Study on bacterial isolation, antibiogram and therapeutic management of canine otitis externa

Introduction

Canine otitis externa (COE) is a non-life-threatening yet distressing condition characterized by acute or chronic inflammation of the external ear in dogs. This condition poses significant challenges, not only for affected dogs and their owners but also from diagnostic and therapeutic perspectives for practicing veterinarians [1,2] . It is a multifactorial disease often associated with various microbial agents, affecting approximately 20% of the canine population [3] . COE is frequently complicated by mixed infections involving both bacteria and yeast. The normal microflora of the ear canal typically includes coagulase-positive and coagulase-negative Staphylococcus species, along with β-hemolytic Streptococcus species, Proteus mirabilis, Escherichia coli, Pseudomonas aeruginosa and others [4] . Despite its incidence, there is a lack of published data on the incidence of otitis externa, the antimicrobial sensitivity patterns of associated bacterial isolates and its management in Gujarat. The present study aimed to address this gap by identifying the bacterial agents responsible for otitis externa, evaluating their antimicrobial susceptibility testing (AST) and providing insights into effective therapeutic strategies to alleviate the suffering of affected dogs.

Materials and Methods

A total of 92 ear swabs from clinical cases of COE were collected. All the samples were collected aseptically by gently rolling the sterile HiCulture collection device (PW 003, HiMedia Laboratories and Mumbai) moistened with sterile 0.9% saline solution from ear canal of affected dogs. They were having complaints of shaking of ears, tilting of the head, dropping of affected ears, ceruminous or purulent ear discharge, pawing of ears and foul smell from ears.

All the dogs underwent a thorough assessment that included, obtaining a detailed history, conducting clinical examinations, performing microbiological analyses and carrying out AST to guide therapeutic management. Primarily Brain Heart Infusion (BHI) agar (M211, HiMedia) was used to isolate bacteria and colonies were subjected to Gram's staining. While, Mueller Hinton (MH) agar (M173, HiMedia) was used for the AST. All the media and antibiotic discs used were procured from Hi-Media.

Results and Discussions Breed-wise Incidence of COE

The breed-wise incidence of otitis externa in the present study revealed the highest incidence in Non-descript (ND) dogs (33.70%, N=31), followed by Labrador Retrievers (23.91%, N=22) and German Shepherds (16.30%, N=15). The incidence of other species was as per (Table 1 and Figure 1). 2). Some samples showed single and mixed infection of two or multiple organisms. These findings highlighted the diverse bacterial population involved in otitis externa, with Staphylococcus species being the dominant pathogens. Kiss et al. [5] also found similar findings for Staphylococcus spp. (39.22%) and Pseudomonas spp. (12.62%). Murugan et al. [2] also observed similar findings for Staphylococcus spp. (48.6%) followed by Pseudomonas spp. (21.5%) and Proteus spp. (14.95%). Luciani et al. [6] also reported similar results for Staphylococcus spp. (48.8%) and Pseudomonas spp. (30.2%). A comparable bacterial distribution was reported by Kumar et al. [7] and Suneja et al. [8] , emphasizing the consistent role of Staphylococcus spp. in COE. Lakshmi & Tirumala [1] reported the presence of similar bacteria in COE in their study. Moreover, Hariharan et al. [9] identified Staphylococcus intermedius as the predominant species in otitic dogs, followed by Pseudomonas spp., which aligns with present findings. Similar observations were made by Mateus et al. [10] , who also highlighted Staphylococcus pseudintermedius as a major pathogen, often found in single or mixed infections. Kania et al. [11] further supported the dominant role of Staphylococcus species, attributing their incidence to their ability to form biofilms and evade host immunity. The Staphylococcus spp. isolates were identified based on their morphological and cultural characteristics (white or golden-yellow colonies). It was confirmed as Gram-positive, catalase-positive, and oxidase-negative. The Pseudomonas spp. isolates in the present study were identified microscopically as Gram-negative, rod-shaped bacteria. It exhibited a characteristic greenish discoloration on BHI agar due to the production of the pigment pyocyanin, a hallmark of the genus [12] . Proteus mirabilis was identified by its swarming growth on agar, Gram-negative microscopic appearance, and biochemical characteristics as catalase-positive and oxidase-negative. Micrococcus spp. was confirmed based on its Gram-positive staining properties and biochemical tests, including catalase-positive and oxidasepositive reactions. Gram-positive bacteria with a distinctive Chinese letter arrangement were identified as Corynebacterium spp. The Gram-positive, typically oval to round and larger yeast cells compared to Staphylococcus species' cocci were identified as Malassezia pachydermatis [13] . Gram-negative bacteria were further cultured on Eosin Methylene Blue (EMB) agar. Escherichia coli (E. coli) produced distinctive metallic green sheen colonies, while Klebsiella spp. formed circular, dome-shaped colonies ranging from pink to purple on EMB agar. Biochemical testing confirmed both E. coli and Klebsiella spp. as catalasepositive and oxidase-negative. Gram-positive cocci in chains were identified as Streptococcus spp. based on their cultural characteristics and biochemical properties, such as catalase-negative and oxidase-negative reactions [13] .

Sex-wise Incidence of COE

The sex-wise incidence of clinical cases of COE was comparatively higher in male dogs (53, 57.61%) than in female dogs (39, 42.39%) (Table-3 and Fig. -3). That might be because people prefer to keep male dogs as pets [14] . Male dogs are often more prone to otitis externa, and various biological and behavioral factors may contribute to this predisposition. Hormonal influences, particularly testosterone, may play a role in immune modulation, affecting males' susceptibility to infections like otitis externa [15] . Additionally, certain behaviors, such as increased outdoor activity or engagement in muddy areas, can lead to greater exposure to environmental pathogens, and moisture is a common risk factor for ear infections [16] . Anatomically, male dogs may have slightly thicker ear canals, potentially altering ear ventilation and trapping moisture, which fosters bacterial and yeast growth, particularly in breeds already predisposed due to ear structure [17] . Agnihotri et al. (2014) [18] observed similar results, with otitis externa being more common in male dogs, with an incidence of 68.71%, compared to 31.29% in female dogs. Subramanian et al. (2015) [19] also reported the same incidence in male dogs (61.03%). Manju et al.

(2018) [20] reported that otitis externa was higher in old age and male dogs. 4 and Figure 4). Suneja et al. [8] reported a higher incidence of canine otitis in the 2-7 years age group, and male dogs were more affected than females. Reddy [21] observed a significant incidence of otitis externa in dogs aged between 5-10 years. Singh et al. [22] reported the highest incidence of otitis externa in dogs aged 3-6 years.

Subramanian et al. [19] also reported a peak incidence of otitis externa in the age group of 5-10 years (31.46%). However, Fraser et al. [23] and Nuttall [24] reported no apparent relationship between the incidence of canine otitis and the age of the affected animals.

Antibiogram of Major Bacteria

Antibiotic susceptibility testing (AST) was performed on Muller-Hinton agar (MHA) as suggested by the Kirby-Bauer disk diffusion method [25] . Three to four distinct pure bacterial colonies (cultured for 24 hours) were suspended in a sterile 0.85% NaCl solution to achieve a concentration corresponding to 0.5 McFarland turbidity. The turbidity was verified using a McFarland densitometer. A cotton swab mounted on a wooden stick was dipped into the suspension and then used to evenly streak the surface of a Muller-Hinton agar plate until the inoculum fully covered the surface. Afterward, antibiotic disks were placed on the agar using a disk dispenser, and the plate was incubated at 37°C for 18 hours. After incubation, the zones of inhibition surrounding each antibiotic disk were measured in millimeters and compared to the reference guidelines provided by the Clinical and Laboratory Standards Institute (CLSI) [26] .

The AST results highlighted distinct susceptibility patterns among Staphylococcus spp., Pseudomonas spp., and Proteus spp. Staphylococcus spp. showed high sensitivity to amoxicillin-clavulanic acid (92.45%), gentamicin (86.79%), and chloramphenicol (83.01%), while resistance was notable for penicillin-G (18.87%). Pseudomonas spp. exhibited significant sensitivity to gentamicin (84.61%) and amoxicillin-clavulanic acid (76.92%) but low response to doxycycline (15.38%) and cefoxitin (0%). Proteus spp. displayed high susceptibility to chloramphenicol (91.67%) and doxycycline (91.67%), with complete resistance to penicillin-G and clindamycin (both 0%) (Table 5). These findings were corroborated by Murugan et al. [2] and Parmar et al. [27] . Bugden [28] also found similar findings of high sensitivity for gentamicin against all three bacterial genera. These findings underscore the importance of AST-guided antibiotic selection to address resistance variability effectively. Shobha et al. [29] also reported the highest resistance against penicillin-G in their study.

Treatment

Ear canals of all affected dogs were cleaned with sterile saline solution and it was advised to clean ears with ear cleanser Virbac EpiOtic (Salicylic acid-0.2%). Patients were treated based on AST results. Amoxicillin-clavulanic acid 12.5-25 mg/kg BW PO or Chloramphenicol 25-50 mg/kg BW BID PO was prescribed for 7 days. In addition to antibiotic therapy, there are several dermatologic conditions where glucocorticoids are helpful, even when antimicrobial treatment or sepsis is present. Glucocorticoids help reduce inflammation and swelling in the ear canal, playing a crucial role in the successful management of these conditions. After one week of treatment, all the affected dogs demonstrated significant improvement, with a noticeable reduction in head shaking, ear scratching, and erythema of the pinna.

Conclusions

The present study's findings reveal COE's intricate nature, highlighting significant trends in its incidence and management. ND breeds, male dogs and those aged 6-10 years were identified as the most susceptible groups, emphasizing the potential influence of genetic and hormonal factors. Among the bacterial pathogens isolated, Staphylococcus spp. emerged as the most prevalent, followed by Pseudomonas spp. and Proteus mirabilis, underscoring their role in disease progression. Antimicrobial sensitivity testing provided critical insights, with notable effectiveness of amoxicillin-clavulanic acid, gentamicin and chloramphenicol against key pathogens, while resistance to penicillin-G and certain other antibiotics was evident. These findings stress the importance of tailoring antibiotic regimens based on sensitivity profiles to combat resistance and optimize therapeutic outcomes. Integrative management, including thorough ear cleaning, strategic antibiotic use and adjunctive glucocorticoids, demonstrated efficacy in alleviating clinical signs and improving recovery rates. The present study underscores the necessity of comprehensive diagnostic and treatment approaches to effectively address COE and enhance the quality of life in affected dogs.

Acknowledgement

AcknowledgmentThe authors cordially thank the Principal and Dean of the College of Veterinary Sciences & Animal Husbandry, Kamdhenu University, Junagadh (Gujarat) for providing the necessary facilities and institutional research funding.

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