The indiscriminate use of antibiotics in human and veterinary medicine has greatly contributed to the spread of microbial resistance and the increase of unexpected adverse reactions and allergies   . Recently, in order to counteract the development and spread of bacterial resistance, promising prospects are essential oils (EOs), of which we already know the potential validity in the prevention and treatment of various infectious diseases    .
EOs are a complex mixture of organic substances, aromatic oily liquids, biodegradable and with low toxicity, synthesized from plants and characterized by their aroma, generally produced from spices, aromatic herbs, fruits, flowers and plant material (buds, seeds, leaves, twigs, bark, wood, roots). These EOs can be obtained by expression, fermentation and extraction but the method of steam distillation is the most commonly used for their commercial production  .
At present more than 3000 EOs are known, of which about 300 are commercially significant. The greatest use of EOs in the European Union (EU) is in food (as flavourings), perfumes (fragrances and aftershaves) and pharmaceuticals (for their functional properties).
Various studies report that EOs of many aromatic plants, in particular Lamiaceae (Zea, Thymus, Melaleuca alternifolia, etc.), possess antibacterial activity against many multi-resistant microbial species, such as methicillin-resistant Sta- phylococcus aureus (MRSA), vancomycin-resistant Enterococcus (VRE), Pseudomonas aeruginosa, Klebsiella pmeumoniae, ESBL E. coli, Acinetobacter spp, Campylobacter jejuni, Listeria monocytogenes, Bacillus cereus, etc.  -  . Furthermore, other studies show that these EOs also have antifungal activity    . Finally, it has been found that the EOs or their components possess antiviral  , antioxidant   , antiparasitic  and anticancer activity   . The mechanism of action of these EOs is due to the presence of terpenoid components, monoterpenoid (C10) and sesquiterpenoid (C15) metabolites, responsible also for anti-inflammatory effects   . The most effective EOs seem to be those with higher content of alcohols and phenols, but also of aldehydes, therefore an accurate knowledge of the dosage and caution in the administration mode is necessary, to avoid sensitivity phenomena, accumulation and toxicity  .
In vitro and preclinical studies report that the most valid EOs in bacterial infections, with or without fungal overlap, are Thymus vulgaris (red thyme), Melaleuca alternifolia (TTO), Corydothymus capitatus (common oregano), Cinnamomum zeylanicum (cinnamon) and Citrus bergamia (bergamot)  .
Thymus EOs have always been considered rich in beneficial properties for human and animal health and the Egyptians also used it for embalming. Today, there is a strong interest in the biological properties of Thymus Eos for its evident effectiveness in several dermatological, uro-gynecological and gastrointestinal diseases   . The main constituents of Thymus vulgaris EO, responsible for many therapeutic properties, are phenols and, in particular, thymol (30% - 70%), recognized as an antiseptic, antispasmodic and vermicide, used in the preparation of products based on thyme for internal and external use, and carvacrol (3% - 15%), an antiseptic widely used in perfumery; other constituents are linalool, cymene, timene, alpha-pinene and luteolin   . Therefore, this EO possesses antimicrobial, antifungal, anti-inflammatory, expectorant and balsamic activity and is used against inflammation of the airways, such as asthma and cough. Finally, for its antiseptic properties it is also used for acne and for the treatment and disinfection of small wounds   .
In recent years, there has arisen particular interest in the use of some EOs in various human diseases, however there are few studies investigating effects on animal pathogens. In our previous studies, the antimicrobial activity of some EOs (Thymus vulgaris and Eucalypsus globus) was tested in the treatment of ovine mammary pustular dermatitis and livestock mastitis documenting the effectiveness of the Thymus vulgaris EO in this disease    .
The aim of this investigation was to study in vivo the therapeutic effects of Thymus vulgaris EO for topical use against pathogen strains in skin infections of dogs housed in a rescue shelter.
2. Materials and Methods
The study was carried out during the summer 2015 on 18 half-breed dogs (3 - 10 years old and body weight 15 - 20 kg), suffering from skin diseases, housed in a rescue shelter located in Messina (Southern Italy). The animals, fed with a commercial dry food and free of antibiotics, were selected based on evident skin lesions among 120 examined dogs.
2.2. Clinical Signs
The dogs affected by skin infections showed clinical signs such aspruritus, alopecia and/or erythema, presence of pustules or crusts, ulcers and/or erosions. The lesions were observed at the level of armpit, abdomen, groin and thigh. General symptoms included slightly raised body temperature, anorexia and depression. Diagnosis of skin infection in dogs was performed by clinical signs, cytological, bacteriological and mycological investigations and skin scrapings for parasites on cutaneous samples.
2.3. Sampling Collection
Dog skin samples were obtained from pustules and/or epidermal collarettes, using sterile swabs and plastic brushes, respectively for bacteriological and mycological surveys, and skin scrapings using sterile scalpels for cytological investigation and scrapings for parasites. The samples were transferred under sterile conditions and examined at the laboratory of infectious diseases of the University of Messina.
2.4. Cytological Examination
Samples of skin scrapings from affected dogs, taken with sterile disposable scalpels, were streaked onto slides, dried, stained with May Grunwald-Giemsa and microscopic examination was performed at high power under oil immersion (1000×).
2.5. Bacteriological Investigations
Within 1 hour from collection, the skin swab samples were transferred into test tubes of Selenite-broth (Biolifes. r. l., Milano, Italy) and incubated to 37˚C for 24 hours. The next day, from each tube was removed the bacterial inoculum and swiped into the plates containing the following selective and differential media, SS-Salmonella-Shigella agar (Oxoid, Basingstoke, Hampshire, UK) e SM-Staphy- lococci 110 medium (Biolifes. r. l., Milano, Italy ), and then incubated to 37˚C for 24 hours, for the isolation of Gram-negative and Gram-positive bacteria respectively. Afterwards, the isolated colonies were transferred into tubes containing BHI-Brain Heart Infusion broth (Biolifes. r. l., Milano, Italy) for the identification and into tube slant containing Mueller Hinton II agar (Biolifes. r. l., Milano, Italy) for the conservation of the strains and incubated at 37˚C for 24 h. The isolated colonies were submitted to rapid bacterial identification by Mass Spectrometry VITEK MS (bioMérieux SA, Marcy l’Etoile, France). The samples for bacteriological investigations were performed before and 3, 5, 7 and 15 days after the treatment.
2.6. Identification of Bacterial Isolates by MALDI-TOF-MS
The bacterial isolates were identified by MALDI-TOF MS (Matrix Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry), designed to provide an accurate and reliable bacterial identification rapidly. This procedure allows the measurement in an extremely accurate molecular weight of biological macromolecules and to determine their identity according to the mass/charge ratio. In particular, MALDI-TOF-MS is valid for the detection of high-abundance soluble proteins, including ribosomal and other structural proteins, directly from microbial cells, resulting in spectra that are analyzed with the VITEK MS system (bioMérieux SA, Marcy l’Etoile, France), using for reading the software Axima (Shimadzu Kyoto, Japan)-SARAMIS database (Spectral ARchive And Microbial Identification System) (AnagnosTec, Berlin, Germany). The spectra were acquired in positive linear mode in the range of m/z 2000 to 20,000. The isolated colonies were seeded in a 48-well metal plate with disposable loops, using as a reference strain Escherichia coli ATCC 8739.
2.7. In Vitro Antimicrobial Activity
The susceptibility of the isolated microorganisms to Thymus vulgaris EO was estimated by the in vitro bacteriological test using the disk diffusion method (Kirby-Bauer) in Mueller Hinton II agar (Biolifes. r. l., Milano, Italy) and measuring the diameter of the growth inhibition area, as recommended by CLSI (Clinical and Laboratory Standards Institute 2015)  . In particular, in vitro activity of Thymus vulgaris EO on the bacterial strains isolated from skin samples of dogs was evaluated in comparison to Citrus bergamia EO and some antimicrobial drugs widely used in veterinary medicine (amoxicillin-clavulanic acid, doxycycline, thiamphenicol and marbofloxacin) (Oxoid). Specifically, paper disks (6 mm diameter) were loaded with 20 µl (18 µg) of antimicrobial drugs, mixed with Tween 80 (Sigma Tau) (1:1 v/v) and then placed on the surface of the Muller Hinton II agar, inoculated with a suspension of the various isolated microorganisms (0.1 ml of 108 CFU/ml). The plates were incubated at 37˚C for 24 h, subsequently the diameter (mm) of the growth inhibition areas were measured. All the assays were performed in duplicate. The microorganisms were considered very susceptible, susceptible, poorly susceptible and resistant when inhibited in vitro by concentrations of 0.56 µg/ml or less, 0.57 - 6.25 µg/ml, 6.25 - 12.5 µg/ml and greater than 12.5 µg/ml respectively.
2.8. Research for Mites and Mycological Investigations
Samples of skin scrapings and hairs were taken from the dogs using a sterile scalpel, then streaked on a specimen slide with the addition of a drop of KOH (10%) and sealed with coverslip and then observed microscopically (40× - 60×).
Afterwards, various cutaneous samples were collected from the dogs using sterile plastic brushes and were sealed in individual sterile bags. Subsequently, the brushes were impressed on Petri dishes containing Potato Dextrose agar (Biolife). The growth of fungal colonies was kept under observation for 7, 15, 21 and 30 days. Suspect colonies which developed (white, smooth, raised, not mucous) only in samples n. 3, 7 and 8 were seeded in Dermatophyte Selective Medium (Biolife).
The EOs tested (Thymus vulgaris L. and Citrus bergamia Risso & Poiteau) were purchased directly from the market (KOS srl, Lab. di Erboristeria, Prato, Italy).
2.10. Pharmacological Treatment
The present investigation with an extra-label use of Thymus vulgaris essential oil in dogs was approved by Commission on Drug Experimentation of the University of Messina and carried out in accordance with local ethical regulations for veterinary practice    and with the prior informed consent of the kennel veterinarian.
The use of topical treatments on dogs were reported in the register of the rescue shelter “Millemusi” (Messina).
The dosage of T. vulgaris EO (20 ml/1000ml H2O) was prepared in accordance with the manufacturer’s instructions. In particular, for this trial the use of a positive control group (treated with Citrus bergamia EO (20 ml/1000ml H2O) rather than an untreated group was applied according to regulations for veterinary practice. Dogs were blocked in pairs and each animal assigned to one or other group at random. The two groups of animals were subjected to topical treatment using a sprayer.
In particular 4 ml of T. vulgaris EO (Group 1: n. 10 animals) and C. bergamiaEO (Group 2: n. 8 animals) respectively were sprayed in the areas affected by lesions for 7 days consecutively. Dogs with complete disappearance of clinical signs of skin infection were subjected to further preventive treatment for 10 days with T. vulgaris EO (Group 1). To prevent licking a collar was applied to each dog.
2.11. Clinical Score
Effectiveness of essential oils was evaluated on the basis of the changes in the animal’s clinical signs, observed before treatment (T0) and 3, 5, 7 and 15 days after the drugs administrations, using a scores system  as follows:
・ pruritus (0 absent; 1 slight; 2 moderate; 3 marked).
・ alopecia and/or erythema (0 absent; 1 slight; 2 moderate; 3 marked).
・ pustules and/or crusts (0 absent; 1 slight; 2 moderate; 3 marked).
・ ulcers and/or erosion (0 absent; 1 slight; 2 moderate; 3 marked).
・ general health (0 good, 1 quite good, 2 fair, 3 poor).
The effectiveness of Thymus vulgaris EO (Group 1) and Citrus bergamia EO (Group 2) was evaluated by comparing the clinical signs in dogs and the microorganisms isolated from skin swab samples before and to 3, 5, 7 and 15 days after the treatment. All animals were kept under observation during the experiment to check for possible side effects.
2.12. Statistical Analysis
The data were expressed as mean ± S.D. elaborated using an analysis of variance (ANOVA) InStat 3.0 software (GraphPad Software San Diego, CA, USA) was applied to assess the significance of the difference between the scores recorded before and 3, 5, 7, and 15 days after the treatment with Thymus vulgaris and Citrus bergamia EOs respectively. Difference with p < 0.05 were considered significant.
3.1. Cytological Examination
The cytological investigation of skin samples performed on 18 dogs with skin diseases highlighted the presence of inflammatory cells (neutrophils, lymphocytes, plasma cells and bacteria) (Figure 1). This cytological pattern, found in all the dogs, is indicative of the presence of a skin inflammation process.
3.2. Bacteriological Test
The bacteria isolated by microbiological test from the skin swab samples of 18 dogs affected by bacterial skin disease were: Staphylococcus pseudintermedius (n. 7 samples), Staphylococcus sciuri (n. 4), ESBL Escherichia coli (n. 3) and
Proteus mirabilis (n. 4).
3.3. In Vitro Antimicrobial Activity
The in vitro susceptibility of the isolated microorganisms in dogs to Thymus vulgaris and Citrus bergamia EOs and other antimicrobials widely used in veterinary medicine amoxicillin-clavulanic acid, doxycycline, thiamphenicol and marbofloxacin) is reported in Table 1. Antimicrobial activity test in vitro showed that Thymus vulgaris EO is the most active antibacterial drug in the comparative analysis.
3.4. Bacteria Findings in Skin Swabs Samples during Pharmacological Treatment
The bacteriological findings in the skin samples of dogs before (T0 baseline) and
Figure 1. Cytological appearance of inflammatory cells (neutrophils, lymphocytes, plasma cells and bacteria) in skin samples of dog with skin diseases.
Table 1. Susceptibility to various antibacterial drugs of Gram-positive and -negative microorganisms isolated from skin samples collected from 18 dogs with skin infections.
+++ Highly susceptible (MIC ≤ 0.56 µg/ml); ++ Susceptible (MIC 0.57 - 6.25 µg/ml); +Poorly susceptible (MIC 6.26 - 12.5 µg/ml); - Resistant (MIC > 12.5 µg/ml).
3, 7 and 15 days after the end oftopical treatment for 7 days with Thymus vulgaris and Citrus bergamia EOs respectively are indicated in Table 2. The severity of the bacterial skin infection of dogs was expressed in the conventional manner (from + to ++++) in relation to the quantity of bacteria present (Table 1). No microorganisms were isolated in all skin samples of dogs at 7 and 15 days after the end of Thymus vulgaris EO treatment. No variation in the bacterial load, instead, was recorded in the animals treated with Citrus bergamia EO.
3.5. Skin Scrapings for Mites and Mycological Investigations
The skin scrapings for mites and mycological investigations for dermatophytes in all skin samples of dogs were negative.
3.6. Variations in Clinical Scores Recorded during Antibiotic Treatments
Table 2. Bacteria isolated from skin samples before treatment (T0 baseline) and 3, 5, 7 and 15 days after topical sprayed for 7 days with T. vulgaris and C. bergamia EOs, respectively, in dogs affected by skin infection.
++++ = 106 colony forming units (cfu); +++ = 105 cfu; ++ = 104 cfu; + = 103 cfu.
Table 3. Effects of topical treatment with Thymus vulgaris EO for 7 days on the mean (±S.D.) scores of clinical signs in 10 dogs with skin infections.
*p < 0.01; **p < 0.05.
in dogs affected with skin infection before and at various time after the treatment with Thymus vulgaris and Citrus bergamia EOs respectively.
In Figure 2 is reported the trend of visible topical effect before, during and after the treatment with Thymus vulgaris EO (Group 1). The dogs with skin infection (Figure 2(a)) showed a moderate decrease of dermic clinical signs after 3 days but significant after 5 days (Figure 2(b)); an evident remission of disease it was observed after 7 days (Figure 2(c)), with complete disappearance of ulcers and erosions and initial regrowth of hair, followed by skin normalization. The treatment with Citrus bergamia EO (Group 2), instead, showed no significant effects in dogs with bacterial diseases of the skin and the clinical signs persisted markedly for the duration of our investigations.
No local or general signs of Thymus vulgaris EO intolerance were recorded in any treated dogs and no relapses occurred in the month following treatment.
Our study, original for the results obtained, documents the potential therapeutic efficacy of Thymus vulgaris EO in skin infections caused by Gram-positive and -negative bacteria of dogs. In particular, this study shows an improvement in the clinical symptoms after 3 days of treatment with complete healing after 7 days. Furthermore, no local or general adverse reactions were observed during the
Table 4. Effects of topical treatment with Citrus bergamia EO for 7 days on the mean (±S.D.) scores of clinical signs in 8 dogs with skin infections.
(a) (b) (c)
Figure 2. Trend of visible topical effect in dog with skin infection (Case N. 2, Group1): (a) Before of Thymus vulgaris EO treatment; (b) 5 days and (c) 7 days after.
various stages of therapeutic treatment. Citrus bergamia EO used, in comparison, as a positive control in dogs for skin infections caused by Gram-positive and -negative bacteria, instead, showed no therapeutic effects.
The mechanism of antimicrobial activity of Thymus vulgaris EO is quite complex and, to date, has not been well elucidated. It is very likely that the antibacterial action of this EO at the cellular level, for the qualitative and quantitative content of various organic compounds, is unrelated to a single mechanism  . No clinical studies are reported in literature on EOs use in animal deseases; the few data available related only to in vitro study are scarce and inadequate to a possible application in veterinary medicine and little is known about their safety after oral or parenteral administration. In our previous investigation, the therapeutic efficacy of Thymus vulgaris essential oils has been documented in ovine mammary postural dermatitis  but currently, there are no studies on the topical use in dog skin infections. Anyway, the topical use of EOs on the skin and mucous membranes appears to be the most promising strategy to prevent or treat skin infections.
The results of our investigations show that the topical use of Thymus vulgaris EO, also for its low cost, could constitute a possible alternative or a support to antibiotic-therapy in dermatological infections, in particular in cases refractory to conventional therapy. In fact, this EO, in addition to showing a good antimicrobial activity, has other biological and therapeutic properties (antifungal, anti-inflammatory, etc.). It is believed also extremely unlikely that bacteria can develop drug resistance to EOs, which consist of several molecular components, compared to an antibiotic consisting of a single active substance  . Moreover, it seems that EOs can effect synergistic activity with antibiotics, improving their therapeutic efficacy   .
In conclusion, this study documents for the first time the effectiveness of Thymus vulgaris EO for topical use in dogs with skin infections. It is important to note that the follow-up with Thymus vulgaris EO (7 days) is quite short as it is not an antibiotic and the speed of therapeutic effect is related to the high content of antioxidant substances, which possess antimicrobial, antifungal, anti-inflam- matory effects, etc., as widely reported in literature. However, to evaluate the optimal use of this EO in skin infections of the dog, further studies are needed, in particular on a greater number of cases.
Conflict of Interest Statement
The authors declare no conflict of interest.
Sources of Funding
Funding was provided by the Istituto Zooprofilattico Sperimentale “A. Mirri” of Palermo (Italy).
The authors are grateful to Dr. Alessandro Taormina, veterinarian in charge of the rescue shelter of Messina (Southern Italy).
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