For many years, a single-dose oral treatment for syphilis has been sought. Benzathine penicillin G, used to treat syphilis for the past 50 years, has many disadvantages, including pain at injection sites, the possibility of an allergic reaction, and the inability of this form of penicillin to achieve treponemacidal levels in the central nervous system (a common site for Treponema pallidum dissemination). Published studies using the rabbit syphilis model demonstrated the effectiveness of azithromycin, an oral azalide antibiotic related to macrolides, for treating syphilis caused by the Nichols strain of T. pallidum. In several human studies, a single 1 or 2 gram dose of azithromycin was effective for treatment of early syphilis. However, recent publications document high frequencies of mutation A2058G in the 23S rRNA genes of circulating T. pallidum strains. In other bacteria, this mutation is associated with macrolide resistance. The Street 14 strain of T. pallidum contains the A2058G mutation, and, in the rabbit syphilis model, is resistant to macrolide and related drugs, including azithromycin. Similarly, azithromycin treatment failure has been documented in patients infected with T. pallidum bacteria that contain the same mutation. Nonetheless, a recent publication has questioned the clinical relevance of these 23S rDNA mutations; similar views have been voiced in discussions related to the development of the new syphilis treatment guidelines by the Centers for Disease Control and Prevention. Rather than continuing to speculate, it is essential to conduct the experiments that will directly determine whether these and other mutations impact the clinical response to azithromycin therapy. Although one could argue that sufficient equipoise exists (or does not exist) to justify a trial of azithromycin for treatment of patients with syphilis whose infecting organism harbors a 23S rDNA mutation, it is unlikely to be conducted in a timeframe that can inform our current clinical practice. The well-established rabbit model of syphilis is ideal for addressing the issue. The goals of the proposed project are 1) to determine the prevalence of A2058G and A2059G point mutations in the 23s rRNA genes of T. pallidum in samples collected from patients in widely disparate geographic regions; 2) to sequence the full 23s rRNA genes (both alleles) in a subset of these samples to identify other mutations that might be associated with macrolide resistance; and 3) to test the correlation of the identified mutations with clinical failure of azithromycin treatment in the well-established rabbit model of syphilis. To effectively treat our patients with syphilis, clinicians urgently need to know whether T. pallidum 23S rDNA mutations confer risk for azithromycin treatment failure. The results of our work will provide an evidence base on which sound treatment recommendations can be made. PUBLIC HEALTH RELEVANCE: The proposed studies will 1) determine whether mutations in the bacterium that causes syphilis affect response to treatment with azithromycin and 2) determine how frequently bacteria containing these mutations are present in syphilis patients from several geographical regions. If these mutations are found to be common and are shown to cause treatment failure, physicians will be advised not to use azithromycin for treating patients with syphilis.