BACTERIAL AGGREGATES WITHIN BODY FLUIDS REPRESENT A METABOLICALLY DISTINCT STATE COMPARED WITH PLANKTONIC AND BIOFILM GROWTH

Introduction & Aim Bacterial pathogens in musculoskeletal infections are traditionally classified as existing in either highly metabolically active planktonic or metabolically quiescent biofilm states. Emerging evidence indicates that bacteria can also interact with host macromolecules in body fluids, such as synovial fluid, to form distinct aggregate structures with unique metabolic profiles. While synovial fluid aggregates are known to exhibit antimicrobial tolerance, their underlying metabolic state remains poorly characterized. This study aimed to compare the relative metabolic activity of bacterial aggregates in bodily fluids, primarily synovial fluid, with planktonic and biofilm growth states using clinically relevant pathogens. Methods Clinical isolates representing four common musculoskeletal pathogens (Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa, Escherichia coli) were evaluated in vitro. Each isolate was assessed under four growth conditions: planktonic, acute biofilm, synovial fluid aggregate, and synovial fluid aggregate exposed to fibrinolytic enzymatic disruption. Relative metabolic activity was quantified using a colorimetric assay over an 18-hour time course, and growth kinetics were analyzed using area under the curve calculations. Results Across all species, planktonic bacteria demonstrated the highest relative metabolic activity. Both biofilm and synovial fluid aggregate states exhibited significantly reduced metabolic activity compared with planktonic growth (p≤0.01). Notably, synovial fluid aggregates formed by S. aureus and P. aeruginosa showed significantly lower metabolic activity than their corresponding biofilm states (p≤0.01), whereas aggregates formed by S. epidermidis and E. coli demonstrated metabolic activity similar to biofilms. Enzymatic disruption of synovial fluid aggregates resulted in restoration of metabolic activity toward planktonic levels for all species, with S. aureus exhibiting metabolic activity exceeding basal planktonic levels following aggregate disruption. Conclusions Bacterial aggregation in synovial fluid represents a conserved, reversible, and metabolically distinct growth state that is not equivalent to classical biofilm or planktonic phenotypes. The metabolic suppression observed in synovial fluid aggregates, particularly among virulent pathogens such as S. aureus, may contribute to diagnostic challenges and therapeutic failure in joint infections. Enzymatic disruption of aggregate structure restores metabolic activity, highlighting aggregation as a modifiable determinant of bacterial physiology. These findings support further investigation of bodily fluid aggregates as a clinically relevant bacterial state with implications for improving the diagnosis and treatment of musculoskeletal infections.