This study highlights a significant correlation between the presence of the icaA gene and strong biofilm formation among MRSA isolates, reinforcing its critical role in initiating the synthesis of the polysaccharide intercellular adhesin (PIA) that constitutes the biofilm matrix. Consistent with our findings, recent studies have demonstrated that icaA-positive S. aureus strains exhibit enhanced adherence and biofilm density due to active production of PIA. A study by Armoon et al.¹⁷; Alibegli et al.,¹² confirmed that icaA expression was significantly associated with increased biofilm biomass and structural integrity in clinical MRSA isolates, particularly in device-associated infections.¹⁷ This finding also aligns with the study by Manandhar et al., who reported that icaA-positive MRSA isolates exhibited significantly enhanced biofilm formation.¹⁵ Similarly, Nourbakhsh and Namvar found that 70% of biofilm-forming MRSA isolates in their study were icaA-positive, further emphasizing the gene’s central role in biofilm development.^18,19

In contrast, the detection of isolates carrying icaB without icaA suggests the presence of ica-independent or compensatory pathways for biofilm formation. The icaB gene encodes a deacetylase that modifies PIA, enhancing its adhesive capacity, but alone may not initiate biofilm synthesis. This observation aligns with the findings by El-Jakee et al.,²⁰ who found that icaB may stabilize the biofilm matrix but is unlikely to independently initiate biofilm formation.¹⁹ More recently, Rimi et al.¹³ also reported discrepancies between ica genotype and biofilm phenotype, demonstrating that some MRSA isolates carrying ica genes remained weak or non-biofilm producers. Moreover, studies by Cafiso et al. and Fitzpatrick et al. have highlighted that ica-independent mechanisms, such as the agr quorum-sensing system and sigB stress response pathways, can facilitate biofilm development, indicating that icaA-negative strains may still possess robust biofilm-forming capabilities.^21,22

Moreover, the existence of weak or moderate biofilm-forming icaA-negative isolates supports the theory of alternative biofilm formation mechanisms. The agr quorum-sensing system, sarA regulon, and sigma factor B (sigB) have been implicated in ica-independent biofilm development. Study demonstrated that MRSA strains lacking icaA could still form biofilms through the action of surface-associated proteins such as fibronectin-binding proteins (FnBPs), clumping factors (ClfA/ClfB), and extracellular DNA release.^22,23

Comparatively, this study’s findings align closely with investigations in developing countries, where a higher prevalence of icaB without icaA has been observed. This variation suggests potential environmental or geographical influences on MRSA biofilm-forming behaviors. For instance, studies in Iran and India have reported similar trends where icaB-positive strains were frequently found among non-biofilm producers, highlighting icaB‘s likely role in biofilm maturation rather than initiation. Additionally, the identification of icaA– / icaB+ isolates suggests that biofilm development in these strains may involve alternative pathways independent of the ica operon. A study by O’Gara has proposed that MRSA may utilize surface proteins like fibronectin-binding proteins (FnBPs) or clumping factor proteins (Clf) to promote biofilm formation in the absence of icaA.²⁴ This alternative pathway may explain the presence of biofilm-forming icaA-negative isolates in this study. The findings underscore the importance of incorporating both phenotypic and genotypic methods for detecting biofilm-forming MRSA strains. Relying solely on genotypic screening for icaA and icaB may overlook ica-independent biofilm pathways, which could compromise diagnostic accuracy and hinder effective treatment strategies. These findings stress the importance of regional surveillance, as local environmental factors and antibiotic usage patterns may drive the evolution of distinct biofilm-forming strategies.

The clinical relevance of biofilm formation in MRSA cannot be overstated. Biofilm-associated infections are notoriously difficult to eradicate due to their resistance to antibiotics and immune clearance, often leading to chronic infections, treatment failures, and device-related complications. Therefore, combining phenotypic biofilm assays with genotypic screening for icaA and icaB provides a more comprehensive understanding of a strain’s biofilm-forming potential.

Overall, this study’s results contribute to the growing body of evidence indicating that icaA plays a crucial role in biofilm initiation, while icaB may serve more of a stabilizing function. The variability observed across different geographic regions and clinical settings highlights the need for region-specific surveillance to guide effective infection control practices and therapeutic interventions.

Routine screening of icaA and icaB genes in MRSA isolates should be integrated into clinical microbiology practices. Anti-biofilm agents and combination therapies should be explored to improve treatment outcomes for biofilm-associated MRSA infections. Further research should investigate alternative biofilm mechanisms to explore regulatory networks controlling ica expression, identify novel genes involved in ica-independent biofilm formation, and assess their implications in virulence, antimicrobial resistance, and persistence.