SILENT SURVIVORS IN COMPLEX FLUIDS: DYNAMICS OF PERSISTER CELL GROWTH IN SYNOVIAL FLUID AND HUMAN SERUM

Introduction and aim Persister cells are a stochastically produced subpopulation of bacterial cells which exhibit a low metabolic status and a non-reproducing phenotype. This results in tolerance to antibiotics and the ability to chronically infect hosts. In musculoskeletal infection (MSKI), especially periprosthetic joint infection (PJI), postoperative wounds filled with synovial fluid and human serum provide a favorable environment for bacteria to persist. Staphylococcus aureus, the leading cause of PJI, poses significant challenges as it can form antibiotic-tolerant aggregates and secrete virulence factors that hinder host tissue repair, frequently requiring prolonged antibiotic therapy and revision surgery. However, the contribution of persister cells to infection persistence in these clinically relevant fluids remains poorly defined. In this work we examined whether Staphylococcus aureus persister cells revert to an awakened state and thrive in these wound-derived fluids, and compared them to actively growing (vegetative) bacteria. Methods Both bacterial populations were isolated and introduced at 10 3 CFU/mL into human serum and equine synovial fluid. Cultures were maintained for three days under physiologic conditions, and samples were collected at 24-hour intervals. We quantified bacterial viability during growth and aggregate formation. Confocal microscopy and image analysis were used to visualize aggregates and quantify biomass, surface coverage, aggregate thickness, and structural organization. Results In human serum, both persister- and vegetative-derived bacteria showed rapid recovery, increasing by 1–5 logs within the first 24 hours. Overall bacterial counts were similar between both bacterial populations over time, although persister-derived cultures initially produced about ten times fewer bacteria on day one. Notably, persister-derived populations continued to increase in biomass, surface coverage, and structural complexity over three days, whereas vegetative populations showed little additional growth after day one. In synovial fluid, both populations increased by 1–6 logs with no meaningful differences in growth or attachment. All measures of aggregate formation were 2- to 20-fold higher than in serum. By day three, gene expression analysis showed no differences in key stress and persistence pathways (relP, gyrA, recA, and lexA), confirming that persister cells had transitioned back to an active state. Conclusions These results demonstrate that persister cells can rapidly “wake up” in fluids present in PJI, particularly synovial fluid, where bacterial growth and aggregation are enhanced. This may help explain infection recurrence despite antibiotic treatment. Current PJI therapies may not adequately address persister cells, highlighting the need to target this population to improve outcomes in MSKI.