In vitro evaluation of combination antibiotic therapy and resistance potential in Treponema pallidum

ABSTRACT Treponema pallidum , the spirochete bacterium that causes syphilis, is responsible for persistent and globally prevalent sexually transmitted infections. There is currently no vaccine for T. pallidum , and treatment relies on antibiotics, with intramuscular administration of benzathine penicillin G as the preferred therapy. However, limitations including drug access, manufacturing constraints, and penicillin allergies highlight the need for alternative treatment strategies. The emergence of resistance to the previously effective antibiotic azithromycin also underscores the need for improved approaches. Combination therapy offers the potential to enhance antimicrobial activity and reduce the likelihood of resistance by targeting multiple bacterial processes. The recent development of an in vitro co-culture system using Sf1Ep rabbit epithelial cells has enabled continuous cultivation of T. pallidum , facilitating evaluation of antibiotic activity under controlled conditions. While minimum inhibitory concentrations for individual antibiotics have been established, the potential benefits of combination therapy remain largely unexplored. In this study, we evaluated combinations of three orally available antibiotics, amoxicillin, cefuroxime, and doxycycline, against T. pallidum (Nichols strain). Two of the antibiotic combinations demonstrated modest additive effects, and no antagonistic interactions were observed for any of the combinations. In parallel, serial passage experiments showed that 12 weeks of exposure to sub-MIC concentrations of amoxicillin did not result in detectable resistance, consistent with prior findings with doxycycline or linezolid (L. C. Tantalo, N. A. P. Lieberman, C. Pérez-Mañá, C. Suñer, et al., Lancet Microbe 4:e994–e1004, 2023, https://doi.org/10.1016/S2666-5247(23)00219-7 and L. C. Tantalo, A. F. Luetkemeyer, N. A. P. Lieberman, B. Ethan Nunley, et al., J Infect Dis 231:729–733, 2025, https://doi.org/10.1093/infdis/jiae381 ). These results support further investigation of combination antibiotic strategies to improve syphilis treatment and mitigate the emergence of antimicrobial resistance. IMPORTANCE Syphilis remains a major and growing global public health concern. Benzathine penicillin G, the preferred therapy for syphilis, has experienced recent shortages, highlighting the need for alternative treatment strategies. Historically, research on Treponema pallidum , the bacterium that causes syphilis, has been limited by the inability to continuously culture the organism in the laboratory. Using a recently developed in vitro cultivation system, we evaluated combinations of commonly available oral antibiotics and assessed resistance potential during prolonged antibiotic exposure. We identified modest additive effects between some antibiotic combinations without evidence of antagonism or detectable resistance development. These findings support further investigation of combination oral antibiotic strategies for syphilis treatment and provide additional insight into antimicrobial susceptibility in T. pallidum .