*Article* **Eradication of** *Staphylococcus aureus* **Biofilm Infection by Persister Drug Combination**

**Rebecca Yee 1,†, Yuting Yuan 1,‡, Andreina Tarff <sup>2</sup> , Cory Brayton <sup>3</sup> , Naina Gour <sup>4</sup> , Jie Feng <sup>5</sup> and Ying Zhang 6,\***


**Abstract:** *Staphylococcus aureus* can cause a variety of infections, including persistent biofilm infections, which are difficult to eradicate with current antibiotic treatments. Here, we demonstrate that combining drugs that have robust anti-persister activity, such as clinafloxacin or oritavancin, in combination with drugs that have high activity against growing bacteria, such as vancomycin or meropenem, could completely eradicate *S. aureus* biofilm bacteria in vitro. In contrast, single or two drugs, including the current treatment doxycycline plus rifampin for persistent *S. aureus* infection, failed to kill all biofilm bacteria in vitro. In a chronic persistent skin infection mouse model, we showed that the drug combination clinafloxacin + meropenem + daptomycin which killed all biofilm bacteria in vitro completely eradicated *S. aureus* biofilm infection in mice while the current treatments failed to do so. The complete eradication of biofilm bacteria is attributed to the unique high anti-persister activity of clinafloxacin, which could not be replaced by other fluoroquinolones including moxifloxacin, levofloxacin, or ciprofloxacin. We also compared our persister drug combination with the current approaches for treating persistent infections, including gentamicin + fructose and ADEP4 + rifampin in the *S. aureus* biofilm infection mouse model, and found neither treatment could eradicate the biofilm infection. Our study demonstrates an important treatment principle, the Yin–Yang model, for persistent infections by targeting both growing and non-growing heterogeneous bacterial populations, utilizing persister drugs for the more effective eradication of persistent and biofilm infections. Our findings have implications for the improved treatment of other persistent and biofilm infections in general.

**Keywords:** *Staphylococcus aureus*; persisters; biofilm; antimicrobial activity; drug combination
