram-negative bacteria, and M. pachydermatis, even though differences were observed in relation to the bacterial or fungal isolate and the tested EO.
Members of Staphylococcus genus are Gram-positive bacteria acting as mammalian commensals that can colonize mucosal membrane, nares, skin, and ears. Different staphylococcal species are frequently isolated from the ears of dogs affected by otitis. This is difficult to resolve because of resistance of these bacteria to many antibiotics. Resistance to methicillin and other antimicrobials are a spread threat with severe implications on animal and human health [23]. The tested staphylococcal isolates were resistant to several antibiotics and most of them were resistant to ampicillin (81.81%) and tobramycin (68.18%) in accordance with other surveys [23][24][25][26][27][28].
Oregano, savory, and thyme EOs were active against the tested Staphylococcus isolates in agreement with data reported by other investigations [3,[29][30][31][32][33]. Low MIC values were found when testing the isolates against the three EOs; moreover, the sensitivity of Staphylococcus spp. strains increased (<0.0195% v/v in 11/22 isolates) when they were assayed with the mixture.
One strain of S. constellatus has been evaluated in this survey. Unfortunately, only one streptococcal isolate was available, therefore it is difficult to verify the real sensitivity of streptococci against the EOs. However, the tested strain was resistant to tetracycline, but wassensitive to oregano, thyme, and savory EOs and their mixture (MIC < 0.0195%). Data about the activity of EOs against this bacterial species are not present in the literature, but in previous studies Origanum compactum, T. vulgaris, S. montana, as well as Cinnamomum verum and Cymbopogon citratus, were active against a human strain of Streptococcus pyogens [34].
Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen able to infect several tissues, including skin and the external ear of pet animals. It is frequently encountered in human hospital-acquired infections, and it has often been found in wastewaters [35]. The natural resistance of this bacterium and the large circulation of multidrug-resistant strains result in the failure of antibiotic therapies in human and veterinary medicine. The 16 P. aeruginosa isolates tested in this survey showed a high level of antibiotic-resistance; these results highlighted that P. aeruginosa strains often represent a serious issue for the health status of dogs for which it is difficult to find an appropriate therapy.
Previous studies investigated different EOs to detect natural antimicrobials with activity against P. aeruginosa antibiotic-resistant strains [35][36][37]. Recently, Van et al. [35] observed antibacterial activity of the thyme EO against P. aeruginosa multidrug-resistant isolates, cultured from human clinical samples and wastewaters. Similar observations have been reported in other investigations. It was observed that red thyme EO was more active against biofilm cells than their planktonic counterparts of both P. aeruginosa and Pseudomonas putida [38]. Moreover, Pandur et al. [39] found a good antioxidant activity of T. vulgaris EOs obtained at different phonologic phases of the plant. In particular, they observed that these EOs and thymol increased catalase and superoxide dismutase activity as well as the antioxidant capacity of the THP-1 macrophages.
Escherichia coli is a Gram-negative bacterium of Enterobacteriaceae family acting as commensal or pathogen in humans and animals. In dogs, it can cause not only intestinal infections, but also infections of genito-urinary tract, skin and external ear canal. Escherichia coli is intrinsically susceptible to the most frequently employed antimicrobials; however, this bacterial species is able to acquire resistance genes, mainly through horizontal gene transfer [40]. The isolates tested in this survey were resistant to two or more antibiotics, confirming that this species is often involved in the antibiotic-resistance issue.
Our results showed that the three selected EOs were active against the tested E. coli strains, except for the strain 856A1 that was not inhibited by T. vulgaris. The best results were obtained when testing E. coli isolates with the mixture; in fact, MIC values were generally lower than those obtained with the singular EOs. To the best of our knowledge, data regarding the activity of EOs against E. coli strains responsible for animal otitis externa are not available in the literature, thus our results are not easily compared to other studies.
Previous papers evidenced the in vitro activity of some EOs against E. coli isolated from other sources. Cinnamomum zeylanicum, O. vulgare, T. vulgaris, and Syzygium aromaticum EOs showed antimicrobial effects when tested against an enteroinvasive E. coli strain [41]. Good activity of O. vulgare and T. vulgaris was also detected against multi-drug resistant E. coli strains isolated from canine urinary tract infections [42].
Klebsiella pneumoniae and S. marcescens are two species belonging to Enterobacteriaceae family, less frequently involved in otitis of animals. The two isolates tested in this study were resistant to different antimicrobials, showing that these bacterial species, even though not frequently involved in canine otitis cases, may be a serious threat for the choice of an effective antibiotic. Few studies about the activity of EOs against these bacteria have been performed [43][44][45][46], and no data about their effectiveness against strains isolated from cases of otitis are available. As a bacteria belonging to the same family of E. coli, and thus having a very similar bacterial cell wall, it is supposable that the same EOs that were active versus E. coli may inhibit K. pneumoniae and S. marcescens, as suggested by our results, which showed the sensitivity of the tested strains versus the three EOs and their mixture.
Our results highlighted sensitivity differences to EOs between Gram-positive and Gram-negative bacteria due to their different cell wall structure. Hydrophobic molecules can get into Gram-positive bacteria and act on the cell wall and cytoplasm, thanks to the cell wall structure [47,48]. Antimicrobial EOs are able to damage the cell wall and cytoplasmic membrane of bacteria, with consequent cell lysis and leakage of intracellular compounds [29]. However, EOs employed in this survey had good activity against all tested isolates and their antimicrobial properties were enhanced when they were used in the mixture.
MIC values of the selected EOs against M. pachydermatis appeared in agreement with the results of studies previously published [33,49]; however, the three EOs in combination did not yield any synergistic effect, suggesting the best efficacy of S. montana alone.
The EO of S. montana were characterized by carvacrol and thymol as major detected compounds. The results partially agreed with different literature studies, all reporting a strong prevalence of the former compound, but also very noticeable amounts of p-cymene and a lower content of the oxygenated monoterpene thymol. This molecule, instead, was the most important one found in the analyzed T. vulgaris EO, whose chemical composition was partially in agreement with the findings observed by Najar et al. [50] for the "Thymol chemotype", in which thymol was found as the most important compound, followed by p-cymene. However, the content of γ-terpinene, which in our work was totally absent, in the cited work reached good amounts, almost accounting for 5%. These findings were in agreement with our previous study [42], also reporting the chemical composition of the EO of O. vulgare. The cited work evidenced a prevalence of carvacrol and p-cymene, consistent with our results, but it did not report the presence of thymol, found, instead, in our sample.

Conclusions
EOs from O. vulgare, S. montana and T. vulgaris were generally active against the main bacterial species involved in otitis externa of dogs. Their in vitro antimicrobial properties were enhanced when they were combined in a mixture, inhibiting the bacterial growth at very low MIC values. Conversely, M. pachydermatis was more sensitive to S. montana EO alone, suggesting an antagonistic effect of the three EOs within the mixture.
Considering that otitis externa is a recurrent or persistent problem in many dogs, due to antimicrobial-resistant bacteria involved in the infections, and thus to the difficulty of finding an appropriate therapy, EOs of O. vulgare, S. montana and T. vulgaris could represent useful alternative remedies. Moreover, the use of a mixture containing the three oils in equal parts may inhibit the bacterial growth using very low EOs concentration.
Previous surveys investigated the antimicrobial properties of oregano, thyme, and savory, but most of them were carried out on reference strains or few clinical isolates; furthermore, data about the sensitivity of bacterial or fungal strains of canine origin to these natural products are almost absent in the scientific literature. Our study gives data about the in vitro activity of oregano, thyme, and savory EOs and their mixture after testing several clinical isolates. The promising results, mainly those regarding the enhanced antibacterial effectiveness of the mixture, suggest the need to perform in vivo studies.

Supplementary Materials:
The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/vetsci10010030/s1, Table S1: Antimicrobial resistance profiles for each examined Gram-positive isolate; Table S2: Antimicrobial resistance profiles for each examined Enterobacteriaceae isolate. Table S3: Antimicrobial resistance profiles for each examined Pseudomonas aeruginosa isolate.