Adjunctive recombinant human thymosin β4 therapy improves antibacterial outcomes against Staphylococcus aureus

Thymosin β4 (Tβ4) is an endogenous multifunctional protein with growing therapeutic applications, including infectious diseases; however, its intrinsic antibacterial activity remains unclear. Previous research with the peptide and related shellfish homologs has suggested broad-spectrum antimicrobial properties, including activity against Staphylococcus aureus . Here, we evaluated the antibacterial activity of recombinant human Tβ4 (rhTβ4) alone and in combination with antibiotics against S. aureus strain 8325-4 and two clinical isolates. Direct antibacterial activity of rhTβ4 (15.625–16,000 µg/mL) was assessed by broth microdilution and colony-forming unit enumeration. Interactions between rhTβ4 and antibiotics were evaluated using checkerboard assays examining total, planktonic, and biofilm-associated bacterial populations. Biofilms were further analyzed using crystal violet staining and SYTO9/PI labeling to assess biomass and membrane integrity, respectively. Moxifloxacin, ciprofloxacin, oxacillin, and vancomycin were evaluated. rhTβ4 alone exhibited modest growth-modulating effects but lacked substantial direct antibacterial activity. In contrast, rhTβ4 demonstrated synergistic interactions with multiple antibiotics, resulting in reduced bacterial growth primarily within total and planktonic populations. Limited effects were observed against established biofilms, as rhTβ4 did not significantly reduce biofilm biomass or membrane integrity, and combination treatments produced antibiotic-dependent biofilm responses. Collectively, these findings indicate that rhTβ4 possesses little intrinsic antibacterial activity against S. aureus but can synergistically augment the effects of select antibiotics in vitro , supporting its utility as an adjunctive therapeutic strategy.