Molecular detection of major coagulase-positive Staphylococcus isolates from the canine otitis externa

Introduction

Canine otitis externa (COE) refers to inflammation of the external auditory canal, which can also involve the ear pinna. It can present as either acute or chronic, with the chronic form defined as persistent or recurring inflammation lasting three months or more. (Huang et al., 2009) [1] . The canal is lined by stratified squamous epithelium similar to skin, containing hair follicles, sebaceous glands and ceruminous (modified apocrine) glands, and it harbours a resident microbiota; these features show considerable inter-breed variation, which represents a major predisposing factor (Miller et al., 2012; Njaa et al., 2012) [2,3] . The prevalence in the general dog population is estimated to be between 8.7% and 20% (Saridomichelakis et al., 2007; Topală et al., 2007) [4,5] . Clinically, it manifests as pruritus, erythema, oedema and otic discharge, frequently accompanied by pain and malodour. Affected dogs typically exhibit behavioural changes, including frequent head shaking, vigorous ear scratching and rubbing of the ears on surrounding objects or surfaces (Summers et al., 2019) [6] . Early and precise diagnosis combined with targeted therapy is critical to achieve resolution, alleviate discomfort and minimize the risk of chronicity or recurrence. Otitis externa remains a common complaint in small animal practice and is often challenging to manage successfully because of its multifactorial and recurrent nature. Breeds with pendulous pinnae, such as Cocker Spaniels, Basset Hounds and Labrador Retrievers, are predisposed owing to poor ventilation, retention of moisture and debris within the ear canal and subsequent promotion of microbial proliferation (Rosser, 2004) [7] . Coagulase-positive staphylococci (CoPS) are gram-positive bacteria that commonly exist as commensals on the skin and mucous membranes of humans and animals while also acting as opportunistic pathogens (Fontana & Favaro, 2018) [8] . These organisms typically colonize areas such as the nasal cavity, throat and perianal region in a symbiotic relationship with their hosts (Nagase et al., 2002; Wertheim et al., 2005) [9,10] . The CoPS group currently comprises ten recognized species: Staphylococcus aureus, S. intermedius, S. pseudintermedius, S. coagulans (formerly S. schleiferi subsp. coagulans), S. hyicus (variable coagulase activity), S. delphini, S. lutrae, S. agnetis (variable coagulase activity), S. cornubiensis and S. ursi (González-Domínguez et al., 2020; Perreten et al., 2020) [11,12] . S. pseudintermedius is the predominant CoPS species causing skin and soft-tissue infections in dogs. It commonly colonizes the skin, mucosa or GIT of over 50% of healthy dogs and fewer healthy cats. Normally commensal, it serves as an opportunistic pathogen and a primary cause of canine pyoderma and otitis externa (Paul et al., 2012) [13] . Several methods exist for bacterial detection, but multiplex PCR (M-PCR) is the most rapid and convenient for identifying coagulase-positive staphylococci (CoPS). (Sasaki et al., 2010) [14] analyzed nuc gene sequences in CoPS and related species to develop an M-PCR assay targeting the nuc locus for reliable species differentiation. Conventional identification involves cultural isolation, biochemical tests, and sugar fermentation patterns, which often require more than 72 hours for genus-and species-level confirmation. To overcome these delays, molecular approaches like M-PCR have become the preferred method for faster and more accurate results. This study is a molecular survey of CoPS isolates from COE, aimed at expanding our understanding of their phenotypic and genotypic identification.

Materials and Methods

Sample collection

Dogs exhibiting head shaking, downward ears, pinna swelling and purulent discharge were selected for sample collection attending the Veterinary Clinical Complex, Veterinary College, Junagadh, Gujarat, India. Sterile cottontipped swabs moistened with sterile 0.9% saline solution were used to collect ear exudate samples from 87 COE patients. These swabs were transported to the Department of Veterinary Microbiology, Kamdhenu University, Junagadh, on the same day while maintaining a cold chain or stored at 4 °C until further processing.

Isolation and biochemical characterization

The samples collected from the clinical cases of COE were streaked onto brain heart infusion (BHI) agar plates and incubated at 37 °C for 24 hours to obtain pure colonies. The bacterial isolates were identified at the genus level by observing colony characteristics, performing Gram's staining, examining microscopic morphology and assessing growth on Mannitol Salt Agar (HiMedia Laboratory, Mumbai). Additional biochemical tests (oxidase, tube coagulase and catalase tests) were performed according to the proper procedures (Quinn et al., 1994) [15] .

Genomic DNA extraction from bacteria

Genomic DNA was extracted from pure Staphylococcus cultures using the conventional Proteinase K-SDS method (Sambrook and Russell, 2001) [16] . The quality and concentration of the isolated DNA were determined using a μDrop™ Plate in a μDrop plate reader (Thermo Scientific).

PCR-based detection of the Staphylococcus genus and species

Primers specific for detecting the genus and species of CoPS were used according to protocols from various researchers. Details of the primers, including their names, oligonucleotide sequences, targeted genes and product sizes are provided in Table 1. The composition of the PCR reaction mixture followed the guidelines established by Mason et al. (2001) [17] and (González-Domínguez et al., 2020) [11] . The PCR reaction mixture was prepared as per Table 2. The PCR conditions for detecting the Staphylococcus genus-specific 16S rRNA gene included an initial denaturation step at 95 °C for 5 minutes, followed by 35 cycles of 45 seconds at 95 °C for denaturation, 40 seconds at 55 °C for annealing and a 45-second extension at 72 °C. The final extension was done at 72 °C for 7 minutes. For Staphylococcus species-specific gene amplification (Pse-nuc, Au-nuc and Sch-nuc), the cycling conditions included an initial denaturation at 95 °C for 5 minutes, followed by 35 cycles of 10 seconds denaturation at 95 °C, 30 seconds annealing at 60 °C and 30 seconds extension at 72 °C, with a final extension at 72 °C for 5 minutes (Table 3). PCR amplifications were performed using a programmable thermal cycler (Verity, Applied Biosystems by Life Technologies, Singapore). The resulting PCR products (4 µL) were analyzed by agarose gel electrophoresis (1.5% w/v), and the gel visualization was done by using a gel documentation system (Bio-PrintST4 ® Vilber Lourmat). [18] and Vanisree et al. (2025) [19] identified Staphylococcus spp. as the most common pathogen in 56.57% and 70.24% of cases, which was slightly higher than observed in the current study, 48.18%. Whereas Hassan et al. (2023) [20] found a lower incidence of Staphylococcus spp. (36%), which was slightly lower than this study. S. pseudintermedius was the most prevalent species, accounting for 67.92% (36/53). S. aureus was identified in 15.09% (8/53), while S. schleiferi subsp. coagulans accounted for 5.66% (3/53). Additionally, 11.32% (6/53) of the isolates were categorized as other Staphylococcus species (Chart 1). Similar results were obtained by Hassan et al. (2023) [20] , who reported 41.6% S. pseudintermedius and 22.2% S. aureus; Prošić et al. (2024) [21] reported 65.8% as S. pseudintermedius, 22.4% as S. aureus, 7.9% as S. coagulans and 3.9% as S. intermedius; Rana et al. (2022) [22] reported 78.16% S. pseudintermedius, 19.71% S. aureus and 2.11% other Staphylococcus spp.; Makwana et al. (2023) [23] also reported S. pseudintermedius (40.63%), S. schleiferi subsp. coagulans (18.75%) and S. aureus (15.63%). Whereas a higher percentage of incidence was reported by Tamakan & Gocmen (2022) [24] , 87.5% S. pseudintermedius, 9.37% S. aureus and 3.12% S. schleiferi. Kadhim & Abdullah (2022) [25] and Núñez et al. (2025) [26] also found the highest prevalence for S. pseudintermedius, 28.5% and 37.5% respectively, among all Staphylococcus species, which was less in percentage for S. pseudintermedius than this study. (2001) [17] were utilized in this study. All 53 isolates that had been presumptively identified by phenotypic methods successfully amplified the expected 16S rRNA gene fragment, thereby confirming their identity as members of the genus Staphylococcus through PCR. Species-level differentiation among the major CoPS was achieved using species-specific thermonuclease (nuc) gene primers for S. aureus, S. pseudintermedius and S. schleiferi subsp. coagulans, as previously reported by González-Domínguez et al. (2020) [11] . Reference strains S. aureus ATCC 43300, S. pseudintermedius ATCC 49444 and S. schleiferi subsp. coagulans ATCC 49545 served as positive controls, whereas Escherichia coli MTCC-722 was included as a negative control to validate the specificity of the nuc genebased PCR assays. Among the 53 phenotypically presumptive staphylococcal isolates, 47 were successfully verified as members of the genus Staphylococcus through amplification of the genusspecific 16S rRNA gene in PCR (Fig. 4). Subsequent species-specific PCR targeting the nuc gene revealed the following distribution: 36 isolates (67.92%) produced the characteristic 99-bp amplicon diagnostic for S. pseudintermedius, 8 isolates (15.09%) showed the 127-bp product indicative of S. aureus and 3 isolates (5.66%) generated the 115-bp fragment specific to S. schleiferi subsp. coagulans. The remaining 6 isolates (11.32%) of the molecularly confirmed Staphylococcus population) did not amplify any of the three species-specific nuc gene targets and therefore could not be assigned to these major coagulase-positive species.

In the present study, all staphylococcal isolates were molecularly confirmed as belonging to the genus Staphylococcus through amplification of the 16S rRNA gene. This complete concordance between phenotypic and genotypic identification is consistent with findings from several previous reports. Notably, Mathapati et al. (2016) [27] , Chaudhary et al. (2021) [28] and Kadhim & Abdullah (2022) [25] similarly achieved 100% positivity for the 16S rRNA gene among their staphylococcal isolates from canine clinical samples. Likewise, Zedan et al. (2023) [29] successfully confirmed all 29 phenotypically presumptive Staphylococcus isolates via genotypic detection of the same marker.

In the present study, 76.60% (36/47) of CoPS isolates were confirmed as S. pseudintermedius through detection of the species-specific nuc gene. This prevalence is notably higher than that reported in several prior investigations using similar PCR-based methodologies. For instance, Weese et al. (2010) [30] and Dziva et al. (2015) [31] identified S. pseudintermedius in 39.2% and 38.5% of isolates, respectively, while Vincze et al. (2014) [32] and Bannoehr et al. (2015) [33] documented rates around 45%. In contrast, Schmidt et al. (2017) [34] reported a substantially higher positivity rate of 89.21%, whereas Kadhim & Abdullah (2022) [25] observed a lower prevalence of 28.5% (10/35) among staphylococcal isolates. More recently, Chehida et al. (2024) [35] detected S. pseudintermedius in 64.4% of 99 CoPS isolates based on thermonuclease gene profiling. The high prevalence of S. pseudintermedius in our study (76.60%) confirms its status as the dominant pathogen in COE and the leading CoPS in canine skin and ear infections. Observed differences among studies likely stem from variations in geographic location, sample size, case type (acute vs. chronic), or specimen collection method, underscoring the value of ongoing molecular surveillance for regional epidemiological insights.

In the current study, 17.02% (8/47) of CoPS isolates were identified as S. aureus based on the detection of the Au-nuc gene. This proportion is somewhat lower than, yet comparable to, rates reported in previous investigations. For example, Weese et al. (2010) [30] documented S. aureus in 32.4% of canine staphylococcal isolates, while Davis & Thompson (2021) [36] reported a 29.2% incidence in cases of COE. Similarly, Nakamura et al. (2020) [37] and Ruzauskas et al. (2016) [38] observed incidences of 25.9% and 25%, respectively, while Chehida et al. (2024) [35] identified S. aureus in 20.2% of CoPS isolates. Although slightly reduced in our cohort, this prevalence falls within the typical range described in the literature and supports the recognition of S. aureus as an important, albeit secondary, pathogen relative to S. pseudintermedius in canine skin and ear infections.

In the present study, S. schleiferi subsp. coagulans accounted for 6.38% (3/47) of staphylococcal isolates. This proportion is moderately lower than the rates documented in prior reports, including 10.2% by (Ruzauskas et al., 2016) [38] , 15% by (Garcia et al., 2022) [39] and 10.1% by (Chehida et al., 2024) [35] .

Conclusions

This study establishes Staphylococcus spp. as the foremost etiological agent in canine otitis externa (48.18% of isolates), with coagulase-positive staphylococci dominating (88.68%). Integrative phenotypic and genotypic analyses confirmed S. pseudintermedius as the predominant pathogen (67.92% overall; 76.60% of CoPS), followed by S. aureus, S. schleiferi subsp. coagulans. PCR-based species-specific nuc gene amplification proved superior in precision and sensitivity to biochemical methods, enabling accurate identification of non-specific isolates. This marked ascendancy of S. pseudintermedius reinforces its primacy in canine otic and cutaneous infections, often exceeding global prevalences. These findings mandate precise species-level diagnostics to guide targeted therapy and antimicrobial stewardship, while geographic variability underscores the need for sustained molecular surveillance to combat evolving resistance in staphylococcal populations.

Acknowledgement

AcknowledgementsThe authors express their sincere gratitude to all the clinicians at the Veterinary Clinical Complex for their invaluable assistance in sample collection. We also extend our heartfelt thanks to the laboratory technician and supporting staff of the Department of Veterinary Microbiology, Kamdhenu University, Junagadh, for their dedicated help with all associated laboratory work throughout this study.

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