Köpeklerde Deri Lezyonlarından İzole Edilen Staphylococcus aureus Suşlarında Biyofilm Oluşturma Kapasitesi ve Antimikrobiyal Direncin Araştırılması

Köpeklerde görülen bakteriyel deri enfeksiyonları sıklıkla stafilokoklarla ilişkilidir ve biyofilm oluşumu ile antimikrobiyal direncin (AMR) birlikteliği tedavi başarısını olumsuz etkileyebilmektedir. Bu çalışmada, deri lezyonu bulunan köpeklerden izole edilen S. aureus suşlarında biyofilm oluşturma kapasitesi, biyofilmle ilişkili icaA ve icaD genlerinin varlığı ve antimikrobiyal duyarlılık profilleri değerlendirildi. Bu amaçla 130 deri svabı kültüre edildi, S. aureus izolatları 23S rRNA PCR ile doğrulandı, biyofilm oluşumu mikrotitre plakta kristal viyole yöntemiyle ölçüldü. icaA ve icaD PCR ile araştırıldı ve antimikrobiyal duyarlılık CLSI kriterlerine göre disk difüzyon yöntemiyle belirlendi. 72 örnekte (%55.4) S. aureus saptandı. İzolatların çoğu biyofilm üreticisi olup %23.6'sı yüksek biyofilm oluşturan grup olarak sınıflandırıldı. Genel olarak izolatların %70.8'inde en az bir icaA veya icaD geni varlığı saptandı. icaA pozitifliği, yüksek biyofilm üreticilerinde düşük biyofilm üreticilerine kıyasla anlamlı derecede daha düşüktü (p=0.002). icaD ile biyofilm kategorisi arasında ise anlamlı bir ilişki bulunmadı. Çoklu ilaç direnci yaygındı (%56.9) ve yüksek biyofilm oluşturan izolatlarda amoksisilin-klavulanata duyarlı olmayanların oranı düşük biyofilm grubuna göre daha yüksekti (p=0.049). Elde edilen bulgular, köpek deri enfeksiyonlarına ait S. aureus izolatlarında biyofilmle ilişkili AMR yükünün belirgin olduğunu ve biyofilm fenotipi ile ica genotipi arasındaki ilişkinin kısmen ica bağımsız mekanizmaları da içeren karmaşık bir yapıda olabileceğini düşündürmektedir.

Canine bacterial skin infections are frequently associated with staphylococci, and biofilm formation together with antimicrobial resistance (AMR) may compromise treatment outcomes. This study investigated biofilm production, carriage of the biofilm-associated genes icaA and icaD, and antimicrobial susceptibility in canine S. aureus isolates from skin infections. Swabs (n=130) from dogs with skin lesions were cultured, and S. aureus was confirmed by 23S rRNA PCR. Biofilm formation was quantified using a microtiter plate assay, icaA/icaD were detected by PCR, and antimicrobial susceptibility was determined by disk diffusion according to CLSI guidelines. 72 (55.4%) samples were confirmed as S. aureus. Most isolates produced biofilm, and 23.6% were classified as high biofilm producers. Overall, 70.8% of isolates carried at least one of icaA or icaD, but icaA positivity was significantly less frequent among high than low biofilm producers (p=0.002). icaD showed no significant association with biofilm category. Multidrug resistance was common (56.9%), and high biofilm producers showed a higher proportion of non-susceptibility to amoxicillin-clavulanate than low producers (p=0.049). These findings indicate a substantial burden of biofilm-associated, antimicrobial-resistant S. aureus in canine skin infections and a complex, partly ica-independent relationship between biofilm phenotype and genotype with implications for empirical therapy.

Introduction

Canine bacterial skin infections are a frequent reason for presentation in small-animal practice, and pyoderma is a major indication for systemic antimicrobial therapy [1]. Staphylococci are the principal pathogens in superficial and deep canine pyoderma, with Staphylococcus pseudintermedius recognised as the primary aetiological agent [2]. However, Staphylococcus aureus is increasingly reported in skin and soft-tissue infections of companion animals, including dogs [3,4]. Close contact between dogs and their owners facilitates bidirectional transmission of S. aureus, including methicillin-resistant lineages, which are now regarded as relevant One Health-associated zoonotic hazards [5].

Biofilm formation is a key virulence trait of S. aureus, promoting persistent colonisation, evasion of host defences, and tolerance to antimicrobial therapy [6]. In staphylococci, a major biofilm matrix component is polysaccharide intercellular adhesin (PIA), a PNAG polymer synthesised by the icaADBC operon, and co-expression of icaA and icaD has been linked to strong biofilm formation [7]. The microtiter plate crystal violet assay remains a standard method for quantifying staphylococcal biofilm biomass in human and veterinary research [8,9]. However, PIA or PNAG is not the sole determinant of the biofilm phenotype, and ica-independent mechanisms involving surface proteins, extracellular DNA, and other matrix components can also mediate biofilm formation in S. aureus [10-12].

Systemic antimicrobial therapy is central in the management of canine pyoderma, and drugs such as amoxicillin–clavulanate, first-generation cephalosporins and clindamycin are commonly used as first-line options [13]. However, increasing reports of AMR and multidrug resistance (MDR) among staphylococci from canine skin and soft-tissue infections raised concerns for treatment success and antimicrobial stewardship [14,15]. Biofilm formation further complicates this picture, as biofilm-embedded S. aureus cells show reduced susceptibility to multiple drug classes and can act as reservoirs for recurrent infection [16]. Although the interplay between biofilm production and AMR has been examined in staphylococci studies jointly assessing biofilm-forming capacity, ica biofilm-associated genes, and antimicrobial susceptibility in S. aureus from canine skin infections remains limited.

In this context, this study investigated S. aureus isolates recovered from canine skin lesions, focusing on their frequency, biofilm-forming capacity, icaA/icaD status, and antimicrobial susceptibility. Associations between biofilm production, ica gene carriage and multidrug resistance, particularly non-susceptibility to amoxicillin–clavulanate, were also evaluated.

Material and Method

Research and Publication Ethics: Ethical review and approval were waived for this study in accordance with national regulations and institutional requirements. The Animal Experiments Local Ethics Committee of Harran University (Meeting No. 2023/005, Decision No. 01-10, document date and number 15.09.2023–260686) concluded that diagnostic skin swab sampling in dogs is not subject to formal ethical approval.

Animals and Sample Collection: This study used clinical samples from dogs with cutaneous lesions presented to Harran University Animal Hospital, Şanlıurfa, Türkiye, between 2022 and 2025. Skin swabs were collected from 130 dogs with suspected bacterial infection using sterile swabs, transported under cold-chain conditions, and cultured on the day of sampling.

Bacterial Culture and Identification: Samples were streaked onto blood agar with 5% defibrinated sheep blood and mannitol salt agar (MSA; Oxoid, Basingstoke, UK) and incubated aerobically at 37 °C for 24-48 h. Colonies with staphylococcal morphology and growth on MSA that were Gram-positive and catalase-positive were regarded as Staphylococcus spp. Preliminary identification of S. aureus relied on colony morphology, mannitol fermentation on MSA, and a latex agglutination test (Phadebact Staph Aureus Test, Bactus AB, Sweden) with S. aureus ATCC 25923 as the positive control.

Molecular Assays: Genomic DNA was extracted from phenotypically identified Staphylococcus isolates using a classical phenol–chloroform method, and the purified DNA was resuspended in nuclease-free water and stored at −20°C until PCR analysis. Species-level confirmation of S. aureus was performed targeting the 23S rRNA gene using the primer pair Sau-327/Sau-1645 as previously described (Table 1) [17]. Amplifications were carried out in 25 µL reaction mixtures containing standard PCR buffer, MgCl₂, dNTPs, Taq DNA polymerase, primers and 2 µL of template DNA, under the following cycling conditions: initial denaturation at 94°C for 2 min; 35 cycles, each composed of 94°C for 45 s, 64°C for 1 min and 72°C for 2 min; and a final extension at 72°C for 10 min.

The presence of the biofilm-associated genes icaA and icaD was assessed using gene-specific primers (Table 1) [18] with the same reaction mixture and a cycling profile of 95°C for 5 min; 33 cycles of 95°C for 30 s, 62°C for 30 s and 72°C for 30 s; and a final extension at 72°C for 5 min. PCR products were separated on 2% agarose gels alongside a FastRuler Middle Range DNA Ladder (Thermo Scientific, USA) and visualised under UV illumination. Staphylococcus aureus ATCC 25923 and Staphylococcus epidermidis ATCC 35984 were used as positive controls for the 23S rRNA and icaA/icaD assays, respectively.

Phenotypic Assessment of Biofilm Formation: Biofilm formation was evaluated using a 96-well microtiter plate crystal violet assay as described by Christensen et al., with minor modifications [19]. Briefly, overnight cultures grown in Tryptic Soy Broth (TSB) were adjusted to a 0.5 McFarland standard, diluted 1:100 in TSB supplemented with 1% (w/v) glucose and dispensed (200 µL) in triplicate into sterile, flat-bottom polystyrene microtiter plates. Plates were incubated statically at 37°C for 24 h, then gently washed with phosphate-buffered saline to remove non-adherent cells, air-dried, stained with 0.1% crystal violet, and rinsed with distilled water. Bound stain was solubilised with 95% ethanol, and the optical density (OD) at 570 nm was measured in a microplate reader (VersaMax, Molecular Devices, San Jose, CA, USA). The mean OD of triplicate wells was used for each isolate. Biofilm production was interpreted using an optical-density cut-off (ODc), calculated as the mean OD of negative-control wells plus three standard deviations (ODc=mean OD_NC + 3 × SD_NC). Isolates with OD ≤ ODc were classified as non-biofilm producers, those with ODc < OD ≤ 2 × ODc as weak, 2 × ODc < OD ≤ 4 × ODc as moderate, and OD > 4 × ODc as strong biofilm producers. Staphylococcus aureus ATCC 35556 was included as a positive control, and wells containing sterile TSB (Oxoid) served as negative controls.

Antimicrobial Susceptibility Testing: Antimicrobial susceptibility testing was performed by the Kirby-Bauer disk diffusion method in accordance with CLSI guidelines [20]. Standardised bacterial suspensions (0.5 McFarland) were spread onto Mueller-Hinton agar supplemented with 5% defibrinated sheep blood. Commercial disks (Oxoid, Basingstoke, UK) containing penicillin (2 U), amoxicillin–clavulanic acid (30 µg), erythromycin (15 µg), tetracycline (5 µg), clindamycin (2 µg), enrofloxacin (5 µg), and trimethoprim–sulfamethoxazole (25 µg) were applied. Plates were incubated aerobically at 37°C for 18-24 h, and inhibition zone diameters were measured and interpreted as susceptible, intermediate, or resistant according to CLSI breakpoints for S. aureus.

Statistical Analysis: Statistical analyses were performed using GraphPad Prism version 10.4.1 (GraphPad Software, San Diego, CA, USA). Associations between categorical variables were assessed using Fisher's exact test (two-tailed), with p<0.05 considered statistically significant. For antimicrobial susceptibility, isolates with intermediate susceptibility were grouped with resistant isolates as non-susceptible, and biofilm production was dichotomised as low (non/weak) versus high (moderate/strong) for comparative analyses.

Results

Characterization of Staphylococcus aureus Isolates and Biofilm Production: Of the 130 skin swab samples from dogs with cutaneous lesions, 95 (73.1%) yielded bacterial growth, and 84 (64.6%) isolates were phenotypically consistent with S. aureus based on mannitol fermentation and latex agglutination (Table 2). PCR targeting the staphylococcal 23S rRNA gene confirmed 72 (55.4%) S. aureus isolates, which were included in subsequent biofilm and antimicrobial susceptibility analyses (Figure 1A).

According to OD-based categories, 1 (1.4%) isolate was a non-biofilm producer, 54 (75.0%) weak, 13 (18.1%) moderate, and 4 (5.6%) strong producers; thus 55/72 (76.4%) were classified as low (non/weak) and 17/72 (23.6%) as high (moderate/strong) biofilm formers.

Among the 72 S. aureus isolates, 21 (29.2%) were negative for both icaA and icaD, whereas 51 (70.8%) carried at least one of these genes. Of these, 27 (37.5%) harboured only icaA, 14 (19.4%) only icaD and 10 (13.9%) both genes (Table 2, Figure 1B, C), giving overall frequencies of 37/72 (51.4%) for icaA and 24/72 (33.3%) for icaD.

Antimicrobial Susceptibility Profiles: Antimicrobial susceptibility testing was performed for 72 PCR-confirmed S. aureus isolates against seven antimicrobial agents, and the results are summarised in Table 3. Non-susceptibility to β-lactams was 44.4% for penicillin and 47.2% for amoxicillin–clavulanic acid (Figure 2, Table 3). Among non-β-lactam agents, the highest non-susceptibility rate was observed for clindamycin (83.3%), followed by tetracycline (52.8%), trimethoprim–sulfamethoxazole (45.8%), and erythromycin (34.7%), whereas enrofloxacin showed the lowest rate (26.4%). Overall, 41 of 72 isolates (56.9%) were multidrug resistant, being non-susceptible to at least three antimicrobial classes.

Correlation Between Biofilm Production, ica Genes and Antimicrobial Resistance: The distribution of ica genes differed between low and high biofilm categories, mainly due to the pattern observed for icaA. Only 3/37 (8.1%) of icaA-positive isolates were classified as high biofilm producers, compared with 14/36 (38.9%) of icaA-negative isolates (p=0.002) (Figure 3A), indicating an inverse association between icaA carriage and high-level biofilm formation. In contrast, 9/24 (37.5%) of icaD-positive isolates and 8/48 (16.7%) of icaD-negative isolates were high biofilm producers, but this difference did not reach statistical significance (p=0.076) (Table 4).

High biofilm-forming isolates showed a slightly higher proportion of multidrug resistance than low biofilm producers (64.7% vs 54.5%), but this difference was not statistically significant (p=0.579). Non-susceptibility rates were broadly similar between low and high biofilm producers for most agents. However, high biofilm producers had a significantly higher non-susceptibility rate to amoxicillin–clavulanic acid than low producers (70.6% vs 40.0%; p=0.049) (Figure 3B), while no significant differences were observed for the remaining antimicrobials. Multidrug resistance was also similarly frequent among icaA-positive and icaA-negative isolates (62.2% vs 51.4%; p=0.476), and among icaD-positive and icaD-negative isolates (62.5% vs 54.2%; p=0.616), indicating no clear association between ica gene carriage and MDR in this collection.

Discussion

Bacterial skin infections in dogs are predominantly associated with staphylococci, with S. pseudintermedius recognised as the primary pathogen, whereas S. aureus is less frequent but noteworthy because of its zoonotic and One Health relevance [1,21]. Biofilm formation and antimicrobial resistance are key traits that promote persistence and treatment failure in staphylococcal infections [22,23]. However, most studies on biofilm and biofilm-associated genes have focused on S. pseudintermedius or non-canine S. aureus, and integrated data on biofilm phenotype, ica status, and resistance profiles in canine S. aureus isolates remain scarce. In this context, the present study examined canine S. aureus isolates to characterise their biofilm phenotype, icaA/icaD carriage and antimicrobial susceptibility, and to explore relationships between these traits.

S. aureus was recovered from 72 of 130 (55.4%) canine skin swab samples, indicating that this species is an important component of the bacterial flora of canine skin lesions in our setting. Almost all isolates produced measurable biofilm and 23.6% were classified as high (moderate/strong) biofilm formers, suggesting that biofilm formation is common among canine S. aureus skin isolates. This high prevalence of biofilm production is consistent with reports of frequent biofilm formation by coagulase-positive staphylococci in companion-animal and human skin infections [21,24]. The study included a broad case mix of skin lesions with suspected bacterial involvement rather than only confirmed pyoderma, which may partly explain the relatively high proportion of S. aureus compared with studies restricted to canine pyoderma.

In this collection, 70.8% of the isolates carried at least one biofilm-associated gene, with icaA and icaD detected in 51.4% and 33.3% of isolates, respectively. Although co-expression of icaA and icaD has traditionally been linked to strong biofilm formation and many studies report a positive association between ica genes and biofilm production [25], icaA positivity in our study was significantly less frequent among high than low biofilm producers, and icaD status was not associated with biofilm category. This discrepancy with the classical ica-dependent model aligns with evidence that S. aureus can form biofilm through ica-independent mechanisms involving surface proteins, extracellular DNA, and other matrix components, and suggests that ica carriage alone does not reliably predict biofilm-forming capacity in canine S. aureus skin isolates [11,26,27].

In addition to widespread biofilm formation, the isolates showed a substantial burden of antimicrobial resistance, with more than half meeting the criteria for multidrug resistance. Non-susceptibility to several first-line drugs for canine skin infections, including amoxicillin–clavulanate, tetracycline, clindamycin and fluoroquinolones, indicates that empirical use of these agents may not always provide adequate coverage in our setting. The MDR proportion appears higher than in some reports on staphylococci from canine pyoderma but comparable to others [28,29], and is consistent with the global trend of increasing multidrug resistance among staphylococci from companion animals. This pattern may reflect the referral nature of our veterinary teaching hospital and prior antimicrobial exposure, which could not be systematically documented, and underlines the need for culture and susceptibility-based therapy, particularly in recurrent or non-responsive cases, as well as the integration of antimicrobial stewardship principles into the management of canine skin infections [13].

In our study, high biofilm-forming isolates showed a higher frequency of multidrug resistance than low producers, although this was not statistically significant. In contrast, non-susceptibility to amoxicillin–clavulanate was significantly more common in high biofilm producers (p=0.049), suggesting particular relevance of biofilm for reduced susceptibility to this first-line agent. While some studies have reported links between biofilm formation, ica genes, and resistance in staphylococci, others have not found consistent correlations [30-32], and our data likewise indicate that the interplay between biofilm phenotype, ica status, and antimicrobial resistance in canine S. aureus is complex and cannot be inferred from any single parameter.

This study has limitations. It was conducted in a single veterinary teaching hospital with a moderate number of isolates, especially in the high biofilm subgroup, which may limit statistical power. Dogs were included based on skin lesions suggestive of bacterial infection rather than confirmed pyoderma, and the culture-based design does not clarify the clinical role of S. aureus (pathogen vs coloniser) in individual cases. Molecular typing and detailed characterisation of methicillin resistance were not performed, so clonal relationships and wider epidemiology could not be assessed. Nevertheless, the high frequency of biofilm formation and multidrug resistance among canine S. aureus skin isolates highlights their clinical and One Health relevance, and supports culture and susceptibility-guided therapy alongside antimicrobial stewardship in canine skin infections.

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