Invasive infections due to Staphylococcus aureus, including 
methicillin-resistant S. aureus are prevalent and life-threatening. Combinations 
of antibiotic therapy have been employed in many clinical settings for improving 
therapeutic efficacy, reducing side effects of drugs, and development of 
antibiotic resistance. Pleuromutilins have a potential to be developed as a new 
class of antibiotics for systemic use in humans. In the current study, we 
investigated the relationship between pleuromutilins, including valnemulin, 
tiamulin, and retapamulin, and 13 other antibiotics representing different 
mechanisms of action, against methicillin-susceptible and -resistant S. aureus 
both in vitro and in an experimental Galleria mellonella model. In vitro 
synergistic effects were observed in combination of all three study 
pleuromutilins with tetracycline (TET) by standard checkerboard and/or time-kill 
assays. In addition, the combination of pleuromutilins with ciprofloxacin or 
enrofloxacin showed antagonistic effects, while the rest combinations presented 
indifferent effects. Importantly, all study pleuromutilins in combination with 
TET significantly enhanced survival rates as compared to the single drug 
treatment in the G. mellonella model caused by S. aureus strains. Taken 
together, these results demonstrated synergy effects between pleuromutilins and 
TET against S. aureus both in vitro and in vivo.