MOLECULAR INSIGHTS AND ANTIMICROBIAL RESISTANCE PATTERNS OF BIOFILM-PRODUCING STAPHYLOCOCCUS AUREUS IN CLINICAL ISOLATES

Abstract

Background: Biofilm-forming Staphylococcus aureus complicates treatment by promoting tolerance and resistance to antimicrobials. This study investigated the association between biofilm formation, biofilm-associated genes, and antibiotic susceptibility in clinical S. aureus isolates from Faisalabad hospitals.

Methods: A total of 3300 samples (wound swabs, catheter tips, sputum, and pus) were collected, yielding non-duplicate 1000 S. aureus isolates, identified phenotypically and through nuc-PCR. Biofilm production was assessed using Congo Red Agar and microtiter plate assays, and isolates were tested for ica operon (icaA, icaB, icaC, icaD) and fibronectin-binding genes (fnbA, fnbB) via PCR. Antimicrobial susceptibility was evaluated using modified Kirby–Bauer disk diffusion method (cefoxitin 30 µg, fusidic acid 10 µg, erythromycin 15 µg, ciprofloxacin 5 µg, linezolid 30 µg, trimethoprim–sulfamethoxazole 25 µg, clindamycin 2 µg, levofloxacin 5 µg) following CLSI guidelines; Vancomycin minimum inhibitory concentrations (MICs) were determined by broth microdilution. Multidrug resistance (MDR) was defined as resistance to three or more antimicrobial classes. Selected isolates showing phenotypic resistance were tested for mecA and vanA, and representative amplicons underwent Sanger sequencing.

Results: Among 1000 isolates, 760 (76.0%) were methicillin-resistant, determined by cefoxitin resistance. High resistance rates were observed for cefoxitin (760/1000, 76.0%), ciprofloxacin (750/1000, 75.0%), erythromycin (700/1000, 70.0%), levofloxacin (650/1000, 65.0%), clindamycin (500/1000, 50.0%), and fusidic acid (590/1000, 59.0%). Resistance to trimethoprim–sulfamethoxazole was 400/1000 (40.0%), while linezolid resistance was rare (20/1000, 2.0%).

Vancomycin MIC testing classified 140/1000 (14.0%) isolates as vancomycin-resistant S. aureus (VRSA; MIC ≥16 µg/mL), 300/1000 (30.0%) as vancomycin-intermediate S. aureus (VISA; MIC 4–8 µg/mL), and 560/1000 (56.0%) as vancomycin-susceptible S. aureus (VSSA).

Biofilm phenotyping identified strong producers (n = 550), intermediate producers (n = 150), and non-producers (n = 300). Strong biofilm producers showed significantly higher MDR prevalence (480/550; 87.3%) compared with non-producers (140/300; 46.7%) (χ² = 32.43, p < 0.001) and a higher rate of vancomycin non-susceptibility (VRSA + VISA: 270/550; 49.1%) than non-producers (60/300; 20.0%) (p < 0.001). The presence of icaA and icaD genes was strongly associated with the strong biofilm phenotype (p < 0.001). Molecular analysis of 150 resistant isolates detected mecA in 50 (33.3%) and vanA in 45 (30.0%), and sequencing confirmed the identity of the amplified genes.

 Conclusions: High MRSA prevalence and significant vancomycin non-susceptibility were found. Strong biofilm formation, particularly in icaA/icaD-positive isolates, associates with MDR and reduced vancomycin susceptibility, highlighting the need for targeted infection control and antimicrobial stewardship.