Even after decades of research and control interventions, mastitis is still among the most expensive diseases in dairy herds (Viguier et al., 2009). The disease occurs in two forms: clinical mastitis and subclinical mastitis (SCM). Clinical form can be easily identified by inflammation of the mammary gland, pain and abnormal milk secretion. SCM, on the other hand, advances silently, without any noticeable external symptoms and is usually identified by an increase in the number of somatic cells or by bacterial identification in the laboratory (Kavitha et al., 2009; Reza et al., 2011; Kumar et al., 2021). Mastitis development is a complex interplay among invading microorganisms, the host immune responses and environmental or management-related stress factors (Bradley, 2002). Despite the wide variety of microorganisms isolated from mastitic milk, bacterial pathogens are the main causative agents (Hawari and Fawzi, 2008). These pathogens are mostly classified as either contagious or environmental, depending on how they are transmitted. Streptococcus agalactiae, Staphylococcus aureus and Mycoplasma bovis are among the most common contagious pathogens, which are highly adapted to survival in the mammary gland and are transmitted primarily during milking. The sources of environmental pathogens, such as Streptococcus uberis, Streptococcus dysgalactiae and coliform organisms such as Escherichia coli and Klebsiella spp., include bedding, soil, faeces and water (Radostits et al., 2000). Although environmental infections often initiate acute inflammatory reactions, contagious pathogens, especially S. aureus, are more likely to be associated with persistent and recurrent intramammary infections (Singh and Baxi, 1982).
       
Biofilm formation is one of the major causes of the enduring nature of S. aureus infections. Biofilms are organised communities of microorganisms surrounded by an extracellular matrix composed mainly of polysaccharides, proteins, extracellular DNA and teichoic acids (Hall-Stoodley  et al., 2004; Lear and Lewis, 2012). Biofilms enable bacteria to colonise mammary tissue, evade immune responses and withstand antimicrobial treatment. Biofilm cells exhibit altered metabolic activity and decreased antibiotic susceptibility, leading to treatment failure and recurrence (Lear and Lewis, 2012). The intercellular adhesion (ica) operon, especially the icaA and icaD genes, mediate biofilm formation in S. aureus and control the expression of polysaccharide intercellular adhesin (Cramton et al., 1999). The indiscriminate use of antimicrobial treatment for mastitis, along with the biofilms, has further exacerbated antimicrobial resistance and diminished treatment efficacy (Giesecke et al., 1994; Ruegg, 2017). Considering the central role of S. aureus in the persistence of chronic mastitis, as well as the increased emphasis on biofilm-mediated persistence, the bacterium’s biofilm-forming potential and underlying genetic determinants require immediate attention. Thus, the aim of the current investigation was to determine the prevalence, antimicrobial susceptibilities and biofilm-forming potential of S. aureus isolates from cases of clinical mastitis.

Study design and bacterial isolation
 
This study was conducted between March and September 2025 in the states of Telangana and Maharashtra, India. All experiments in the current study were conducted at PVNR Telangana Veterinary University, Hyderabad, India. A total of 200 milk samples were collected from lactating dairy cows clinically diagnosed with mastitis. Clinical mastitis was confirmed using the California mastitis test following the recommendations of the National Mastitis Council (Hogan et al., 1999). Milk samples were collected aseptically from affected quarters after proper teat disinfection. Samples were transported to the laboratory under refrigerated conditions and processed immediately. Bacteriological examination was performed using standard microbiological procedures (Cruickshank et al., 1975). For bacterial isolation, milk samples were cultured on Nutrient Agar, MacConkey Agar, Eosin Methylene Blue Agar and Mannitol Salt Agar. Streptococcus Selective Broth was used to selectively enrich Streptococci spp. All plates were incubated aerobically at 37°C for 18-24 hours. Representative colonies were subcultured to obtain pure isolates. Preliminary identification was based on colony morphology, Gram staining and standard biochemical tests including catalase, coagulase, oxidase, indole, methyl red, Voges-Proskauer, citrate utilisation and carbohydrate fermentation assays (Cruickshank et al., 1975; Jawetz et al., 2004).
 
Antimicrobial susceptibility testing
 
Antimicrobial susceptibility of Staphylococcus isolates was determined using the Kirby-Bauer disk diffusion method on Mueller-Hinton Agar, following Clinical and Laboratory Standards Institute guidelines (CLSI, 2023). Bacterial suspensions were adjusted to a 0.5 McFarland standard and evenly spread onto agar plates. Commercial antibiotic discs (HiMedia Laboratories, India; Oxoid, UK) were used, including ampicillin (10 µg), ceftiofur (30 µg), ceftriaxone (30 µg), enrofloxacin (30 µg), gentamicin (30 µg), methicillin (30 µg) and tetracycline (30 µg). After incubation at 37°C for 18-24 hours, zones of inhibition were measured and interpreted as susceptible, intermediate, or resistant according to CLSI guidelines.
 
Assessment of biofilm formation and molecular detection of the icaD gene
 
Biofilm production was screened using Congo Red Agar (CRA) and 96-well microtiter plate (MTP) assay (Freeman et al., 1989; Stepanovic et al., 2003). Isolates that formed black, dry, crystalline colonies on CRA were considered biofilm producers, whereas red colonies were classified as non-producers. For MTP assay, overnight cultures grown in Tryptic Soy Broth (TSB) were inoculated into sterile microplates in triplicate. Wells containing only TSB served as negative controls. Following incubation at 37°C for 24 hours, wells were gently washed with phosphate-buffered saline to remove planktonic cells. The remaining adherent biomass was stained with 0.1% crystal violet. After solubilising the bound dye, the optical density (OD) was measured at 630 nm using a microplate reader. The cut-off OD (ODc) was defined as three standard deviations above the mean OD of the negative control. For genotypic characterisation, genomic DNA was extracted from Staphylococcus isolates using the phenol–chloroform method. PCR amplification targeting the icaD gene was performed using previously described primers, yielding an expected amplicon of approximately 381 bp (Vasudevan et al., 2003).
 
Scanning electron microscopy
 
To visualise biofilm architecture, selected isolates confirmed as biofilm producers by phenotypic and genotypic methods were examined using scanning electron microscopy. Cultures were grown in TSB and incubated with sterile glass coverslips placed in 6-well tissue culture plates at 37°C with shaking for 18 hours. After incubation, coverslips were gently washed with PBS, fixed in 2.5% glutaraldehyde at 4°C for 24 hours and then post-fixed in 1% osmium tetroxide. Samples were dehydrated through graded ethanol concentrations, dried using a critical-point dryer, mounted on stubs and sputter-coated with gold. Specimens were examined using a JEOL JSM-5600 scanning electron microscope to assess bacterial adherence, aggregation and extracellular matrix formation.

Isolation and identification of bacterial pathogens from milk samples
 
From the 200 clinical mastitis samples, 214 bacteria were isolated, indicating that intramammary infections are often polymicrobial. The dominant species were Staphylococcus spp. (58.8%), then Escherichia coli (24.2%), Pseudomonas spp. (11.4%) and Streptococcus spp. (5.6%) (Fig 1A). This is in line with the global epidemiological trends that indicate that S. aureus is among the most successful pathogens in the mammary tract (Lasa and Penadés, 2006; Taponen and Pyörälä, 2009). Nonetheless, its significance goes beyond frequency. Intracellular survival, immune evasion and biofilm formation are some of the adaptive mechanisms developed by S. aureus that have allowed it to survive in the long term in the mammary gland; often with subclinical or recurrent infections which are challenging to eradicate (Pedersen et al., 2021; Ruegg, 2023; Song et al., 2024). S. aureus unlike many other environmental pathogens which cause acute inflammation and are often eliminated quickly, is often linked with long term colonisation, subclinical persistence and incomplete bacteriological cure following antimicrobial therapy (Pedersen et al., 2021; Taponen and Pyörälä, 2022; Ruegg, 2023). E. coli and Pseudomonas spp. are commonly associated with exposure to the environment and herd-hygiene behaviour (Bradley, 2002; Kromker and Leimbach, 2014). Collectively, the observed distribution pattern indicates interactions among host susceptibility, pathogen adaptiveness and management conditions in the farm.
 
Antimicrobial susceptibility and emerging therapeutic challenges
 
The antimicrobial susceptibility test showed a dynamic therapeutic situation. Ceftiofur was the most sensitive, with 58% of Staphylococcus isolates identified as susceptible (Fig 1B). Even though this is consistent with prior reports indicating retained efficacy of third-generation cephalosporins (Gupta et al., 2019; Basanisi et al., 2021), a sensitivity rate of less than 60% indicates a diminished reliability. Tetracycline (40%), gentamicin (38.5%) and enrofloxacin (35%) were found to be moderately susceptible. These values indicate some effectiveness, but could also indicate increasing resistance pressure, which might be due to their empirical or repeated use in dairy practice. The decreased effect of aminoglycosides and fluoroquinolones is also alarming considering their frequent use in the treatment of mastitis. The lowest sensitivity to ampicillin (19%) and methicillin (18.5%), was the most alarming. The developing resistance of methicillin increases the likelihood of livestock-related MRSA, which has both therapeutic and zoonotic consequences (Holmes and Zadoks, 2011; Shakya et al., 2025). These patterns of extensive â-lactam resistance in dairy herds have been reported elsewhere (Bardiau et al., 2013) indicating the deleterious impact of long-term exposure to antimicrobials. It should be noted that disc diffusion is a test of the planktonic bacterial susceptibility in laboratory conditions. Biofilm-associated bacteria could be much more tolerant in vivo. Biofilms limit penetration of antibiotic, form nutrient and oxygen gradients and support metabolically dormant cells all of which decreases treatment efficacy (Costerton et al., 1999; Arciola et al., 2001). Consequently, routine antibiogram results may underestimate the risk of therapeutic failure in biofilm-mediated intramammary infections.

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Phenotypic expression, genetic determinants and ultrastructural characterisation of S. aureus biofilms
 
Of the 126 S. aureus isolates tested, 75 formed biofilms in the microtiter plate (MTP) assay (Fig 2A), whereas only 50 were detected by Congo Red Agar (CRA) (Fig 2B). The increased sensitivity and quantitative reliability of MTP is supported by the higher rates of detection in other studies (Mathur et al., 2006; Stepanovic et al., 2007). The icaD gene was detected in all MTP-positive isolates, indicating a genetic basis for biofilm formation in these strains (Fig 3). The ica operon produces the enzymes required to synthesise the polysaccharide intercellular adhesin which is an important structural element of the biofilm’s extracellular matrix (Cramton et al., 1999). The uniform occurrence of icaD in biofilm producers indicates that the matrix-forming strains have a high level of selective advantage in the mammary environment. Biofilm formation is a community-based change of lifestyle for bacteria. Such changes are characterised by changes in gene expression, slowing metabolism and changes in resistance to environmental stresses (Hall-Stoodley  et al., 2004). These adaptations probably allow S. aureus to avoid immune clearance and survive even when antimicrobial treatment is initiated in the mammary gland. The visual confirmation of the biofilms was done through scanning electron microscopy. The isolates with biofilm forming properties were represented by dense bacterial aggregates surrounded by a thick extracellular matrix and the isolates that were not biofilm producers did not have the organised structure (Fig 4). These ultrastructural variations reflect previous findings and depict the physical obstruction that biofilms pose to penetration of therapeutic agents (Melchior et al., 2006; Mack et al., 2007). S. aureus that forms biofilms within the mammary gland creates organised microbial communities within an extracellular polymeric matrix that limits the entry of the antibiotic, creating metabolic heterogeneity and enhancing persistence of dormant persister cells. As a result, antimicrobial concentrations that are effective against planktonic bacteria are unlikely to eliminate biofilm populations leading to low bacteriological cure rates, chronic inflammation and frequent intramammary infections (Pedersen et al., 2021; Demontier et al., 2024). Recently published experimental and field-based studies also support that ica-positive and strong biofilm-producing strains have a higher survival following antimicrobial exposure despite exhibiting in vitro sensitivity (Rychshanova et al., 2022; Song et al., 2024). This lack of correlation between in vivo therapeutic and in vitro susceptibility profiles emphasises shortcomings of traditional antibiogram-based therapy. Furthermore, studies on biofilm-targeting interventions have demonstrated that targeting biofilm-related tolerance mechanisms can be a highly effective method to enhance antimicrobial activity (Lin et al., 2023). Together, these data demonstrate that the biology of biofilms is a key determinant of treatment failures in Staphylococcus-mediated mastitis and highlight the need for integration of biofilm measurement and adjunctive antibiofilm measures in mastitis disease management programs to enhance long term treatment success (Pedersen et al., 2021; Demontier et al., 2024). The interplay between high Staphylococcus prevalence, high antimicrobial resistance and a widespread biofilm-forming ability supports the fact that persistent mastitis is not only a result of the inappropriate use of therapy but of microbial adaptation strategies. S. aureus that forms biofilms are adapted to resist host immunity and exposure to antibiotics, which in turn makes the intramammary infection chronic and recurrent (Pedersen et al., 2021; Demontier et al., 2024; Song et al., 2024). These results point to the necessity of modifying the traditional treatment paradigms. Good mastitis management must include specific antimicrobial management, regular evaluation of resistance profiles and consideration of biofilm-related pathogenicity. New methods such as enzymatic biofilm disruption, quorum-sensing inhibitors, bacteriophage therapy and nanotechnology-based drug delivery are promising adjunctive methods (Hoiby et al., 2010; Basanisi et al., 2021; Sandhu et al., 2025).

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To conclude, results of the current study indicate that a significant percentage of mastitis causing S. aureus isolates exhibited icaD mediated biofilm-forming ability. This virulence attribute is probably the basis of persistence, low susceptibility to antibiotics and frequency of infection. To enhance therapeutic responses and promote sustainable control of mastitis in dairy systems, there is an urgent need to integrate measures to target biofilm-mediated pathogenicity along with appropriate antibiotic therapy.

No specific funding was received for this study. The authors would like to acknowledge PVNRTVU, Hyderabad and Acharya Nagarjuna University, Guntur, for providing resources to conduct the study.
 
Disclaimers
 
The views and conclusions expressed in this article are solely those of the authors and do not necessarily represent the views of their affiliated institutions. The authors are responsible for the accuracy and completeness of the information provided, but do not accept any liability for any direct or indirect losses resulting from the use of this content.
 
Informed consent
 
No animal experimentation that requires permission of IAEC was performed in this study.
 

The authors declare that there are no conflicts of interest regarding the publication of this article. No funding or sponsorship influenced the design of the study, data collection, analysis, decision to publish, or preparation of the manuscript.