Study on biofilm characterization and its association with antibacterial resistance in Staphylococcus pseudintermedius isolated from skin infections of dogs

Introduction

Canine skin, due to exposure to allergens, parasites, microbes, irritants, etc. is highly susceptible to dermatological disorders. Further, other factors such as breed predisposition, immune status, and underlying ailment also contribute to many skin diseases, including pyoderma and atopic dermatitis (Zahri et al., 2024) [14] . Thus, skin diseases constitute a major portion of the health problems encountered in companion animals. In the infectious category, bacterial pathogens contribute significantly to canine skin diseases. In addition to obligate pathogens, resident bacteria of the normal canine skin flora can also act as opportunistic pathogens, especially when dogs' immune status is compromised. Among these, S. pseudintermedius is a major commensal inhabitant of canines that causes a wide spectrum of clinical diseases in dogs. Although it is a commensal organism, it has a repertoire of resistance genes and frequently exhibits MDR characteristics, thereby prolonging treatment regimens. Further, rising pet ownership and close contact with dogs pose a significant risk of zoonotic transmission of MDR S. pseudintermedius to humans (Guimaraes et al., 2023; Somayaji et al., 2016; Moses et al., 2023) [4,15,16] . In addition to antimicrobial resistance phenotype, S. pseudintermedius isolates also exhibit biofilm formation, which hampers successful treatment. Biofilms are aggregates of microbial communities protected by an outer polymeric layer that is largely impermeable to antibiotics. Within the biofilm, bacterial communities communicate through quorum sensing and genetic exchange, disseminating resistance genes. The biofilms have the potential to enhance the tolerance of bacterial pathogens to antimicrobial substances up to 1,000-fold, thereby facilitating the selection and persistence of MDR bacteria (Nahum et al., 2025) [10] . With this background, the current study was conducted to understand the AMR pattern, biofilm-forming potential, and association between these two phenotypes in S. pseudintermedius isolated from dogs.

Methods

S. pseudintermedius isolates

Fifty isolates recovered from dogs with clinical dermatological conditions from Andhra Pradesh, India, were used in the study. The isolates were previously characterized using conventional laboratory and molecular techniques.

Biofilm characterization

The isolates were tested for their biofilm using the microtiter plate method (Stempelova et al., 2022) [12] . The optical density (OD) at λ = 570 nm was measured using a microplate reader. Biofilm was measured using the OD and the cut-off value (ODc). ODc is 3 SD above the mean optical density value of the negative control. The OD value for all isolates was calculated as the average OD minus the ODc value. The isolates were then categorized as weak (ODc < OD ≤ 2×ODc), moderate (2×ODc < OD ≤ 4×ODc), and strong (OD > 4×ODc) biofilm producers.

Antimicrobial Susceptibility Testing (AST)

The AST profiles of the S. pseudintermedius isolates were assessed using the disc diffusion assay (Kirby-Bauer method) in accordance with CLSI guidelines (CLSI, 2023) [3] . Briefly, single colonies of the S. pseudintermedius isolates were inoculated into BHI broth and incubated (for 24 hours at 37 °C). Further, inoculum was prepared by adjusting the culture turbidity to 0.5 McFarland standards. Using a sterile cotton-tipped applicator, the inoculum was plated onto Mueller-Hinton agar plates to form bacterial lawns. Once the plates were dry, Penicillin (P), Ampicillin (AMP), Amoxicillin-clavulanic acid (AMC), Ceftriaxone (CTR), Gentamicin (GEN), Amikacin (AK), Ciprofloxacin (CIP), Enrofloxacin (EX), and Tetracycline (TE) discs were placed on the bacterial lawns using sterile forceps, followed by incubation for 24 h at 37 °C . The Zone of Inhibition (ZOI) was measured and recorded in millimeters. Afterwards, isolates were categorized as resistant or susceptible as per the CLSI guidelines.

Statistical Analysis

The association between the AMR profile and biofilm production potential was assessed by performing Fisher's Exact Test. The strength of the association, where applicable, was estimated using the odds ratio with 95% confidence intervals. A p-value of < 0.05 was considered statistically significant. The analysis was conducted using IBM SPSS v.30.

Results

Biofilm Production Assay

Forty isolates produced biofilm, with 11 being moderate and 29 weak biofilm producers (Table 1; Figure 1). No isolate showed the strong biofilm-forming phenotype.

Antimicrobial Susceptibility Testing

Based on ZOI values obtained in the disc diffusion assay, the susceptibility of the S. pseudintermedius isolates was determined (Table 1&2; Figure 2). Amikacin was showed to be effective in vitro, as 100% susceptibility was observed. High levels of resistance were detected to ciprofloxacin (86%) and penicillins (78%). MDR was observed in 74% of the isolates.

Association between biofilm and AMR Among the 50 isolates studied, 37 (74.0%) were multidrugresistant (MDR) and 13 (26.0%) were non-MDR. Based on biofilm production strength, the MDR group comprised 22 (59.5%) weak biofilm producers, 8 (21.6%) moderate biofilm producers, 7 (18.9%) non-biofilm producers, and no strong biofilm producers. Similarly, among the non-MDR isolates, 7 (53.8%) were weak producers, 3 (23.1%) were moderate producers, and 3 (23.1%) were non producers, while none showed strong biofilm formation. Weak biofilm production was the predominant phenotype in both MDR and non-MDR isolates. The association between biofilm and AMR was investigated using Fisher's Exact Test and odds ratio analysis. Statistical analysis revealed no significant relationship between biofilm and resistance to the evaluated antimicrobial agents, namely penicillin, ampicillin, amoxicillin-clavulanic acid, ceftriaxone, enrofloxacin, ciprofloxacin, gentamicin, amikacin, and tetracycline (p > 0.05). Moreover, the 95% CIfor all odds ratios included one, indicating no significant difference in AMR profiles of biofilm-producing and non-biofilm-producing isolates (Table 3).

Discussion

Staphylococcus pseudintermedius is a commensal bacterium in canine skin microbiota, yet it is a frequent opportunistic pathogen in skin infections. Antimicrobial resistance, frequently MDR patterns, and biofilm-forming capability are significant challenges in treating S. pseudintermedius associated dermatological conditions in dogs. Biofilm formation will increase the tolerance of the isolates to various commercially available antibiotics and the selection pressure on resistant isolates. In present study, evaluation of association between biofilm and AMR patterns among 50 S. pseudintermedius isolates were done. The isolates examined demonstrated AST patterns, with many showing resistance to commonly available drugs used to treat infections. In agreement with this, Kim et al. (2025) [6] reported 78.5% resistance to penicillin in the S. pseudintermedius population recovered from canines. In addition, Teixeira et al., 2023 [13] reported a 76% rate of Penicillin resistance in S. pseudintermedius isolates, further confirming our findings. Apart from this, the AST pattern observed for other antibiotics was also in agreement with previous studies. Arefin et al., 2025 [1] reported that 60% of isolates were resistant to erythromycin, ampicillin, and ciprofloxacin. Lord et al. (2022) [7] reported that the majority of isolates were ampicillin resistant (84.3%), while substantial proportions were gentamicin (27.0%) and tetracycline (47.9%) resistant. Similar to these findings, Morais et al. (2023) detected high resistance rates to penicillin (85.2%), tetracycline (55.5%), erythromycin (36.8%), clindamycin (36.8%), trimethoprim-sulfamethoxazole (30.3%), gentamicin (25.2%), and fluoroquinolones (25.2%). However, among all the drugs tested, amikacin was consistent with high effectivity in vitro, with 100% susceptibility (Calabro et al., 2024) [2] . The biofilm production potential of S. pseudintermedius was evaluated using a microtitre platemethod, and 80% of the isolates were weak or moderate producers of biofilm. Although no strong producers of biofilm were detected, the high potential for biofilm formation among S. pseudintermedius isolates poses a significant challenge to the management of canine skin ailments. However, the association between AMR and biofilm formation is yet to be fully elucidated. In this study, biofilm production was observed in 30 MDR and 10 non-MDR isolates. However, no significant association between biofilm production and AMR could be detected. In conjunction with this finding, Stefanetti et al. (2017) [11] also reported no significant correlation between AMR and biofilm production in S. pseudintermedius isolates. However, in contrast, certain studies have reported a positive relation between these two phenotypes. Meroni et al., 2019 [8] reported that MDR isolates of S. pseudintermeidus exhibits greater biofilm production potential on comparison to the susceptible isolates. Similarly, Jantorn et al., 2021 [5] observed that strong biofilm production is frequent in MDR isolates, whereas moderate biofilm production is common in susceptible isolates. The contrasting significance of the associations reported in these studies shows that biofilm production in bacteria is regulated by many factors, including the genetic repertoire and prevailing environmental conditions. There is a limited possibility of categorizing an isolate as a weak-, moderate-, and strong biofilm producer based on its AMR profile, though further studies are warranted. Further, the high frequency of weak biofilm producers and low number of non-biofilm producers may have limited the subtle differences detection and correlation between AMR and biofilm. To conclude, the study detected a high level of MDR among the S. pseudintermedius isolates of canine origin. Most of the isolates were biofilm producers, though no strong biofilm producers were detected. No significant association could be detected between AMR and biofilm production in S. pseudintermedius. Further studies involving a larger number of isolates are warranted to unravel the correlation between AMR and biofilm in S. pseudintermedius.

Acknowledgement

AcknowledgementsThe authors gratefully acknowledge financial support from the Indian Council of Agricultural Research (ICAR) under the All India Network Program on Advanced Research Project on Canines (AINP-ARCC). The authors also thank the Dean of Veterinary Science, Sri Venkateswara Veterinary University, Tirupati; the Officer-in-Charge, Super Specialty Veterinary Hospital, Visakhapatnam; and ICAR-IVRI, Izatnagar, Bareilly, for their support and assistance in sample collection.

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