In vitro and in vivo evaluation of a new phytotherapic blend to treat acute externa otitis in dogs

Abstract Canine otitis externa is frequently encountered in veterinary practice, caused by primary factors with bacteria and yeast overgrowth acting as secondary and perpetuating factors. The pharmacological support includes anti‐inflammatory, antimicrobials, and antimycotic drugs, but therapeutic failure and antimicrobial resistance are leading to alternative strategies based on phytotherapic products. This study aimed to evaluate an essential oil blend (Otogen®) to treat otitis externa in dogs. The experimental design was divided in: (a) an in vitro approach, based on the European Normative UNI EN 1275:2006, to assess the efficacy of the product against the most frequently isolated microorganisms during otitis externa. (b) an in vivo part, 12 owned dogs presenting with acute otitis externa were enrolled. A significant growth reduction (>99.9%) of Malassezia pachydermatis and Candida albicans after 15 min of contact and Pseudomonas aeruginosa after 1 h of incubation was recorded. For Staphylococcus pseudintermedius, 50% of growth reduction were appreciated after 15 min. Results obtained in vivo after 7 days of blend administration, noted a significant improvement of all the considered parameters (most important were head shaking, erythema, and scraping). The results obtained may support the usefulness of the tested phytotherapic blend to manage acute otitis externa in dogs.


| INTRODUC TI ON
Canine otitis externa (OE) is an inflammatory pathology commonly reported in veterinary clinical practice. During the early stages, inflammation results in erythema of the pinnae, external meatus, and lining of the external canal. Subsequently, there can be a wide range of clinical signs, such as head shaking, ear scratching, ceruminous, or purulent discharge, excoriations due to self-trauma, malodor, swelling, and pain. In recurrent or chronic cases, clinical signs may progress to proliferative changes leading to stenosis of external ear canal, and ultimately to occlusion (Guarda et al., 2013).
In such a scenario bacteria or yeasts act as perpetuating causes of the inflammatory process, since they are not responsible for the initiation of the OE but permit to continue once established and can lead to pathology chronicity (Bajwa, 2019;Guaguère & Prèlaud, 2005). Staphylococcus and Pseudomonas are bacteria able to produce | 911 VERCELLI Et aL. biofilm that acts as protection and can lead to therapeutic failure (Bajwa, 2019), while among yeasts, the species by far more prevalent is Malassezia pachydermatis (Guillot & Bond, 2020).
In clinical practice, most cases of acute OE are managed using polyvalent topical ear products that include a glucocorticoid (used to control mild acute inflammation), and antimicrobial agents used to treat concurrent infections (Nuttall, 2016). Various detergents, such as ethylenediaminetetraacetic acid (TrizEDTA) and chlorhexidine, are largely used to clean the ear canal, remove debris, and excessive cerumen production and to disrupt biofilm (Guardabassi et al., 2009;Pye, 2018). Antibiotics, especially fluoroquinolones, aminoglycosides, and polymyxins, should be used only after the identification of etiological agents and only after ascertaining the integrity of the tympanic membrane (Ghibaudo et al., 2015). Treatments against M.
pachydermatis include antifungal drugs like azole derivatives (thiabendazole, clotrimazole, miconazole, and itraconazole), nystatin, and terbinafine. The main causes of therapeutic failure are incorrect patient medication management, and the lack of identification of primary conditions (Nuttall, 2016) and progressing to otitis media force to switch to systemic therapy (Six et al., 2000).
Antimicrobial resistance (AMR) is a global threat for humans and animals, with a significant public health risk due to AMR transmission between these two populations considering the side-by-side style of life among humans and pets (WHO, 2015). Antimicrobial stewardships have been recently proposed in veterinary medicine and should be emphasized for frequently diagnosed pathologies (Vercelli et al., 2021), such as canine otitis externa (Chan et al., 2020). Resistance has been demonstrated for bacteria that can alter target sites, increased drug outflow, and enhance enzymatic degradation (Wright, 2005), and other mechanisms have been recently described also for M. pachydermatis (Angileri et al., 2018;Peano et al., 2017;Peano et al., 2020). Considering all the aforementioned factors, it does not surprise the increasing interest in alternative therapies. Medicinal plant-derived products represent today between 25% and 50% of pharmaceutical products (Gerwick, 2013;Gupta & Birdi, 2017).
The antimicrobial properties of medicinal plant extracts come from the large variety of secondary metabolites. These are intermediate or final products of plant metabolism, not fundamental for life plant processes, playing a defensive role toward bacteria, fungi, protozoa, and viruses (Gorlenko et al., 2020). Secondary metabolites include quinines, alkaloids, lecithins, polypeptides, flavones, flavonoids, coumarin, terpenoids, essential oils, and tannins (Chandra et al., 2017). The applications of plant-derived products are increasing, and mainly direct to treat parasitic disease and skin pathologies (López et al., 2019;Tresch et al., 2019). Some studies have been performed in the last years to investigate the efficacy of essential oils (EOs) in otitis externa in dogs. Still, the majority are in vitro, and only one is in vivo Sim, Khazandi, Pi, et al., 2019;Song et al., 2020).
The present study aimed to investigate the efficacy of a new phytotherapic blend containing essential oils with a double approach: first an in vitro evaluation on the most frequently diagnosticated microorganisms in case of canine otitis externa was performed. Then an in vivo trial was organized, evaluating the efficacy the same phytotherapic blend in dogs presenting with spontaneous acute otitis externa.

| In vitro assays
The in vitro efficacy of Otogen ® blend was assessed following the method of the European Normative UNI EN 1275 for the evaluation of fungicidal or yeasticidal activity of chemical disinfectants and antiseptics (Anon., 2005), with some modifications. The method consists in evaluating the number of living microorganisms after the contact with antiseptics at different time points.
The organisms tested included one clinical strain of M. pachydermatis, Pseudomonas aeruginosa, and Sthaphylococcus pseudintermedius and an ATCC strain of Candida albicans (ATCC strain 90028).
The microorganism inoculums (Test Suspension -TS) were prepared by picking some bacterial or yeast colonies and suspending them into sterile tubes with HSO. The TS was vortexed for 5 min to obtain a preparation as homogeneous as possible. The use of HSO instead of distilled water recommended in the Normative (Anon., 2005), was necessary since the phytotherapic blend under test is a mix of oils. The possible effects of HSO on microorganisms were assessed in preliminary experiments, and it was established that HSO does not affect microorganism viability (data not shown).

| In vivo evaluation
To perform the in vivo evaluation of the phytotherapic blend, we enrolled owned dogs presenting with clinical symptoms of acute otitis externa. Owners signed an informed consent before the beginning of the trial. Dogs could be of any breed, weight, sex, or neuter status, provided that they were at least 8-week-old. Exclusion criteria consisted in the administration of systemic or topical drugs within the last 2 months. It was hypothesized to withdraw patients during the study for the following reasons: adverse events, administration of concomitant therapy, owner incompliance, or any other documented reason.
Each ear was considered separately, as a single case: this is because each ear can have different anatomy and a unique microenvironment. Table 2 reports the description of the 12 dogs enrolled in the study.
At the first visit, a complete physical examination was performed by a veterinarian. All the information regarding the dog, past and recent anamnesis, general and objective examinations, signs of otitis and findings at the otoscopic evaluation were recorded. At the same time, a sample was collected for a cytological exam. Otogen ® was administered once a day for seven consecutive days, using cotton soaked in the product.
At the end of the treatment, a complete physical examination, including otological exam, and a new cytological exam were performed. Scores for each parameter were given on a severity scale of 0-3 (0 = none; 1 = mild; 2 = moderate; 3 = marked). The sum of the scores yielded the total score for each ear (maximum score 27).

| Ear examination
The presence of mites, ulcers, and foreign bodies was also recorded.

| Cytological exam
Cerumen samples were collected using a swab prior to the first and after the last administration. Slides were prepared by rolling the swabs on their surface. They were stained by the Wright's technique (Merchant, 2005) and observed microscopically for the presence of yeasts (Malassezia) and bacteria (cocci and rods).
The presence of inflammatory cells (neutrophils and macrophages), eventually with bacteria within them, was also recorded as an evidence of actual infection.

| Effectiveness evaluation criteria
At the control visit, a complete cure (ear "recovered") was defined as a return to normal of all parameters (sum of scores = 0).
Secondary criteria were also considered: • an improvement between 80% and 100% of the initial severity score was considered as "strong improvement"; • between 60% and 80% "clear improvement"; • between 40% and 60% "improvement"; • between 0% and 40%: the dog was considered as "steady"; • if total score of the final visit was higher than that of the first examination the condition was considered as "worsened".
According to cytological findings, pathogens were considered normalized in case the score went to 0. Other evaluations were possible (improved, unmodified, worsened) basing on the comparison of the score pre-and post-treatment.

| Contact assays
The results obtained are presented in Table 3 and Figures 1-4. As regards the BTS 90, the highest efficacy was shown against M.
pachydermatis (99,99% reduction already after 5 min of incubation) followed by C. albicans and P. aeruginosa (99,99% reduction after 1 h of incubation). For S. pseudintermedius, the activity was good, though the reduction did not reach 99.99%. The activity of the blend diluted at 50% was generally inferior, but anyway around 99% in many cases after 15 min or 1 h of contact (Table 3).  Table 4 shows data about the assessment of treatment efficacy. Main outcome (complete recovery) is considered together with secondary criteria. Mites, ulcers, and foreign bodies were not found either during the first visit or during the control visit.

| In vivo study
No adverse effects were recorded and none of the cases was withdrawn.

| Cytological exam
Inflammatory cells were not seen in any of the cases. As regards microorganisms, the presence of cocci and Malassezia was noted in 18 and 12 cases, respectively. Rods were not present. decreased till the normalization in 9 cases, unchanged in 2 cases and worsed only in 1 case out of 12 (details are available in Appendix S3).  Among the different EOs contained in the phytotherapic blend,

| DISCUSS ION
Melaleuca alternifolia and Salvia officinalis have been considered as potent antibacterial agents and confirmed efficacy obtained in the present study (Tresch et al., 2019). Essential oils could thus be included in the treatment, as an alternative therapeutic option, alone or in combination with allotherapic approach (Ebani et al., 2017).
The essential oils are known to possess antimicrobial activity (Mickienė et al., 2011): particularly TTO is the most effective contrasting the growth of streptococci, enterococci, staphylococci, as well as having an antifungal effect against yeasts and dermatophytes (Carson et al., 2006). Some studies reported that TTO could have a sensitizing potential if used pure or in high concentrations, or in case of oxidation of its components terpinene-4-ol and α-terpinene can counteract the growth of Candida spp. (Oliveira, Vilela, et al., 2019) while lavender has a possible application against parasite infestations, including those caused by mites (Cavanagh & Wilkinson, 2002). The control of the inflammatory process can be mediated by the extracts of the Eucalyptus which, thanks to its high content of 1 -8 cineole, is able to suppress the production of pro-inflammatory cytokines (Juergens et al., 1998). To control pain, the essential oil of Rosmarinus officinalis is able to achieve an antinociceptive effect, and demonstrates antibacterial effect against Streptococcus spp., Staphylococcus spp., Pseudomonas aeruginosa, and Candida albicans, all involved in the development of the ear inflammatory process .
A limit of the present study may be the inoculum size employed in in vitro tests. The number of CFUs was indeed different from organism to organism, and in none of the experiments, we could reach the size indicated in the standard procedure followed (Anon., 2005).
Due to the fact that we tested the final commercial product, we had to suspend the germs in oil, which did not allow to prepare a homogeneous and reproducible inoculum. Using "classical" broth-dilution methods (whose results are expressed as MIC values) would have allowed us to overcome this technical problem and obtain a more accurate and reproducible evaluation of the activity of the blend components. We decided to use contact tests instead, because we think that they are more predictive of in vivo outcome of topical treatment. Contact tests allow to take into account the main factors which influence the efficacy of antimicrobial topical products, namely the product formulation effects and the duration of contact (Russel & McDonnel,2000). This in turn allows simulating what happens when the final marketed formulation is applied on skin or in the ears. Lloyd and Lamport (1999) demonstrated that formulation is an important factor affecting the antimicrobial efficacy of topical products (chlorhexidine in that case). In marketed products, other principles (i.e., surfactants) can interact with the active principles.
Contact tests have been employed in another study on the activity of topical formulations employed to treat dermatitis and otitis in dogs (Nebbia et al., 2008 investigations on the efficacy of plant-derived compounds. The tested bacteria demonstrated in the last years increasing antimicrobial resistance phenomena. The total or partial inhibition obtained by the phytotherapic blend used in the present study suggests a good efficacy of EOs against these pathogens.
All the above considerations come from selected subjects that do not present chronic otitis, and it is not possible to extend these considerations to complicated otitis externa. The Authors' advice is to consider Otogen to clean and regulate the wetting of the ear environment in case of complicated otitis externa but further studies are needed to collect information specifically addressed to this condition.

| CON CLUS ION
The phytotherapic blend tested solution could be regarded as valuable support to cure acute otitis externa in dogs.
The improvement shown for both the clinical and cytological examinations in most of the enrolled cases leads to the hypothesis that the product has good effectiveness in restoring and maintaining homeostasis of the auricle environment.

CO N FLI C T O F I NTE R E S T
The authors declare that the study received financial support by Nutrigen LG Distribuzione S.r.l., necessary to purchase materials.
No other conflicts of interest have to be declared in publishing this work.

AUTH O R CO NTR I B UTI O N S
CV and AP designed the experimental procedures, coordinate the different experimental phases and wrote the draft of the paper, and CV performed the visits, MP and SV performed the analysis, and enrolled patients, GG 1 , GG 2 , GR, and MG supervised all the procedures, revised the results, checked the draft of the paper.

DATA AVA I L A B I L I T Y S TAT E M E N T
All relevant data are within the paper and supporting information files.