Biofilm Detection in Bacterial Isolates From Musculoskeletal Infections: A Comparative Study of Tissue Culture Plate and Congo Red Agar Methods With Antimicrobial Susceptibility Correlation

Background Musculoskeletal infections are a significant cause of morbidity and are frequently associated with prolonged hospital stay, increased healthcare costs, and poor functional outcomes. A major contributing factor to the persistence and recurrence of these infections is the ability of microorganisms to form biofilms. Biofilm-associated bacteria exhibit enhanced resistance to host immune responses and antimicrobial agents, making their detection clinically important. This study aimed to evaluate the prevalence of biofilm formation in bacterial isolates from musculoskeletal infections, compare phenotypic detection methods, and assess their antimicrobial susceptibility patterns. Methods This prospective study was conducted over a period of 12 months in the Bacteriology division of a tertiary care hospital. A total of 105 non-duplicate bacterial isolates obtained from 90 cases of musculoskeletal infections were included. Biofilm detection was performed using two phenotypic methods: Congo red agar (CRA) and the tissue culture plate (TCP) method, a standardized microtiter plate assay. The TCP method was considered the reference standard. Antimicrobial susceptibility testing was carried out using the Kirby-Bauer disc diffusion method in accordance with Clinical and Laboratory Standards Institute (CLSI) guidelines. Results The most common clinical condition was infected/open fractures (18; 20.0%), followed by trauma (7; 7.8%), osteomyelitis, implant infections, and joint infections (6; 6.7% each), while miscellaneous cases accounted for 37 (41.1%). Staphylococcus aureus was the most frequently isolated organism (26; 24.8%), followed by Klebsiella pneumoniae (22; 21.0%) and Pseudomonas aeruginosa (15; 14.3%). Biofilm formation was detected in 16 (15.2%) isolates by TCP method, compared to seven (6.7%) by CRA method, with CRA missing nine biofilm-producing isolates. Among organisms, biofilm production was highest in Enterobacter spp. and Proteus spp. (33.3% each), followed by Acinetobacter baumannii (28.6%), coagulase-negative staphylococci (25.0%), and S. aureus (19.2%). Biofilm-producing isolates were more commonly observed with joint infections (50.0%) and spine infections (40.0%). Among Gram-negative isolates, maximum sensitivity was observed to carbapenems (40; 64.0%) and gentamicin (34; 55.0%), whereas resistance was high for cephalosporins and fluoroquinolones. Among Gram-positive isolates, all were sensitive to linezolid and teicoplanin (100%), followed by gentamicin (78.0%). Biofilm-producing isolates demonstrated comparatively higher resistance to commonly used antibiotics than non-biofilm producers; however, this difference was not statistically significant. Conclusion Biofilm formation is an important virulence factor in musculoskeletal infections and contributes to antimicrobial resistance and chronicity. The TCP method is a more sensitive and reliable technique compared to CRA for the detection of biofilm production. Routine screening for biofilm formation, along with appropriate antimicrobial stewardship, can aid in better clinical management and improved patient outcomes.