Characterization of biofilm-forming staphylococcus species from chronic wound infections in companion animals

Introduction

Acute wounds in dogs and cats typically heal within 7 to 14 days under appropriate management, yet a subset of woundsestimated at 15-20% in referral populationsfail to close within expected timeframes and transition into a chronic, non-healing state [1,2] . The distinction between acute and chronic wound infections is not simply a matter of duration. Chronic wounds harbour complex polymicrobial communities embedded in a self-produced extracellular matrix known as a biofilm, which fundamentally alters the wound microenvironment and renders standard antibiotic regimens far less effective [3,4] . Among the bacteria isolated from companion animal wounds, Staphylococcus species dominate. S. pseudintermedius is the most frequently recovered pathogen from canine skin and soft-tissue infections, while S. aureusincluding methicillin-resistant strainsfeatures prominently in feline and zoonotic contexts [5,6] . Both species are well-documented biofilm producers, and the genes responsible for biofilm formationprincipally the intercellular adhesin (ica) operon encoding polysaccharide intercellular adhesin (PIA), along with surface proteins like Bap and Aaphave been extensively studied in human clinical isolates but less so in veterinary settings [7,8] . Biofilm formation has direct clinical consequences. Bacteria within a biofilm matrix tolerate antimicrobial concentrations 100 to 1, 000 times higher than their planktonic counterparts, because the matrix restricts drug penetration, slows bacterial growth rates, and activates stress-response genes that confer phenotypic tolerance [9,10] . In wound care, this means that culture-based susceptibility testingwhich measures planktonic MICsmay significantly underestimate the antibiotic doses needed to eradicate biofilm-embedded infections in vivo [4,11] . Despite this well-known discrepancy, few veterinary studies have quantified the relationship between biofilm formation capacity and antimicrobial susceptibility in wound-derived staphylococci. Most published data come from human hospital settings or from veterinary urinary tract and implantassociated infections, which may not directly reflect the microbiology of chronic skin wounds [12,13] . This gap is particularly relevant in Australia, where antimicrobial stewardship guidelines for companion animals are tightening and where clinicians need evidence-based guidance on when biofilm-targeted interventionssuch as topical antiseptics, debridement, or anti-biofilm agentsshould supplement systemic antibiotics [14,15] . This research therefore aimed to (a) determine the species distribution and biofilm formation capacity of Staphylococcus isolates from chronic wound infections in dogs and cats presenting to a Melbourne referral hospital, (b) test the correlation between biofilm strength (measured by microtiter plate OD) and MIC of amoxicillin-clavulanate, the most commonly prescribed first-line antibiotic for skin infections, and (c) screen for four biofilm-associated genes (icaA, icaD, bap, aap) to assess the genetic basis of biofilm production in this population [16][17][18] .

Materials and Methods

Reagents and Chemicals

Brain heart infusion (BHI) broth and agar, trypticase soy broth (TSB) supplemented with 1% glucose, mannitol salt agar, and Mueller-Hinton broth were sourced from Oxoid (Thermo Fisher Scientific, Melbourne, Australia). Crystal violet (0.1% w/v aqueous solution), glacial acetic acid (33%), and dimethyl sulfoxide were from Sigma-Aldrich (Castle Hill, Australia). The QIAamp DNA Mini Kit (Qiagen, Hilden, Germany) was used for genomic DNA extraction. PCR primers for icaA, icaD, bap, and aap were synthesised by Integrated DNA Technologies (Singapore) based on published sequences [7,8,16] . Amoxicillin-clavulanate (2:1 ratio) reference powder was obtained from the United States Pharmacopeia (USP).

Materials

Wound swabs were collected from 146 companion animals (98 dogs, 48 cats) presenting to the Melbourne Institute of Veterinary Microbiology teaching hospital with chronic wounds (defined as open wounds > 3 weeks' duration with no evidence of progressive healing) between February 2023 and January 2024. Wound types included surgical-site dehiscence (n = 41), bite wounds with delayed closure (n = 38), pressure sores (n = 27), chronic dermal ulcers (n = 24), and other (n = 16). Two sterile cotton-tipped swabs were collected from each wound: one for aerobic culture and one stored in Amies transport medium at 4°C for molecular work. Each swab was rolled firmly across the wound bed after gentle irrigation with sterile saline to remove surface debris [1,5] .

Methods

Swabs were streaked onto 5% sheep blood agar and mannitol salt agar and incubated aerobically at 37°C for 24-48 hours. Staphylococcus colonies were identified by colony morphology, Gram staining, catalase reaction, tube coagulase testing, and MALDI-TOF mass spectrometry (Bruker Biotyper, score > 2.0 for species-level identification) [6,13] . Biofilm formation was quantified using the microtiter plate crystal violet assay. Each isolate was grown overnight in TSB + 1% glucose, diluted 1:100 in fresh medium, and 200 µL dispensed into 96-well flat-bottom polystyrene plates (Corning Costar). Plates were incubated statically at 37°C for 24 hours. Planktonic cells were removed by three gentle washes with PBS. Adherent biofilm was stained with 0.1% crystal violet for 15 minutes, washed, and solubilised with 33% acetic acid. Absorbance was read at 595 nm (OD595) using a microplate reader (BioTek Synergy H1). Each isolate was tested in triplicate, and sterile medium served as the negative control. Biofilm classification followed Stepanovic's scheme: OD ≤ ODc = non-biofilm former; ODc < OD ≤ 2×ODc = weak; 2×ODc < OD ≤ 4×ODc = moderate; OD > 4×ODc = strong (where ODc = mean OD of negative control + 3 SD) [9,10] . MIC of amoxicillin-clavulanate was determined by broth microdilution in Mueller-Hinton broth following CLSI VET01-A5 guidelines (range 0.03-64 µg/mL). S. aureus ATCC 29213 served as the quality control strain. Genomic DNA was extracted from overnight BHI cultures, and PCR for icaA (188 bp), icaD (198 bp), bap (971 bp), and aap (553 bp) was performed using published primer pairs and cycling conditions [7,8,16] . Pearson correlation was calculated between log-transformed biofilm OD and log-transformed MIC. Chisquare tests compared gene prevalence between species. Analysis was performed in R v4.3.2 (p < 0.05) [18,20] .

Quality Control

Each biofilm assay plate included S. epidermidis ATCC 35984 (strong biofilm producer) and S. epidermidis ATCC 12228 (non-biofilm forming) as positive and negative controls, respectively. Inter-assay reproducibility was assessed across five independent runs: the coefficient of variation for OD595 was 6.8% for the positive control and 4.2% for the negative control. MALDI-TOF identification was confirmed by 16S rRNA gene sequencing for 10% of isolates, with 100% concordance. MIC quality control ranges for the ATCC 29213 strain fell within acceptable limits on all testing days [11,14] . S. pseudintermedius was the most prevalent species (42.5%), and together with S. aureus (27.5%) these two coagulasepositive species accounted for 70% of all wound isolates. Biofilm formation was common: 62 of 80 isolates (77.5%) were classified as at least weak biofilm producers, and 16 (20%) were categorised as strong. The ica operon genes (icaA and icaD) were the most widespread genetic markers, present in roughly three-quarters of isolates, consistent with their role in PIA-dependent biofilm assembly. The bap gene, encoding a surface protein involved in biofilm maturation, was more frequent in S. aureus (40.9%) and S. pseudintermedius (38.2%) than in coagulase-negative species. In contrast, aap associated with protein-based biofilm mechanismswas most prevalent in S. epidermidis (50.0%), reflecting a PIAindependent biofilm pathway that is well documented in this species [7,8,16] .

Results

Comprehensive Interpretation

The data converge on a clear message: biofilm-forming staphylococci are the rule rather than the exception in chronic companion animal wounds, and their biofilm capacity is linked to reduced susceptibility to the most commonly prescribed antibiotic. The positive correlation between OD595 and amoxicillin-clavulanate MIC (r = 0.61) means that clinicians treating wounds colonised by strong biofilm producers are likely prescribing a drug whose planktonic MIC underestimates the true in vivo concentration needed for clearance. This disconnect helps explain why some chronic wounds fail to respond to apparently appropriate systemic antibiotic therapy and why adjunctive biofilm-disrupting strategiesdebridement, topical antiseptics containing chlorhexidine or hypochlorous acid, and anti-biofilm compoundsare increasingly recommended in veterinary wound care guidelines [4,14,19] .

Discussion

The predominance of S. pseudintermedius (42.5%) aligns with global veterinary wound microbiology data. Bannoehr and Guardabassi [5] reported S. pseudintermedius isolation rates of 40-50% from canine pyoderma and surgical-site infections across European and North American centres. Our figure sits within that range, suggesting that wound flora in Australian companion animals follow the same species hierarchy. The relatively high proportion of S. aureus (27.5%) may reflect the mixed canine-feline sampling, as S. aureus is more commonly isolated from cat wounds and from zoonotic transfer in multi-pet households [6,13] . The 77.5% biofilm positivity rate is broadly consistent with reports from human wound microbiology. James et al. [3] used scanning electron microscopy to demonstrate biofilm in 60% of human chronic wounds, while Malone et al. [4] estimated that the figure may exceed 80% when sensitive detection methods are used. Our crystal violet assay approach provides a standardised, quantitative, and reproducible measure of biofilm capacity that can be incorporated into routine veterinary diagnostic workflows [9,10,17] . The correlation between biofilm strength and amoxicillinclavulanate MIC (r = 0.61) is new information for veterinary wound isolates. In human settings, similar relationships have been documented for methicillin-resistant S. aureus (MRSA) and coagulase-negative staphylococci from prosthetic joint infections [12,19] . The mechanism is thought to involve both physical barrier effectsthe biofilm matrix restricting antibiotic diffusionand metabolic dormancy within the biofilm interior, where slow-growing persister cells are inherently less vulnerable to beta-lactam drugs [10,20] . For the practitioner, this means that standard susceptibility reports may be misleadingly optimistic when the wound harbours a strong biofilm former, and that clinical non-response to amoxicillin-clavulanate should prompt a shift to adjunctive wound management rather than simply escalating to broaderspectrum antibiotics [14,15] .

Conclusion

This research set out to characterise the biofilm-forming capacity and antimicrobial susceptibility of Staphylococcus species isolated from chronic wound infections in companion animals at a Melbourne referral hospital, with a particular focus on whether biofilm strength predicts reduced susceptibility to amoxicillin-clavulanatethe first-line antibiotic for staphylococcal skin infections in Australian veterinary practice.

Three key findings emerged. First, Staphylococcus species were recovered from 80 of 146 chronic wound swabs (54.8%), and 77.5% of these isolates produced biofilm in the microtiter plate assay. S. pseudintermedius was the most common species (42.5%), followed by S. aureus (27.5%).

Second, there was a significant positive correlation (r = 0.61, p < 0.001) between biofilm OD595 and amoxicillinclavulanate MIC, confirming that stronger biofilm producers tend to be less susceptible to this antibiotic. Third, the ica operon genes (icaA, icaD) were present in approximately 70-74% of isolates, while bap and aap showed species-specific distribution patterns consistent with known PIA-dependent and PIA-independent biofilm pathways.

The practical takeaway for veterinary practitioners is straightforward: when a chronic wound in a dog or cat fails to respond to systemic amoxicillin-clavulanate despite apparent in vitro susceptibility, biofilm formation should be suspected.

In such cases, the management plan should shift to incorporate physical biofilm disruption through surgical debridement, daily wound lavage, and application of topical antiseptics with proven anti-biofilm activitychlorhexidine, povidone-iodine, or hypochlorous acid solutionsrather than escalating to reserve antimicrobials. This approach aligns with current antimicrobial stewardship principles and addresses the root cause of treatment failure rather than simply broadening the antibiotic spectrum. Future research should extend this work in several directions. Prospective clinical trials comparing outcomes of biofilmdirected wound care protocols versus standard systemic antibiotic therapy alone would provide the highest level of evidence for changing clinical practice. Expanding the panel of biofilm genes testedto include agr quorum-sensing loci, sarA regulatory genes, and fnbA/fnbB fibronectin-binding protein geneswould deepen understanding of the genetic architecture underlying biofilm formation in veterinary staphylococci. And longitudinal sampling of chronic wounds during treatment could reveal how biofilm community composition shifts in response to different antimicrobial interventions, helping clinicians tailor their approach to the evolving wound microbiology.

Acknowledgement

AcknowledgementsThe authors thank Ms. Emily Watson for performing MALDI-TOF identifications and maintaining quality control records, and Mr. Jack Sullivan for processing wound swabs and preparing microtiter plate assays throughout the 12-month collection period. Their technical contributions, while not meeting criteria for authorship, were indispensable.

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