Emerging threats of high biofilm formation and antibiotic resistance in clinical methicillin-resistant Staphylococcus aureus (MRSA) isolates from Pakistan

Objectives: This multicenter study, conducted from a One Health perspective, aimed to comprehensively examine the prevalence of methicillin-resistant Staphylococcus aureus (MRSA) infections and their biofilm -forming capabilities in Pakistan. Phylogenetic analysis of the study isolates was also performed. Methods: A total of 150 MRSA isolates were screened from various clinical samples using Cefoxitin antibiotic discs. Genotypic confirmation was conducted through mecA , S. aureus -specific nuc , and 16S rRNA genes. Biofilm formation was assessed using Congo red agar (CRA) and slime layer quantification methods. The intercellular adhesion ( ica ) operon genes, specifically icaA and icaD , were detected. Phylogenetic analysis utilized the 16S rRNA sequences. Statistical associations between various parameters were determined using chi-square analysis. Results: The presence of the mecA gene was observed in 131 out of 150 isolates (87.3%). CRA identified 28% and 40% of isolates as strong and moderate biofilm producers, respectively, while 9.3% were classified as non -biofilm producers. The slime layer assay exhibited higher sensitivity, classifying only 4.7% of isolates as non -biofilm producers. Biofilm -forming genes icaA and icaD were detected in 85.3% and 86.7% of the isolates, respectively. Antibiotic resistance was more prevalent among biofilm -forming isolates, particularly against ciprofloxacin, levofloxacin, erythromycin, trimethoprim-sulfamethoxazole, and fosfomycin. Ceftaroline demonstrated efficacy irrespective of biofilm -forming abilities. Conversely, non -biofilm producers exhibited complete susceptibility to clarithromycin and tigecycline. Conclusions: Clinical MRSA strains exhibit a substantial potential for biofilm formation, contributing to a resistant phenotype. Routine antibiotic testing in clinical settings that overlook the biofilm aspect may lead to the failure of empiric antibiotic therapy.