Molecular confirmation of Pseudomonas aeruginosa in dogs affected with otitis externa

Introduction

Otitis externa (OE) is a commonly encountered and clinically important inflammatory disorder of the external ear canal in dogs which may extend from the auricle to the tympanic membrane. It significantly affects animal welfare and can predispose to otitis media or interna if inadequately treated. The overall prevalence of canine OE has been reported between 5-20 % of the canine population in various studies (Rougier et al., 2005; Korbelik et al., 2019) [1,2] . It is a multifactorial disease influenced by predisposing, primary, secondary, and perpetuating factors, with microbial infection being a critical component in most chronic or recurrent cases (Hariharan et al., 2006) [3] . Among the bacterial pathogens, Pseudomonas aeruginosa has emerged as a predominant cause of chronic or treatment-resistant OE (Barnard et al., 2017) [4] . This opportunistic Gramnegative bacterium is capable of forming biofilms, producing tissue-damaging enzymes, and expressing multiple antibiotic resistance mechanisms, making infections difficult to eradicate (Garcias et al., 2025) [5] . Studies have demonstrated that P. aeruginosa accounts for approximately 15-20 % of bacterial isolates from canine OE and is frequently associated with severe, purulent otic discharges, ulceration, and persistent inflammation (Morris et al., 2021; Raus et al., 2023) [6,7] . The empirical use of antimicrobials without culture and sensitivity testing remains common in veterinary practice, which can contribute to the emergence of various multidrug-resistant (MDR) and extended-spectrum β-lactamase (ESBL)-producing strains (Pye, 2022; Kashyap et al., 2023) [8,9] . The identification of β-lactamase genes, including bla TEM , bla SHV and bla CTX-M in P. aeruginosa isolates obtained from dogs underscores the growing issue of antimicrobial resistance and raises concerns regarding possible zoonotic transmission (Kadhim et al., 2023) [10]. Considering the rising incidence of resistant otic infections, molecular confirmation and characterization of P. aeruginosa and associated resistance determinants are essential for accurate diagnosis, effective therapy, and antimicrobial stewardship. Hence, the present study was undertaken to confirm P. aeruginosa isolates molecularly from dogs affected with otitis externa through 16S rRNA gene amplification and to detect ESBL-producing genes.

Materials and Methods

Study location:

Sample collection:

Cotton ear swabs (Astra Bioscience Ltd., Kerala, India) were used for collecting samples from dogs with clinical otitis externa. Under aseptic conditions, each sterile swab was gently introduced into the external auditory canal and rotated for 10-15 seconds at the point where the vertical and horizontal canals meet to ensure proper sample collection. Samples were collected irrespective of age, sex or breed and transported to the laboratory for bacteriological examination following Wilkinson and Harvey (1994) [11] .

Media for isolation of bacterial isolates

Media used for bacteriology included nutrient broth, nutrient agar, mannitol salt agar, MacConkey agar, Pseudomonas isolation agar and eosin-methylene blue agar (HiMedia Laboratories Pvt Ltd., Mumbai) for initial culturing.

Morphological and biochemical identification of isolates

Gram's crystal violet, Gram's iodine, Gram's decolourizer and safranin (0.5 % w/v; HiMedia Laboratories, Mumbai) were used for Gram staining. Presumptive identification of P. aeruginosa was done according to the methods of Goto and Enomoto (1970) [12] .

Isolation of Pseudomonas aeruginosa

The collected ear swabs were initially inoculated into CFC (Cephalothin-sodium fusidate-cetrimide) broth base enriched with CetriNix supplement and incubated at 25 °C for 48 hours. After incubation, the cultures were streaked onto cetrimide agar or Pseudomonas agar base supplemented with CetriNix and further incubated at 37 °C for 24-48 hours. Colonies producing characteristic bluegreen or brown pigmentation were provisionally considered as P. aeruginosa and subsequently subjected to biochemical tests for confirmation Goto and Enomoto (1970) [12] .

Phenotypic confirmation of ESBL

Isolates were screened phenotypically for ESBL production using ESBL agar (HiMedia) with ESBL-selective supplement (FD278). Pink or purple colonies indicated ESBL-producing Pseudomonas spp.

Genotypic characterization of Pseudomonas aeruginosa

DNA extraction, PCR amplification, and gene detection were carried out as follows: 2.7.1 Genomic DNA extraction: Pure bacterial isolates were cultured in 5 ml of Luria-Bertani (LB) broth and incubated at 37 °C for 12-16 hours. From this culture, 1.5 ml was centrifuged at 8, 000 rpm (approximately 6, 800 ×g) for 6 minutes. The resulting pellet was washed twice with phosphate-buffered saline (PBS), resuspended in 300 µl of distilled water, and subjected to boiling at 100 °C for 10 minutes. The samples were then immediately chilled on ice for 20 minutes and centrifuged at 10, 000 ×g for 2 minutes. The supernatant containing DNA was collected and preserved at-20 °C. The concentration and purity of the extracted DNA (A260/280 ratio between 1.8 and 2.0) were assessed using a NanoDrop spectrophotometer (Nicklas et al., 2003) [13] .

16S rRNA PCR for species confirmation:

Each PCR assay for detection of 16S rRNA gene (Table 1) was carried out in a total volume of 25µl containing 12.5 µl of DreamTaq PCR Master Mix, 0.5 µl each of forward and reverse primers (0.5 mM), 1 µl of DNA template, and 10.5 µl of nuclease-free water. The amplification protocol involved an initial denaturation step at 94 °C for 2 minutes, followed by 25 cycles of denaturation at 94 °C for 20 seconds, annealing at 58 °C for 20 seconds, and extension at 72 °C for 40 seconds, with a final extension at 72 °C for 5 minutes. The amplified products were resolved on 1.5% agarose gel to detect the expected 956 bp fragment (Spilker et al., 2004) [14] .

Detection of ESBL genes by multiplex PCR

Multiplex PCR for bla TEM , bla SHV and bla CTX-M gene detection (Table 2) used reaction mixture of 25 µl: 3 µl 10× PCR buffer, 2.5 µl dNTP mix, 6 µl primers, 0.25 µl Taq polymerase, 3 µl genomic DNA and 10. 25 [15,16,17] . Amplified products were resolved on 1.6 % agarose gel in 1× TBE buffer containing ethidium bromide (0.5 µg/ml), electrophoresed at 70 V for 45 min and visualised using Gel Doc™ XR system.

Results and Discussion

Molecular confirmation of Pseudomonas aeruginosa

All presumptively identified Pseudomonas spp. isolates (n = 19) on the basis of cultural and biochemical characteristics were subjected to molecular characterization by polymerase chain reactio using species-specific 16S rRNA primers. A clear amplification of a 956 bp fragment (Fig. 1) specific to P. aeruginosa was obtained in all isolates (n = 19) recovered from canine ear swab samples, thereby confirming their molecular identity. The presence of a single distinct band of the expected amplicon size indicated the assay's specificity and reliability.

Universal primers designed to amplify conserved segments of the bacterial 16S rRNA gene are commonly employed for accurate species-level identification of Pseudomonas isolates (Tyler et al., 1995) [18] . The present results are consistent with earlier reports on molecular confirmation of P. aeruginosa from canine otitis externa (Serrano et al., 2016) [19] , human infections (Elfadadny et al., 2024) [20] , bovine mastitis (Rawat et al., 2021; Huang et al., 2024) [21,22] , and environmental water samples (Kaszab et al., 2019) [23] . The outcome of the present study substantiates that PCR targeting the 16S rRNA gene provides a sensitive as well as specific molecular method for the confirmation of P. aeruginosa. This approach overcomes the limitations of phenotypic identification, which often requires laborious and time-consuming biochemical assays that can be influenced by the organism's variable phenotypic traits. Similar observations have also been stated by Spilker et al. (2004) [14] , who demonstrated the reliability of 16S rRNA gene-based PCR for the accurate identification and differentiation of Pseudomonas species. The adoption of molecular diagnostic tools in Veterinary bacteriology enables early detection and precise identification of P. aeruginosa in otitis infections, facilitating rational antimicrobial selection and improved clinical management. Considering the high recurrence rate and antimicrobial resistance frequently associated with P. aeruginosa, the use of 16S rRNA-based PCR offers a valuable diagnostic advancement for veterinary clinical laboratories.

Detection of Extended-Spectrum β-Lactamase (ESBL) producing P. aeruginosa 3.2.1 Phenotypic detection of ESBL producers

A total of 19 Pseudomonas aeruginosa isolates recovered from dogs affected with otitis externa were subjected to phenotypic screening for extended-spectrum β-lactamase (ESBL) production using ESBL agar (HiMedia Laboratories, Mumbai). All isolates (100%, 19/19) were found to be presumptive ESBL producers based on the appearance of characteristic pink to purple colonies on selective media (Fig. 2) The high rate of ESBL positive samples observed in our study is indicative of the widespread antimicrobial resistance among P. aeruginosa strains infecting the canine ear canal. Similar findings were reported by Sharif et al. (2017) [24] , who documented 91.3% (42/46) ESBLproducing Pseudomonas isolates from diarrhoeic dogs in Andhra Pradesh. The results obtained in the present investigation are thus consistent with earlier studies suggesting that P. aeruginosa possesses intrinsic resistance mechanisms and a remarkable ability to acquire and disseminate β-lactamase-encoding genes (Raus et al., 2023; Pye, 2022) [7,8] . The 100% phenotypic ESBL positivity in the present study further suggests the influence of prior or empirical antibiotic exposure, as well as the chronicity of otitis externa cases, which favour the selection of multidrug-resistant bacteria (Kashyap et al., 2023) [9] . Previous studies have also emphasized the role of irrational or prolonged antimicrobial therapy in the emergence of ESBL-producing Pseudomonas isolates in companion animals (Damborg et al., 2016; Gomez-Santamarian et al., 2021) [25,26] . Such findings underline the need for continuous monitoring of antimicrobial susceptibility profiles to guide appropriate therapeutic decisions in clinical practice.

Molecular detection of ESBL genes

All 19 P. aeruginosa isolates were screened for the presence of ESBL-encoding genes (bla TEM , bla SHV and bla CTX-M) by multiplex PCR which revealed that prevalence of ESBL genes was 36.84% (7/19) for bla TEM , 21.05% (4/19) for bla SHV , and 5.26% (1/19) for bla CTX-M (Fig. 3) The detection of these genes provides molecular confirmation of the phenotypic resistance observed and demonstrates that P. aeruginosa from canine otitis externa can act as a significant reservoir for β-lactamase genes.

Similar gene distributions were reported in P. aeruginosa isolates from canine infections and environmental sources (Raus et al., 2023; Kadhim et al., 2023 and Mohamed et al., 2022) [7,10,27] , reflecting the increasing prevalence of βlactamase-mediated resistance in veterinary and environmental contexts. The predominance of bla TEM among the isolates in this study aligns with reports from other animal-origin Pseudomonas isolates, where bla TEM was found to be the most frequently occurring β-lactamase gene (Apaka et al., 2010; Boyd et al., 2004) [15,17] . The relatively lower detection of bla CTX-M may be due to its sporadic occurrence in Pseudomonas spp., as it is more commonly associated with Escherichia coli and Klebsiella pneumoniae (Paterson et al., 2003) [16] . The simultaneous presence of multiple ESBL genes in Pseudomonas species has been reported to increase resistance to a wide range of β-lactam antibiotics (Ranjbar et al., 2020) [28] . Hence, the findings from this study emphasize the urgent need for prudent antimicrobial use, regular surveillance of resistance determinants, and molecular monitoring to prevent the dissemination of resistant clones in veterinary clinical settings.

Conclusion

The study confirmed Pseudomonas aeruginosa as a major pathogen in canine otitis externa through molecular characterization of the 16S rRNA gene. Phenotypic testing indicated that all 19 isolates produced extended-spectrum βlactamase (ESBL) while PCR revealed the presence of bla TEM (36.84%), bla SHV (21.05%) and bla CTX-M (5.26%) genes. The predominance of bla TEM suggests its primary role in β-lactam resistance. These findings highlight the emergence of multidrug-resistant P. aeruginosa in dogs and stress the importance of molecular surveillance and judicious antimicrobial use in veterinary practice.

Acknowledgement

The authors wish to acknowledge Teaching and nonteaching staff of Teaching Veterinary Clinical Complex, Padmanabhpur and Private Veterinary Practitioners in and around Durg district for providing clinical cases and ear swab samples for the research work. Authors are also grateful to Dean, Veterinary College, Anjora for providing necessary laboratory facilities for conducting this research work.

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