Antimicrobial Activity of Essential Oils against Staphylococcus and Malassezia Strains Isolated from Canine Dermatitis

Staphylococcus spp. bacteria are the most frequently involved agents in canine cutaneous infections. Treatment of these infections is based on antibiotic therapy, that often is not effective because of the antibiotic-resistance of the bacterial strains. Cutaneous staphylococcal infections are often complicated by Malassezia yeasts, that may be resistant to the conventional antifungal drugs. The present investigation was aimed to evaluate the in vitro antimicrobial activity of some essential oils (EOs) in view of a potential cutaneous application. In detail, EOs obtained from lemon verbena (Aloysia triphylla L’Hèr. Britton), cinnamon (Cinnamomum zeylanicum J. Presl), myrrh (Commiphora myrrha (Nees) Engl. var. molmol), lemongrass (Cymbopogon citratus (DC.) Stapf), litsea (Litsea cubeba (Lour.) Pers.), lemon balm (Melissa officinalis L.), oregano (Origanum vulgare L.), savory (Satureja montana L.), and thyme (Thymus vulgaris L.) were assayed against Staphylococcus spp. and Malassezia pachydermatis strains previously isolated from dogs with dermatitis. All EOs were tested by agar disk diffusion and minimum inhibitory concentration methods to verify the antistaphylococcal activity, and by a microdilution method to evaluate the activity against M. pachydermatis. O. vulgare, T. vulgaris, and S. montana showed the best antibacterial activity against all the selected strains, with MICs ranging from 0.29 to 0.58 mg/mL, from 0.58 to 1.16 mg/mL, and from 0.56 to 1.12 mg/mL, respectively, whereas A. triphylla (1.03 mg/mL) and S. montana (1.8 mg/mL) were the most active against M. pachydermatis. After a proper in vivo evaluation, O. vulgare, T. vulgaris, and S. montana EOs could be a promising treatment to combat canine cutaneous mixed infections.


Introduction
Bacterial infections are responsible for the most frequent disorders of the skin of companion animals, mainly dogs. In particular, pyoderma is a pyogenic infection that can affect epidermis and hair follicle units or dermis and subjacent fatty tissue. Atopic dermatitis is a genetically predisposed inflammatory and pruritic allergic skin disease in which the skin microbiome may cause secondary infections that can influence its severity [1,2]. In both cases, Staphylococci are the most frequently involved agents: S. pseudointermedius is considered the primary canine cutaneous pathogen, but other Staphylococcus species may be found in skin infection, as well as Escherichia coli, Proteus spp., and Pseudomonas spp.
Treatment of canine bacterial skin infections is usually based on antibiotic therapy, which is often not effective because of the involvement of antibiotic-resistant bacterial strains.
All EOs (FLORA ® , Pisa, Italy), were maintained in dark glass vials at 4 • C until used in the different experiments.
Quality control for antibacterial and antimycotic activity was tested for each EO before the analyses. For this purpose, each EO was streaked onto a blood agar plate, and the plates were incubated at 37 • C for 48 hours. Absence of colonies after the incubation period confirmed the EOs sterility.

Essential Oils Analysis
The hydrodistilled essential oils were diluted to 0.5% in HPLC-grade n-hexane and then injected into a GC-MS apparatus. Gas chromatography-electron impact mass spectrometry (GC-EIMS) analyses were performed with an Agilent 7890B gas chromatograph (Agilent Technologies Inc., Santa Clara, CA, USA) equipped with an Agilent HP-5MS (Agilent Technologies Inc., USA) capillary column (30 m × 0.25 mm; coating thickness 0.25 µm) and an Agilent 5977B single quadrupole mass detector (Agilent Technologies Inc., USA). Analytical conditions were as follows: injector and transfer line temperatures of 220 and 240 • C, respectively; oven temperature programmed from 60 to 240 • C at 3 • C/min; carrier gas helium at 1 mL/min; injection of 1 µL (0.5% HPLC grade n-hexane solution); split ratio 1:25. The acquisition parameters were as follows: full scan; scan range: 30-300 m/z; scan time: 1.0 s. 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) and laboratory-developed mass spectra library built up from pure substances and components of known oils and MS literature data [13][14][15][16][17][18].

Bacterial Strains
A total of eight Staphylococcus spp. strains were tested in vitro for antimicrobial sensitivity. All strains were previously isolated from skin of dogs with dermatitis and typed using the API Staph system (BioMerieux, Milan, Italy). In detail, the isolates were 1 S. aureus, 1 S. pseudointermedius, 1 S. hyicus, 2 S. chromogenes, and 3 S. xylosus.
The isolates were kept in collection at −80 • C in glycerol broth. Each strain was inoculated into brain hearth infusion broth (BHIB, Oxoid Ltd., Basingstoke, Hampshire, UK) and incubated at 37 • C for 24 h. Cultures of 1-2 × 10 7 CFU/mL, corresponding to 0.5 McFarland standard, were employed in the tests.

Agar Disk Diffusion Method
Antibacterial activity of the selected EOs was tested by Kirby-Bauer agar disk diffusion method following the procedures reported by Clinical and Laboratory Standards Institute (CLSI) [19]. Briefly, EOs were 5% diluted in dimethyl sulfoxide (DMSO, Oxoid Ltd.), and one absorbent paper disk was impregnated with 10 µL of each dilution, respectively, and tested against each isolate.
A paper disk impregnated with 10 µL of DMSO was included as negative control. A commercial disk impregnated with chloramphenicol (30 µg) (Oxoid) was used as positive control. Growth inhibition zones were evaluated after incubation at 37 • C for 24 h. All tests were performed in triplicate.
The in vitro sensitivity of all Staphylococcus isolates to chloramphenicol (30 µg) (Oxoid) was assayed by the same method, and the results were interpreted as indicated by CLSI [20].

Minimum Inhibitory Concentration
Minimum inhibitory concentration (MIC) was determined for all EOs with the broth microdilution method, following the guidelines of CLSI [21] and the protocol previously described [22]. Briefly, the test was performed in 96-well microtiter plates in a total volume of 200 µL/well including 160 µL of BHIB (Oxoid), 20 µL of each bacterial suspension, and 20 µL of each EO. The MIC value was determined as the lowest concentration, expressed in percentage and mg/mL, of each EO at which staphylococci show no visible growth. The same assay was performed simultaneously for bacterial growth control (tested agents and media) and sterility control (tested oil and media). Positive control using chloramphenicol (Oxoid) was also included. All tests were performed in triplicate.

Antifungal Activity
Five M. pachydermatis clinical isolates were tested in vitro for the antimicrobial sensitivity. The strains were previously cultured from skin of dogs with atopic dermatitis.
The antifungal activity of selected EOs was assessed by microdilution method, using liquid m-Dixon medium for preparing yeast suspensions, as reported elsewhere [23]. The yeasts were tested against ketoconazole by E-test (AB Biodisk, Solna, Sweden) to evaluate the efficacy of a conventional antimycotic drug, currently employed for the treatment of Malassezia infections [6].

Essential Oil Composition
Taken in consideration compounds present in percentage equal to or greater than 1% in at least one of the investigated EO, 49 compounds were identified (Table 1), representing 99.4%-100% of the total identified fraction. The oxygenated monoterpenes was the main class of compounds in seven out of nine species tested in this work (A. triphylla, C. citratus, T. vulgaris, L. cubeba, S. montana, M. officinalis, and O. vulgare), with a percentage ranging from 34.7% (A. triphylla) to 88.2% (C. citratus). T.vulgaris and S. montana shared the same amount of oxygenated monoterpenes and monoterpene hydrocarbons, with a slight predominance of oxygenated monoterpenes (53.1/35.2 and 47.6/39.8, respectively).
Among the Lamiaceae plants, only the A. triphylla sample differed from the others because monoterpene hydrocarbons were the main class of constituents (62.2%). Limonene (31.1%) and sabinene (26.0%) were the principal compounds.
Both of L. cubeba and C. zeylanicum belong to Lauraceae family, but they evidenced a great difference in their composition. In fact, L. cubeba EO was characterized by a high percentage of oxygenated monoterpene compounds (80.8%), while phenylpropanoids prevailed in C. zeylanicum EO (66.7%) where (E)-cinnamaldehyde (63.2%) was the main component.

Agar Disk Diffusion Method
The nine EOs tested in this study showed different degrees of growth inhibition against the assayed Staphylococcus isolates. The strongest antibacterial activity was observed with O. vulgare and T. vulgaris EOs: the overall inhibition zone ranged from 9.0 to 13.0 mm and from 7.0 to 22.0 mm, respectively. On the other hand, the lowest activities were shown by C. zeylanicum and C. myrrha EOs.
C. myrrha EO was not active against the three S. xylosus isolates, and the remaining EOs showed weak effectiveness against them.
No growth inhibition zone was observed with DMSO as negative control, whereas chloramphenicol, included as positive control, resulted effective against all isolates. Results are summarized in Table 2.

Minimum Inhibitory Concentration
The minimum inhibitory concentration (MIC) values, expressed both as percentage and mg/mL, testing EOs versus the Staphylococcus spp. isolates are reported in Table 3. O. vulgare, T. vulgaris and S. montana showed good antibacterial activity against all the selected strains, with MICs ranging from 0.29 to 0.58 mg/mL for O. vulgare, from 0.58 to 1.16 mg/mL for T. vulgaris, and from 0.56 to 1.12 mg/mL for S. montana.
C. zeylanicum and C. myrrha had the lowest antistaphylococcal activity with MIC of 10.2 mg/mL versus six isolates and of 10.0 mg/mL versus five isolates, respectively.
A. triphylla showed not high MICs varying from 2.17 to 8.7 mg/mL in relation to the tested isolate. The remaining EOs showed a weak activity: 0.55-2.23 mg/mL for C. citratus, 1.10-4.42 mg/mL for L. cubeba, and 1.11-2.22 mg/mL for M. officinals.
No growth inhibition was observed with the negative control, whereas chloramphenicol resulted active against all strains.

Antifungal Activity
Selected EOs showed different degrees of efficacy against M. pachydermatis isolates (Table 3). In detail, C. myrrha and L. cubeba were not effective at 5% dilution. A. tryphilla was the most active with MICs of 0.87 and 1.03 mg/mL, followed by S. montana with MIC of 1.8 mg/mL and C. zeylanicum with 3.06 and 4.08 mg/mL.

Discussion
The results obtained in the present survey showed different antimicrobial activity degrees in relation to the EO and the bacterial or fungal isolates assayed.
Different staphylococcal species, all previously isolated from dogs with skin infections, were examined in our study. Even though S. pseudointermedius is considered the primary canine cutaneous pathogen [24], other staphylococcal species may be involved in pyoderma and atopic dermatitis. In fact, bacteriological examinations for some canine clinical cutaneous cases allowed us to isolate, other than S. pseudointermedius, also S. aureus, S. chromogenes, S. hyicus, and S. xylosus, which are usually related to infections in other animal species.
S. chromogenes, a coagulase-negative species, causes mastitis in dairy animals [25]. S. hyicus, considered a coagulase-variable species, is mainly found in pigs, but it is also frequently isolated from canine specimens [26]. S. xylosus is a coagulase-negative staphylococcal species considered as commensal and able to colonize the skin of mammals and birds [27]. Moreover, it is reported as the most frequently isolated coagulase-negative staphylococcus from skin and mucous membrane of healthy dogs [28]. S. aureus is the main pathogen responsible for mastitis in dairy animals [29], as well as it is involved in infections of different anatomic districts in humans, as well as farm and companion animals [30]. Moreover, it is a major food poisoning microorganism posing risk to consumer health, mainly through its production of heat-stable enterotoxins [31].
O. vulgare and T. vulgaris EOs resulted very active against all staphylococcal strains tested in the present survey. These results are corroborated by other studies that found high antimicrobial activity against several bacterial species, including Staphylococcus spp. [11,12,32,33].
The antibacterial effect has been related to the major compounds of these two oils, in particular carvacrol and thymol [34,35]. Exposure of bacterial cells to carvacrol has resulted in increases in the membrane fluidity and leakage of protons and potassium ions, leading to a decrease in pH gradient across the cytoplasm membrane, a collapse of the membrane potential, an inhibition of ATP (adenosine triphosphate) synthesis, and ultimately cell death [36].
As regards thymol, it has been speculated that its antimicrobial effect may result, at least in part, from a perturbation of the lipid fraction of the bacterial plasma membrane resulting in the leakage of intracellular materials [37].
S. montana EO showed a very good anti-staphylococcal activity, too. Antimicrobial effectiveness of S. montana EO against some Gram-positive and Gram-negative bacteria was previously observed, and it was related to major compounds, such as carvacrol. In particular, Vitanza et al. [38] found that S. aureus, submitted to the action of S. montana EO, showed collapse of cell wall without breaks.
Our study confirmed thymol and carvacrol as main constituents of the EOs with the best antibacterial activity: 40.5% of thymol in T. vulgaris and 38.2% and 66.5% of carvacrol in S. montana and O. vulgare, respectively. Moreover, T. vulgaris and S. montana EOs had the highest amount of p-cymene (19.4% and 14.8%, respectively), a monoterpene compound with antimicrobial properties [39].
EOs from C. myrrha and C. zeylanicum showed the lowest activity against the bacterial isolates, mainly against the three S. xylosus strains. No relevant differences were observed testing C. myrrha and C. zeylanicum against the remaining staphylococcal isolates. In detail, C. zeylanicum was active against six isolates of the eight tested with high MIC value (10.2 mg/mL). This result is in contrast with the good antibacterial activity of C. zeylanicum EO previously observed against S. aureus [40]. Similarly, C. myrrha EO was effective against five of the tested isolates with 10 mg/mL MIC, and it was not active against the three tested S. xylosus strains. Mahboubi and Kazempour [41] found relevant activity of C. myrrha against a S. aureus ATCC strain, whereas Adam and Selim [42] observed slight sensitivity of S. aureus to myrrh oil.
C. citratus, L. cubeba, and M. officinalis EOs showed quite similar effectiveness. Anti-S. aureus activity of these EOs was previously reported by other authors. In particular, lemongrass and lemon balm were found more active against Gram-positive bacteria, such as S. aureus, than Gram-negative ones [43,44]. Litsea was demonstrated to be an effective bacterial inhibitor and bactericide against methicillin-resistant S. aureus for a destructive effect on the bacterial cell membrane [45].
Scant information about the anti-staphylococcal activity of A. tryphilla is available; however, its effectiveness against reference S. aureus strains has been observed [46][47][48]. A. triphylla EO does not provoke whole cell lysis of S. aureus but compromises the structural integrity of the plasmic membrane and induces a loss of the cytoplasmic contents, with consequent cellular death [48].
A. triphylla EO showed a strong anti-Malassezia activity. To the best of our knowledge there are no studies regarding the activity of this EO against Malassezia. Nevertheless, lemon verbena EO has been reported as active against different fluconazole-resistant Candida spp. isolated from human patients, with MIC values higher (35-140 mg/mL) than the value observed in the present study. Moreover, this EO showed a good activity against Aspergillus fumigatus in a previous study [49] and a poor effectiveness versus the probiotic yeast Saccharomyces cerevisiae [50], indicating a variable efficacy against different species of both molds and yeasts. The antifungal activity seems to be related to a higher content of limonene and sabinene, in comparison with the other EOs. For these reasons A. triphylla EO would appear of great interest when used as antimycotic compound, paying attention to the fungal species involved.
S. montana showed MIC values of 1.8 mg/mL, resulting more effective in comparison with the well-known antimycotic compounds from O. vulgare and T. vulgaris. These results are not in agreement with a previous study [51], where O. vulgare showed a very low MIC against malassezia isolates from canine dermatitis.
This finding is of interest, considering that this EO also appeared active against staphylococci. S. montana has been recently reported as active against M. pachydermatis recovered from canine otitis [52], Candida albicans [53], and Candida glabrata [54] and moderately effective against dermatophytes [55], suggesting a good activity against yeasts.
Another interesting feature is the high sensitivity of Malassezia to C. zeylanicum. These results are in agreement with findings reported by Bismarck et al. [52] and Sim et al. [56] in otologic canine isolates. This EO, in fact, although poorly effective against molds such as A. fumigatus [49] and dermatophytes [55], showed a strong antimicrobial activity versus Salmonella enterica serotype Typhimurium and Escherichia coli isolated from poultry [50].
On the other hand, Malassezia yeasts showed a marked variability in their sensitivity to EOs. For these reasons, a sensitivity assay of the fungal isolates is recommended, as suggested by Bismarck et al. [52].

Conclusions
The overuse of antibiotics has led to the extensive antibiotic resistance in pathogenic bacteria, including staphylococci, of human and veterinary concern. In this view, natural products such as EOs with antimicrobial properties could represent a suitable alternative in the treatment of infections, mainly when conventional drugs resulted not effective.
Our results underlined and corroborated the variability of the EOs' activity in relation not only to the bacterial species, but also to the isolates [10]. Consequently, there is not always a correspondence between results obtained with reference and wild strains. Even though the antimicrobial activity of a given EO has been previously determined, an in vitro antibacterial/antifungal sensitivity test should always be performed to better verify the effectiveness of the EO against the studied strains.
Our in vitro study showed the activity of O. vulgare and T. vulgaris EOs against cutaneous staphylococcal isolates, and the good effectiveness of S. montana EO against both staphylococcal and M. pachydermatis strains. After a proper in vivo evaluation, these EOs could be a promising treatment to combat canine cutaneous mixed infections due to these pathogens.
To the best of our knowledge, this is the first study that found, among EOs of which antimicrobial activity has already been defined, the natural product active versus both staphylococcal and Malassezia strains involved in canine cutaneous infections.