A large screen identifies beta-lactam antibiotics which can be repurposed to target the syphilis agent

Syphilis, caused by *Treponema pallidum*, is a significant global health concern with increasing incidence rates, particularly among men who have sex with men and female sex workers. The infection presents diverse symptoms, and untreated primary syphilis can progress to more severe secondary and tertiary stages, causing irreversible organ damage and even death. Congenital syphilis, transmitted from mother to child, is also a major concern. Historically, research on *T. pallidum* has been hampered by the lack of a reliable long-term in vitro culture system. However, recent breakthroughs have enabled continuous in vitro cultivation, opening new avenues for research, including the identification of new therapeutic strategies. Benzathine penicillin G is the current treatment of choice, but challenges such as penicillin allergies, supply shortages, and the potential for developing antibiotic resistance necessitate the identification of alternative therapies. This study aimed to screen a large panel of β-lactam antibiotics to identify potential new treatments for syphilis by leveraging the newly available continuous in vitro culture system for *T. pallidum*.

The literature review section discusses the limitations of previous studies due to the lack of a reliable, continuous *T. pallidum* in vitro culture system. It highlights the importance of benzathine penicillin G as the primary treatment but also emphasizes its limitations: penicillin allergies, supply chain issues, and the theoretical risk of antibiotic resistance. Existing alternative treatments, such as doxycycline, have shortcomings like the need for multiple doses, ineffectiveness against late-stage disease, and contraindications in pregnant individuals. The review sets the stage for the current study by highlighting the critical need for novel effective therapies for syphilis.

The study utilized a recently developed continuous in vitro cultivation system for *T. pallidum*, allowing for the high-throughput screening of nearly 100 β-lactam antibiotics. The growth kinetics and characteristics of *T. pallidum* in the in vitro culture were first characterized by monitoring cell density, calculating doubling time, and analyzing cell length distribution using automated cell detection software (Outif). The relative in vitro efficacy of the β-lactams (penicillins and cephalosporins) was determined at a concentration of 5 nM, assessing the percentage of remaining cells compared to untreated controls. The top 25% of β-lactams were further evaluated using quantitative RT-PCR to analyze mRNA levels of two genes involved in cell division and motility (tp47 and flaA) to gain insights at the molecular level. Minimum inhibitory concentration (MIC) values were determined for the top-performing β-lactams via serial titration and cell enumeration. To investigate the mechanism of action, the researchers employed 7-hydroxycoumarin-3-carboxylic acid amino-D-alanine (HADA), a fluorescent derivative of D-alanine, to label peptidoglycan (PG) synthesis sites. This process was done for both whole cells and purified peptidoglycan to determine the impact of the top β-lactams on PG synthesis.

The researchers demonstrated stable, prolonged in vitro growth of *T. pallidum*, achieving over 10 cumulative generations in a continuous culture. The analysis of the β-lactam screen revealed that penicillins generally exhibited greater efficacy than cephalosporins at 5 nM, with first and fourth-generation penicillins performing best. Quantitative RT-PCR analysis of the top 25% of β-lactams provided additional validation of their effectiveness. The MIC analysis identified several β-lactams with low nM MIC values, significantly outperforming doxycycline. Azlocillin and mezlocillin showed the lowest MICs, exceeding the efficacy of benzathine penicillin G. Nafcillin displayed an MIC comparable to benzathine penicillin G. Among the cephalosporins, ceftriaxone, cefmenoxime, cefazolin, and cefepime exhibited notable activity. HADA labeling of whole cells and purified peptidoglycan sacculi confirmed that the top-performing β-lactams effectively inhibited peptidoglycan synthesis, validating their activity at the cellular level. The study also revealed that *T. pallidum* exhibits lateral peptidoglycan synthesis, differing from the known mechanism in Borrelia species.

The identification of several β-lactams with comparable or superior efficacy to benzathine penicillin G addresses the critical need for alternative syphilis treatments. The study's findings suggest potential replacements for benzathine penicillin G, particularly azlocillin, mezlocillin, and nafcillin, considering their potency, cost, and potential for reduced allergenicity compared to benzathine penicillin G. The cephalosporins identified also represent promising alternatives, especially considering their long half-life and ability to penetrate the central nervous system. The study also highlighted the importance of developing new technologies and tools for studying *T. pallidum* which has previously been challenging to study.

This study successfully identified several β-lactam antibiotics with high efficacy against *T. pallidum* in vitro. These findings provide strong candidates for further in vivo testing and potential repurposing as novel syphilis therapeutics. Future research should focus on in vivo efficacy studies, pharmacokinetic and pharmacodynamic analyses, and investigation of potential mechanisms of resistance. This research also underscores the value of the newly developed continuous in vitro culture system for future *T. pallidum* research.

The study's limitations include the in vitro nature of the experiments; therefore, in vivo efficacy must be verified. The cost analysis of the antibiotics should be considered with care, as bulk pricing does not reflect real-world clinical costs. The potential for cross-reactivity with penicillin allergies in some individuals remains a consideration even with the structural differences observed. It is important to note that only a subset of all β-lactams were tested in this high-throughput study.