In vitro evaluation of the antimicrobial activity and diffusion capacity of solutions used for canine ear cleaning

Otitis is a common condition among dogs and requires appropriate treatment. Given the importance of the otitis externa and the use of products to combat this condition, this study evaluated the in vitro antimicrobial abilities of six commercial ear cleaners solutions (EC1 to EC6) and solutions of 3% boric acid, 3% lactic acid, 0.11% salicylic acid, 0.5% chlorhexidine, and 3% propylene glycol against Staphylococcus spp., Pseudomonas spp., Proteus spp., and Malassezia spp. The in vitro diffusion capacity in synthetic cerumen was also assessed. Dogs with clinical signs of otitis externa were selected and samples were collected from the external ear canals. The microbiological study was performed to identify the microorganisms from samples collected and the microorganisms isolated were used in the study. One ceruminolytic for human use and five commercial solutions used for canine ear cleaning were selected based on the diversity of the components used in each formula. The results show a variation of antimicrobial activity and diffusion capacity ear cleaners and its compounds. Lactic acid, chlorhexidine, EC1, EC2, EC4, and EC5 showed the best results for microbiological growth inhibition; boric acid, salicylic acid, propylene glycol and EC6 had little or no effect on microorganism growth. The ECs tested demonstrated diffusion capacity using the SSC. EC1 was the solution with the most significant responses, both as an antimicrobial agent and with regards to diffusion capacity. Among the commercial veterinary products tested, EC4 was found to have the best results.


Introduction
Management of otitis require appropriate topical ear cleaners and prescription of ear drops. Diagnose and manage primary skin diseases, treat secondary infections and recognize any perpetuating or predisposing factors and manage them are the key to successful therapy of otitis (Paterson, 2016). The use of systemic medications to treat otitis is often associated with a low efficacy due to the inadequate penetration of these drugs into the lumen of the ear canal. Most of the topical medications comprise antimicrobial and antifungals agents, and glucocorticoids in their formulations, increasing the selective pressure and facilitating the emergence of multidrug-resistant bacterial strains (Guardabassi et al., 2004;Guardabassi et al., 2010).
There are different cleaning products with a range of active ingredients including ceruminolytics, surfactants, astringents, antimicrobials and anti-inflammatories. Ceruminolytics dissolve cerumen and dried debris in the ear canal, while surfactants emulsify debris and keep it in solution, and astringents dry the surface of the ear canal. Anti-inflammatories are often to inhibit inflammation and pruritus and antimicrobial activity limits the proliferation of bacteria and yeast (Nuttall & Cole, 2004;Swinney et al., 2008). Moreover, topical treatment should be performed after chemical removal of cerumen, but it may remain in the external ear canal, affecting the treatment successful. Therefore, otic preparations with active ingredients should persist inside the external ear canal for adequate effects and ceruminolytic activity and diffusion through cerumen residues are beneficial for high efficacy (Stahl et al., 2013).
Antimicrobial resistance is a growing concern in human and veterinary healthcare and effective use of antiseptics to reduce antibiotic use is often studied (Rafferty et al., 2019). The use of antiseptics as an alternative therapy in the control of multidrug-resistant bacteria has gained importance in veterinary medicine, provided there is no evidence of resistance to these agents. This approach would lead to a lesser selection of resistant strains of commensal and pathogenic microbiota (Banovic et al., 2013). Another advantage of using cleaning solutions is the absence of substances with potential to cause adverse reactions, such as glucocorticoids. Thus, the use of these antiseptic ear solutions may be considered as an important tool in the treatment of otitis, as they could reduce the use of antibiotics (Mason et al., 2013).
There are few reports of antimicrobial efficacy for most ear cleaners (Swinney et al., 2008), therefore studies on ear cleaners efficacy is important to improve treatment of canine otitis. Stahl et al. (2013) performed a detailed study of the composition of canine cerumen and developed a standardized synthetic cerumen (SSC), which allowed the assessment of the diffusion activities of eight commercial solutions in ear cleaning and the treatment of otitis. Therefore, the objectives of this study were to evaluate the in vitro antimicrobial activity of six commercial ear cleaners and five compounded products against main five microorganisms involved in canine otitis externa and to assess the in vitro diffusion capacity of these 11 solutions using the SSC.

Selection of samples.
The microorganisms were selected based on the studies of Bugden (Bugden, 2013) and Lyskova et al. (Lyskova et al., 2007). Dogs with clinical signs of otitis externa presented to the Department of Dermatology of the Veterinary Hospital of the UFMG from April 2013 to November 2014 were selected. All animals were evaluated by veterinarians experienced in dermatology and the inclusion criteria for the study were the following: (i) presence of two or more clinical signs of otitis; and (ii) presence of bacteria and/or yeasts in the cytological examination of the cerumen, performed at the exam room, following sample collection. The study design was reviewed and approved by the Animal Care and Use Committee at the Federal University of Minas Gerais (CEUA/UFMG, protocol 246/2013).
The samples were collected from the external ear canals with a sterile swab and stored in Stuart transport media for up to 48 hours. The samples were forwarded to the Applied Microbiology Laboratory of the School of Veterinary Medicine of UFMG, where they were inoculated onto Petri plates containing blood agar culture medium, and then incubated at 37°C for 24 to 48 hours, in order to obtain isolated colonies. After incubation, the isolated colonies were subjected to Gram staining for the identification of two main groups, Gram-positive and Gram-negative bacteria.
Gram-positive bacteria with an arrangement similar to grape bunches were then inoculated onto Petri plates containing Mueller-Hinton agar culture medium and incubated at 37°C for 24 to 48 hours. Bacteria of the genus Staphylococcus were identified by biochemical tests for phenotypic identification as proposed by Bannoehr & Guardabassi (Bannoehr & Guardabassi, 2012) and Quinn et al. (Quinn et al., 2011). After phenotypic identification, all Staphylococcus spp.
were identified using molecular methods, with specific primers for S. pseudintermedius, as previously described Sasaki et al. (Sasaki et al., 2010) and then subjected to polymerase chain reaction (PCR) analysis for detecting the presence of the mecA gene, according to Mehrotra et al. (Mehrotra et al., 2000).
From the PCR results, 10 samples of S. pseudintermedius were selected for the in vitro study, five methicillin-resistant S. pseudintermedius (MRSP), and five methicillin-susceptible S. pseudintermedius (MSSP). Five additional samples of S. schleiferi coagulans (SC) were also selected for analysis.
The isolates characterized as Gram-negative rods were seeded onto MacConkey agar Petri plates and incubated at 37˚C for 24 to 48 hours. The isolated colonies were characterized by biochemical tests for the phenotypic identification of bacterial species from the Pseudomonas and Proteus genera, according to the methods described by Oplustil et al. (Oplustil et al., 2000); five samples each of Proteus spp. (PRT) and Pseudomonas spp. (PSM) were selected for the in vitro studies.

Selection of ear cleaners (ECs).
One ceruminolytic for human use (EC1), and five commercial solutions (EC2-EC5) used for canine ear cleaning were selected based on the diversity of the components used in each formula. The composition of the commercial formulations is detailed in Table 1. Knowledge of the components of the ear cleaning solutions used is in the present study is important to evaluate the results obtained and compare with literature. Five single substances used in canine ear cleaning were also selected: 2% boric acid, 3% lactic acid, 0.11% salicylic acid, 0.5% chlorhexidine, and 3% propylene glycol, compounded in aqueous solutions.  1% miconazole for M. pachydermatis (Rojas et al., 2016) and 200µL of the negative control (water base) were added to the wells. After incubation for 24 hours, the sizes of the halos formed around the wells were measured using a millimeter ruler.

Production of standardized synthetic cerumen (SSC) and evaluation of the diffusion capacity of EC
The production of the SSC was performed according to the method described by Sánchez-Leal et al. (Sánchez-Leal et al., 2006); the final composition of the prepared SSC is described in Table 2. One of the objectives of the present study was to determine the diffusion activity of otic preparations through cerumen. Therefore, to analyze this, we used an in vitro method using synthetic canine cerumen. Because an in vivo study to test different products simultaneously would be too expensive, an in vitro method was developed based on the average composition of natural canine cerumen as described in the literature.
In order to evaluate the EC diffusion capacity, 4mm of the SSC produced was added to 60×15-mm Petri plates and, in each plate, a well was made using an 8mm biopsy punch.

Statistical analysis
The present study is an descriptive and experimental research with a qualitative and quantitative approach, through the analysis of numerical data and interpretation of the data obtained (Pereira et al., 2018). Data were analyzed using SAS (version 9.2). Descriptive analyses were performed, and principal component analysis was based on a correlation matrix and was calculated using a fitted mean input, and a biplot was obtained to illustrate the relationships between the ECs, microorganisms, and diffusion in SSC after 12 and 24 hours. The biplot was generated by tracing the entries according to their scores of the first and second major components. For the variable halo size in the SSC, where the EC × time interaction was significant, unfolding was performed, and the means for the ECs and time factors were compared by the Scott-Knott test. Statistical significance was set at P ≤ 0.05.

Results
The results of the inhibition of microorganism growth in the presence of the substances tested in the study are shown in Table 3, where the mean size of the inhibition halos measured in shown, together with the results of negative and positive controls. The largest halos of growth inhibition were formed by the action of EC1 on all microorganisms, followed by EC2, EC4, chlorhexidine and lactic acid. Salicylic acid had no action on all microorganisms, boric acid slightly inhibited the growth of only SC, and EC6 and propylene glycol had low inhibition on all microorganisms. The halo diffusion of oil red supplemented with the ear cleaners tested through the SCC after 12 and 24 hours are shown in table 4, in which the mean size of the halos measured for each ear cleaner tested are shown and compared. All ear cleaners tested showed diffusion capacity in the SSC used. In the EC×time interaction, the most significant diffusion activities after 12 hours were observed with boric acid, salicylic acid, chlorhexidine, propylene glycol, EC1, EC4, EC5, and EC6. After 24 hours, the same ECs, except for propylene glycol and EC5, displayed the most significant activities (Table 4). Research, Society andDevelopment, v. 10, n. 10, e539101019285, 2021 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v10i10.19285    Ear cleaner 5 (EC5); EC13. Ear cleaner 6 (EC6

Discussion
The present study assessed if an ECs, by itself or as a part of a commercial solution, can inhibit the growth of specific microorganisms, verified by the formation of growth inhibition halos, and also if these solutions have the ability to diffuse in a medium that simulates canine cerumen, visualized by the diffusion of oil red supplemented with era cleaners tested. The solution with highest activity against all microorganisms was EC1, which also showed diffusion capacity in the SCC. The active compound of EC1 is carbamide peroxide, a ceruminolytic that release oxygen in situ to help break up debris (Paterson, 2016). Carbamide peroxide decomposes into hydrogen peroxide and urea when in contact with cerumen (Thickett & Cobourne, 2009  while hydrogen peroxide decomposes to water and oxygen with generation of heat, and its oxidant activity acts as an antimicrobial agent and disinfectant (Marrero et al., 2017). Marrero et al. (2017) evaluated the in vitro activity of 1.5% hydrogen peroxide, a decomposition product of carbamide peroxide, and also found activity against microorganism growth.
According to Marrero et al. (2017), although hydrogen peroxide does not produce reactions in the ear at concentrations of up to 5%, its ototoxicity is controversial, being recommended only when there is no rupture of the tympanic membrane. Therefore, EC1 demonstrated good activity against common microorganisms that cause otitis and had a good diffusion in the SSC, but it is for human use only.
EC2, EC4, chlorhexidine and lactic acid also showed inhibition for the microorganisms tested and good diffusion capacity, although lactic acid and EC2 apparently do not have good diffusion compared to all ear cleaners tested. Moreover, EC4 demonstrated positive factor correlated with diffusion, and chlorhexidine and lactic acid correlated positively with inhibition halo. Organic acids, such as boric acid, lactic acid and salicylic acid are astringents, help buffer the ear canal environment, and have a moderate antimicrobial effects (Marrero et al., 2017;Swinney et al., 2008). In a study conducted by Swinney et al. (2008), the presence of specific organic acids was not associated with antimicrobial efficacy, and the authors highlighted that although they have some antimicrobial effect, dilution and interaction with other ingredients in commercial ear cleaners may reduce the antimicrobial efficacy. Chlorhexidine is an antimicrobial compounds frequently incorporated in ear cleaners to retard microbial proliferation (Nuttall & Cole, 2004) and is an antiseptic commonly used in the dermatological treatment of companion animals with superficial pyoderma caused by S. pseudintermedius (Borio et al., 2015).
Salicylic acid had no growth inhibition on microorganisms tested, since no halo formation was observed, although diffusion was observed in the SSC. Salicylic acid and EC4 had positive factor on diffusion principal component analysis and lactic acid with inhibition halo formation. Salicylic acid is a bacteriostatic agent that is keratoplastic at low concentrations and keratolytic at higher concentrations (above 2%) (Nuttall & Cole, 2004). These results indicate that salicylic acid might have no activity when used alone, since EC4 has salicylic acid and lactic acid, and had significant activity and might justify the use of salicylic acid in association with lactic acid. Considering the activity of lactic acid on the microorganism's growth and the diffusing capacity of the salicylic acid, solutions that combine these two components can possibly act more effectively if we consider a synergistic effect. If salicylic acid penetrates the cerumen; consequently, lactic acid can increase its area of activity.
This is a common association used in commercial ECs such as EC4 and EC5.
The commercial ear cleaner EC6 had no inhibition halo formation on growth for PSM and MLZ and no inhibition growth halo was observed against PTR and PSM for EC3. EC6 displayed good activity when its diffusion capacity in SSC was analyzed and also was positive corelated with diffusion in PCA. In a previous study, ear cleaners showed variable antimicrobial activity against different isolates of Pseudomonas and EC4 resulted in variable and inconsistent inhibition of the Pseudomonas isolates, while an ear cleaner with 0.15% chlorhexidine showed excellent activity against Pseudomonas, suggesting that chlorhexidine at this concentration is a safe choice as a flush in otitis (Steen & Paterson, 2012). In case of M.
pachydermatis, EC1 generated a larger growth inhibitory halo, as did lactic acid and solutions containing lactic acid. These findings the activity of chlorhexidine, lactic acid and EC2 for Pseudomonas spp. and M. pachydermatis.
Boric acid did not show activity against all microorganisms tested. A study on in vitro antimicrobial activity of acetic acid/boric acid impregnated cleansing wipes found no antimicrobial activity, although they are marketed as antiseptic, antibacterial and/or antifungal cleansing wipes (Rafferty et al., 2019). Propylene glycol showed no activity against PRT, PSM and MLZ, despite reports of its antimicrobial (Nalawade et al., 2015) and antifungal actions (Lloyd et al., 1998). Moreover, its diffusion capacity is lesser than that of other ECs; in addition, there are reports of its ototoxicity (Vassalli et al., 1988). A study with a commercial product with propylene glycol in its composition, demonstrated poor antimicrobial action of this compound (Marrero et al., 2017). Propylene glycol is a ceruminolytic commonly included in ear cleaners to soften and dissolve cerumen (Nuttall & Cole, 2004). Altogether, our study indicates that boric acid, salicylic acid, propylene glycol are not indicated for use alone and commercial solutions EC6 and EC3 should be used with caution.
Our study has some limitations. Antimicrobial efficacy of available commercial ear cleaners is especially variable and it is very difficult to draw conclusions about the their importance, as the cleaners contain a great variety of ingredients in different combinations and concentrations (Swinney et al., 2008). It is not possible to evaluate the diffusion of the ear cleaners into the surrounding medium for antimicrobial and diffusion studies, therefore, the study is more descriptive than analytical.
Also, many factors affect the response of in vivo therapy, therefore, in vitro studies may not represent in vivo responses, rather, indicate the use of certain products. We selected the strains for the study very carefully, but different strains may show different biological characteristics. But studies like ours provide knowledge of ear cleaners properties and help to choose the best product for specific clinical situations (Marignac et al., 2019).

Conclusion
The current study demonstrates the inhibitory activity of ECs on the growth of important microorganisms associated with canine otitis, in conjunction with their diffusion capacities, and indicates that EC1 presented the best results. The commercial solution EC4 was the best veterinary solution for combating microorganisms involved in otitis, followed by EC2, chlorhexidine and lactic acid. Salicylic acid had no halo formation, while and boric acid, EC3, EC6 and propylene glycol showed low inhibition on all microorganisms. The results show a variation of antimicrobial activity and diffusion capacity ear cleaners and its compounds. Further studies are necessary to establish clinical efficacy and if the results found can be replicated with larger bacterial/fungal samples and different synthetic cerumen, with the addition of other components of natural cerumen like keratinocytes or proteins.