Syphilis remains a serious global health concern, underscoring the need for better control strategies. In the absence of treatment, the syphilis agent, Treponema pallidum subsp. pallidum (T. pallidum), can persist for the lifetime of the host and syphilis can progress to its later stages. To combat the increase in syphilis incidence, doxycycline post-exposure prophylaxis (doxy-PEP) can be used to reduce the likelihood of infection with T. pallidum. However, the widespread use of doxy-PEP raises concerns about the possibility that this pathogen might become resistant, as seen in the past when azithromycin was used to treat syphilis. We wanted to explore whether continuous in vitro exposure to doxycycline could induce resistance in T. pallidum. To test our hypothesis, cultures of T. pallidum Nichols or SS14 strain were exposed to either increasing concentrations of doxycycline, azithromycin, or grown without antibiotics. Darkfield microscopy (DFM) was used to quantify the treponemal yield in cultures weekly. DNA was also extracted from T. pallidum cultures to evaluate bacterial presence by PCR, targeting the tp0574 gene. We found no sign of doxycycline resistance in T. pallidum SS14 cultures. Darkfield microscopy counts were detectable for up to three weeks in Nichols, whereas they lasted for five weeks in SS14. DNA extractions and PCR analysis showed no significant differences between strains, suggesting that albeit no strain developed resistance, one might be intrinsically more tolerant to the antibiotic. The results from this research provide encouraging evidence that T. pallidum may not easily develop resistance to doxycycline.

Syphilis, caused by Treponema pallidum (T. pallidum), remains a significant global health concern, with increasing cases worldwide. Doxycycline post-exposure prophylaxis (Doxy-PEP) has emerged as a potential strategy to prevent infection. However, widespread use raises concerns about the possibility that doxycycline-resistant T. pallidum strains might emerge and spread. This issue is alarming since doxycycline is a second-line therapeutic for syphilis and is often used in patients with allergies to beta-lactams or when beta-lactams are unavailable due to shortages. If genetic resistance to doxycycline were to develop in T. pallidum, it could undermine the effectiveness of Doxy-PEP and further narrow the range of treatment options for syphilis. To address this concern, I developed a restriction fragment length polymorphism (RFLP) assay to detect potential doxycycline resistance mutations in T. pallidum. This assay analyzes the 16S rRNA gene region of T. pallidum where most likely mutations could develop based on the analysis of other resistant pathogens. The assay was optimized using three synthetic 16S rRNA gene constructs containing the resistance-associated mutations and DNA from a wild-type T. pallidum strain (Nichols) as controls. The presence of mutations in the amplified control DNA was assessed by restriction digestion with the AluI, RsaI, and SfaNI enzymes, which can selectively cut wild type and mutant sequences and reveal specific mutations. The analysis of 60 archived samples from syphilis patients collected in the US, Madagascar, Argentina, and Sri Lanka is ongoing. Results will provide data on the frequency of doxycycline resistance mutations in T. pallidum, if any are found in this selected group of specimens. Developing a rapid, cost-effective surveillance tool is essential for monitoring potential resistance and preventing treatment failures when doxycycline is used.