Molecular Detection of cna, fnbA, icaA, and icaD Genes and Antibiotic Resistance Patterns in Biofilm-Forming Methicillin-Resistant Staphylococcus aureus from Orthopedic Wound Infections in Abakaliki, Nigeria

Abstract

Background: Methicillin-resistant Staphylococcus aureus (MRSA) remains a formidable pathogen in orthopedic surgery, with biofilm formation and adhesion genes facilitating chronic infections. The icaADBC operon (particularly icaA and icaD) encodes polysaccharide intercellular adhesin (PIA), essential for biofilm accumulation, while cna (collagen-binding) and fnbA (fibronectin-binding) mediate initial tissue attachment, yet molecular data on adhesion and biofilm genes in orthopedic MRSA isolates from southeastern Nigeria remain limited. Objective: This study aimed to provide the first molecular investigation of cna, fnbA, icaA, and icaD genes among all phenotypically confirmed biofilm-forming MRSA isolates from chronic orthopedic wounds with comprehensive antibiotic susceptibility testing, and statistical analysis of resistance patterns in gene-positive versus gene-negative isolates. Methods: A cross-sectional study involving 200 wound swabs was conducted from April 2025 to November 2025. S. aureus was isolated and identified using standard methods. MRSA was detected phenotypically using cefoxitin-oxacillin disk diffusion and chromogenic agar. Biofilm formation was assessed using Congo Red Agar. All 48 biofilm-forming MRSA isolates were subjected to antibiotic susceptibility testing using the Kirby-Bauer disk diffusion method, and screened for cna, fnbA, icaA, and icaD genes via polymerase chain reaction (PCR). Statistical analysis (Chi-square, Fisher's exact, and logistic regression for linezolid resistance only) assessed resistance patterns between gene-positive and gene-negative isolates. Results: Among 200 samples, 148 (74.0%) were S. aureus. Phenotypic screening identified 78 MRSA isolates (39.0% prevalence). Biofilm production was detected in 48 (61.5%) of MRSA isolates. Molecular analysis of all 48 biofilm-forming MRSA isolates revealed: cna in 29 (60.4%), fnbA in 31 (64.6%), icaA in 41 (85.4%), icaD in 43 (89.6%), and co-carriage of all four genes in 22 (45.8%). Regarding antibiotic resistance, all 48 isolates were resistant to ampicillin and amoxicillin (100%). Resistance to clindamycin was 87.5%, vancomycin 70.8%, and linezolid a critical reserve antibiotic showed 20.8% resistance. Gene-positive isolates exhibited significantly higher resistance to clindamycin (p = 0.010), vancomycin (p = 0.018), and linezolid (p = 0.031) compared to gene-negative isolates. Logistic regression revealed fnbA positivity as an independent predictor of linezolid resistance (OR = 8.2, 95% CI: 1.6–41.3, p = 0.012). Logistic regression for MDR was not performed because all 48 biofilm-forming MRSA isolates were multidrug-resistant (100%), resulting in no outcome variation. Mean MARI was 0.66 ± 0.12. Conclusion: This first report from Abakaliki confirms a high prevalence of cna (60.4%), fnbA (64.6%), icaA (85.4%), and icaD (89.6%) genes in biofilm-forming MRSA from orthopedic wounds. The complete presence of icaAD genes explains the strong biofilm phenotype, while the significant association between fnbA carriage and linezolid resistance is a novel finding with serious clinical implications. These findings underscore the urgent need for molecular surveillance and antimicrobial stewardship in Nigerian orthopedic practice.