EBioMedicine 65 (2021) 103281 Contents lists available at ScienceDirect EBioMedicine journal homepage: www.elsevier.com/locate/ebiom Article Efficacy of linezolid on Treponema pallidum, the syphilis agent: A preclinical study Austin M. Haynesa,y, Lorenzo Giacania,b,*,y, Marti Vall Mayansc, Maria Ubalsc,d, Carles Nietoe, rez-Man Clara Pe ~a  h,i,1, Emily Romeisa, Oriol Mitja f,g, Llorenç Quinto c,j,y,* a Department of Medicine, Division of Allergy and Infectious Diseases, University of Washington, Seattle, WA, USA b Department of Global Health, University of Washington, Seattle, WA, USA c Fight Aids and Infectious Diseases Foundation, Hospital Universitari Germans Trias i Pujol, Barcelona, Spain d Facultat de Medicina, Universitat de Barcelona, Barcelona, Spain e Laboratorio Reig Jofre, Barcelona, Spain f Clinical Pharmacology Unit, Hospital, Universitari Germans Trias i Pujol, Institut de Recerca Germans Trias i Pujol (HUGTiP-IGTP), Badalona, Spain g Department of Pharmacology, Therapeutics and Toxicology, Universitat Auto noma de Barcelona, Barcelona, Spain h Barcelona Institute for Global Health, Hospital Clínic-Universitat de Barcelona, Barcelona, Spain i Manhiça Health Research Institute (CISM), Maputo, Mozambique j Lihir Medical Centre-International SOS, Lihir Island, Papua New Guinea A R T I C L E I N F O A B S T R A C T Article History: Background: Penicillin G, the current standard treatment for syphilis, has important drawbacks, but virtually Received 5 January 2021 no preclinical or clinical studies have been performed to identify viable alternatives. We tested, both in vitro Revised 10 February 2021 and in vivo, three marketed antibiotics with adequate pharmacological properties to treat syphilis. Accepted 26 February 2021 Methods: We used an in vitro culturing system of T. pallidum to perform drug susceptibility testing and Available online xxx applied quantitative PCR targeting the tp0574 gene to measure bacterial growth. To confirm in vivo efficacy, fifteen rabbits were infected intradermally with T. pallidum at eight sites each and randomly allocated to an Keywords: experimental treatment (linezolid, moxifloxacin, clofazimine) or a control arm (benzathine penicillin G Treponema pallidum Syphilis [BPG], untreated). The primary outcome was treatment efficacy defined as the time to lesion healing mea- Yaws sured from the date of treatment start. Secondary outcomes were absence of treponemes or treponemal Repurposing mRNA in injection sites, absence of seroconversion, and cerebrospinal fluid (CSF) abnormalities and negative Linezolid rabbit infectivity tests (RIT). Preclinical Findings: Linezolid showed in vitro bactericidal activity at concentrations of 0.5 mg/mL or higher. When administered orally to experimentally infected rabbits, it induced healing of early lesions at a time similar to BPG (hazard ratio 3.84; 95% CI 2.057.17; p < 0.0001 compared to untreated controls). In linezolid-treated animals, dark-field microscopy and qPCR assessment showed no presence of treponemes after day 3 post- treatment start, serologic test did not convert to positive, CSF had no abnormalities, and RIT was negative. Moxifloxacin and clofazimine failed to inhibit bacterial growth in vitro and could not cure the infection in the rabbit model. Interpretation: Linezolid, a low-cost oxazolidinone, has in vitro and in vivo activity against T. pallidum, with efficacy similar to BPG in treating treponemal lesions in the animal model. Our findings warrant further research to assess the efficacy of linezolid as an alternative to penicillin G to treat syphilis in human clinical trials. Funding: European Research Council (ERC) under the European Union’s Horizon 2020 research and innova- tion program (Grant agreement No. 850450). © 2021 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/) Introduction * Corresponding author. Syphilis, a multi-stage, chronic, sexually transmitted infection ). E-mail addresses: giacal@uw.edu (L. Giacani), omitja@flsida.org (O. Mitja caused by the spirochete Treponema pallidum subsp. pallidum (T. pal- y These authors contributed equally 1 lidum) still represents a significant global health problem. The infec- Please direct any inquiries to Lorenzo Giacani: Department of Medicine, Division of Allergy and Infectious Diseases, University of Washington, Seattle, WA, USA. tion has been steadily resurgent in many high-income nations in https://doi.org/10.1016/j.ebiom.2021.103281 2352-3964/© 2021 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/) 2 A.M. Haynes et al. / EBioMedicine 65 (2021) 103281 activity. However, only three randomized clinical trials have com- Research in context pared penicillin to other antibiotics (i.e., ceftriaxone and azithromy- cin) [2022]. Conversely, the efficacy of doxycycline, the second-line Evidence before this study treatment for syphilis, is mainly supported by two prospective stud- In December 2020, we searched PubMed database for articles ies [23,24] and other small retrospective studies [2527]. reporting on preclinical studies about antibiotics to treat syphi- Overall, additional effort is needed to uncover alternative treat- lis. Search terms included “syphilis”, “Treponema pallidum”, ment options for syphilis that allow overcoming the limitations of “antimicrobial treatment”, and “susceptibility testing”. The penicillin G. The ideal compound to treat syphilis infections should drug susceptibility profile of Treponema pallidum (T.p.) was achieve effective tissue concentrations in all anatomical regions, unknown because the microorganism could not be grown in including the CNS, be able to be delivered orally, and not have culture until very recently. In animal models, penicillin, some reported shortages. In this study, we used an in vitro cultivation sys- cephalosporins, aztreonam, and macrolides had shown curative tem for T. pallidum [28,29] and the rabbit model of syphilis to evalu- results, while clindamycin and ofloxacin did not cure syphilis. ate the efficacy of linezolid (a protein synthesis inhibitor), Strains of T.p. resistant to azithromycin had appeared quite rap- moxifloxacin (a DNA gyrase inhibitor), and clofazimine (a bacterial idly after its introduction, therefore current therapeutic options respiratory chain blocker) to treat syphilis, compared to penicillin G for syphilis are restricted to parenteral penicillin (preferred (in vitro) and BPG (in vivo) as positive controls. drug) and doxycycline or ceftriaxone (second-line drugs). Methods Added values of this study Compounds selection and dosage For the first time, we have screened multiple antibiotics to treat treponemal disease using a novel approach based on a tissue Candidate molecules for the treatment of syphilis were screened culture system that supports long-term multiplication of T.p. In from FDA-approved antibiotics based on their pharmacological prop- addition, we have confirmed drug effectivity in the rabbit erties and activity against other pathogenic spirochetes. We used model, which is a mandatory step because the clinical relevance genome-based prediction of resistance mechanisms to choose drugs of in vitro susceptibility testing is uncertain. In addition, we against which T. pallidum was unlikely to develop resistance. The have employed sensitive molecular methods to evaluate the dose for experiments on rabbits was established as the human equiv- burden of live treponemal cells in animals following treatment, alent systemic exposure based on bodyweight dosing. Pharmacoki- rather than more archaic approaches that are prone to error. netic parameters (AUC, Cmax, and t1/2) were obtained from the literature; [3033] the variation in metabolic systems, including Implications of all the available evidence metabolic rates and enzymatic inhibition, were also considered. Our preclinical efficacy studies provide strong evidence on the clinical promise of linezolid to treat syphilis. Oral linezolid, if In vitro assessments shown efficacious to treat neurosyphilis and ocular syphilis in human clinical trials, would overcome the need for 10-to- The SS14 strain of T. pallidum used to inoculate culture plates was 14 days hospital admission to using intravenous aqueous crys- obtained from a frozen stock of treponemes propagated intratesticu- talline penicillin every 4 hours. larly in New Zealand White (NZW) rabbits [34]. We tailored the cul- turing system of T. pallidum [28] to perform the susceptibility tests in our lab (Supplementary Methods, Appendix). Briefly, treponemes North America and Europe during the last two decades [14]. Glob- were sub-cultured into three 96-well cell culture plates to allow a ally, most syphilis cases occur in Sub-Saharan Africa, South East Asia, total of nine replicates for each antibiotic concentration to be tested. and South America, where congenital syphilis accounts for up to 50% Tissue-culture grade antibiotics were obtained from Sigma- of stillbirths [14]. In adults, the bacteria disseminates to virtually Aldrich (St. Louis, MO). Linezolid and clofazimine were solubilized in every organ and affects the patient’s cardiovascular and central ner- DMSO, while moxifloxacin and penicillin G were dissolved in sterile vous systems (CNS) [5]. Hence, the choice of treatment for syphilis water. Final DMSO concentration in the wells was 1% (v/v). Wells needs to consider the outstanding invasiveness of this pathogen and without antibiotic and wells containing only solvent were included its ability to colonize so many microenvironments. as negative controls. After seeding the treponemes and adding the Parenteral penicillin G is currently the recommended treatment antibiotics, the culture plates were incubated at 34°C for seven days for syphilis [6,7]. Although the T. pallidum genome encodes for at in the microaerophilic incubator. Two of the three plates were then least one enzyme with b-lactamase activity (i.e., the lipoprotein used to determine treponemal growth after a week of exposure to Tp0574), no genetic resistance to this antibiotic has ever been each antibiotic concentration, while one of the three plates was used observed [8]. Nonetheless, the use of penicillin G has important limi- to reseed another plate containing antibiotic-free media to assess the tations. First and foremost, because penicillin has low penetration to viability of treponemes and confirm treponemocidal activity of the the CNS, [9,10] treatment of neurosyphilis and ocular syphilis tested drugs. The plates were processed for DNA extraction using a requires patient admission for the administration of intravenous (IV) Quick DNA-96 kit (Zymo Research, Irvine, CA) according to the manu- aqueous crystalline penicillin every four hours, for 1014 days. Sec- facturer’s protocol, and we used a qPCR approach targeting the ond, benzathine penicillin G (BPG), used to treat uncomplicated syph- tp0574 gene [34]. ilis, must be delivered by trained personnel via an intramuscular (IM) injection. Third, penicillin-allergic patients need to be either desensi- Rabbit infection and treatment tized before treatment or treated with second-line drugs [7,11]. Finally, the supply of BPG is being compromised by product shortages Adult male NZW rabbits, an established animal model for syphilis (currently experienced by 39 countries, according to the WHO) [35,36], were used for experimental infections for antibiotic testing [12,13]. (Appendix). Studies to support the use of alternatives for treating syphilis are For evaluation of antibiotic efficacy, 15 animals were infected overall limited. In the preclinical setting, cephalosporins, [1416] intradermally on the same day in eight sites/animal on their shaved aztreonam, [17] and macrolides [18,19] have shown anti-syphilis backs. One million (106) viable T. pallidum cells were inoculated at A.M. Haynes et al. / EBioMedicine 65 (2021) 103281 3 each injection site. Following intradermal inoculation, animals were For the in vivo study, no sample size estimate was conducted randomly assigned to one of the five treatment arms, including three because this was a proof-of-concept study. A sample size of 12 in experimental arms (groups 1-3) and two control arms (groups 4-5). each arm should suffice for pilot studies; [38] our sample size Group 1 received oral linezolid (75 mg/Kg) every 8 h for 5 days; group amounted to 120 lesions (15 rabbits, 8 lesions each). The effect of 2 received oral moxifloxacin (40 mg/Kg) every 12 h for 5 days, group treatment on rabbits was described using the mean and SE of the 3 received oral clofazimine (75 mg/Kg) once daily for three days, fol- diameter of the lesions and the T. pallidum lesion burden, determined lowed by a reduced dose of 25 mg/Kg once daily for four additional by DFM analysis and by qPCR. The primary endpoint for the in vivo days. Group 4 (positive control) received a single IM injection of BPG assessment was treatment efficacy, defined as the time to disease- (200,000 units, equivalent w/w to 2.4 million units for humans), free event (lesion healing), measured from the date of treatment start while group 5 (negative control) was left untreated. Rabbits belong- (day 7) or the day of the appearance of a skin lesion (whatever ing to different treatment groups were housed in different racks in occurred last) to lesion healing. The primary analysis was performed the room to avoid confounders. Study arms were blinded to experi- for two definitions of lesions: (1) lesions that were either indurated menters and analysts. Antibiotic treatment was initiated when at or ulcerated, with a measurable diameter, and (2) lesions in the least two injection sites per animal were found to harbour trepo- broadest sense, including also erythema observed in the days preced- nemes upon analysis of needle aspirates by Dark Field Microscopy ing or following induration/ulceration. The cumulative incidence of (DFM). treatment success among treatment groups was estimated using Fine and Gray competing risk regression models for clustered data and Ethics considering biopsies as the competing events; results were reported as the Sub-Hazard Ratios (HR) and their 95% Confidence Intervals (CI) Animal care was provided in accordance with the Guide for the [39,40]. The equivalence of the cumulative incidence functions Care and Use of Laboratory Animals, and all experimental procedures between treatment groups was assessed by the weighted log-rank were conducted under protocol #4243-01 (PI: Lorenzo Giacani), test [39]. The significance threshold was set at a two-sided alpha approved by the University of Washington (UW) IACUC. All animal value of 0.05 for all analyses. Descriptive and comparative analyses procedures were conducted in compliance with the ARRIVE guide- were performed using Prism 8 (GraphPad Software, San Diego, CA), lines. Details on animal care are provided in Appendix 1. whereas time-to-event analyses were performed using Stata version 16 (StataCorp. 2019. Stata Statistical Software: Release 16. College Clinical monitoring and sample collection Station, TX: StataCorp LLC). Intradermally-infected rabbits were shaved daily until day 40 Role of funders post-inoculation to allow monitoring of lesion progression and facili- tate collection of needle aspirates to evaluate treponemal burden by The funder of the study had no role in the study design, data col- DFM and collection of lesion biopsies to evaluate burden by qPCR. In lection, data analysis, data interpretation, or writing of the report. all animals, challenge sites were monitored by recording lesion pro- The corresponding author had full access to all the data in the study gression and healing. Both the diameter of induration or ulceration and had final responsibility for the decision to submit it for publica- and the diameter of erythema were measured using a calliper every tion. day for 40 days post-intradermal inoculation. Lesion aspirates and lesion biopsies were collected at days 3, 8, 12, and 16 post-treatment Results initiation. Aspirates were examined by DFM and T. pallidum cells were counted blindly in a total of 200 fields. Biopsies were collected, Antimicrobial drug selection minced using sterile scalpel and forceps, resuspended in 400 mL of TRIzol reagent, used for RNA extraction, and tested by qPCR targeting Based on the screening of antimicrobial drugs, we identified two the tp0574 mRNA [34,37]. drugs, linezolid [41] and moxifloxacin [42] that met the predefined Serum was also obtained from each animal every week to per- pharmacokinetic criteria [43] (Table S2, Appendix) and were active form Venereal Disease Research laboratory (VDRL) and fluores- against other spirochetes [4446]. CNS penetration of linezolid (75%) cent treponemal antibody absorption (FTA-ABS) tests to monitor and moxifloxacin (45%) is higher than penicillin G (5%) [22]. The pos- the development of humoral immunity in response to infection. sibility of linezolid resistance occurring as a result of point mutation VDRL data were collected for each week, while FTA-ABS tests of 23S ribosomal RNA in T. pallidum was deemed to warrant consider- were performed only up to when sera became reactive. Nine ation [47]. T. pallidum lacked putative genes encoding for intrinsic weeks post-treatment initiation, animals underwent spinal tap to resistance mechanisms for moxifloxacin. We also included clofazi- obtain CSF and were subsequently euthanized. Approximately mine [48], despite its inability to cross the blood-brain barrier, 900 mL of CSF were sent to Phoenix Laboratories (Mukilteo, WA) because of its long half-life, low cost, and strong safety profile [49]. to be evaluated for red blood cell contamination, presence of Additional drugs that were not prioritized will be tested in a second nucleated cells, glucose, and total protein, while 100 mL were round of experiments, including cefixime, isoniazid, pyrazinamide, used to perform CSF-VDRL in our laboratory. dalbavancin, and zoliflodacin. To conduct the rabbit infectivity test (RIT), naïve recipient rabbits were injected intratesticularly with minced popliteal lymph nodes In-vitro effects from untreated, BPG-treated and linezolid-treated donor rabbits. Serum was collected monthly to assess seroconversion of these recip- Based on clinically relevant concentrations of antibiotics (i.e., ient animals via VDRL testing (Appendix 1). maximum plasma concentration achievable in humans), we tested the anti-treponemal activity of linezolid at a concentration range of Statistics 0.25 to 4.0 mg/mL, and clofazimine and moxifloxacin at 0.06 to 2.0 mg/mL. Penicillin G was tested at a concentration range of 0.1 to Results regarding the molecular assessment of T. pallidum in vitro 60.ng/mL (Table S1). growth were described as the mean and standard error (SE). Differen- Linezolid inhibited treponemal replication at all concentrations ces in growth between no treatment wells and each antibiotic dose tested, except 0.25 mg/mL (Fig. 1a). Clofazimine failed to completely were compared using one-way ANOVA. inhibit treponemal replication at all concentrations tested, except 2.0 4 A.M. Haynes et al. / EBioMedicine 65 (2021) 103281 Fig. 1. qPCR amplification of the tp0574 gene in treated and control treponema cultures. Sample size was 192 wells distributed in four antibiotic groups, each group having eight samples (with six different drug concentrations, and two controls  no antibiotic and no solvent), and each sample having six biological replicates (Table S1). Following DNA extraction from each replicate, treponemal burden was evaluated for each replicate in triplicate using a qPCR approach targeting the tp0574 gene to obtain a total of 18 data points (6 replicates x 3 qPCR reading) for each antibiotic concentration tested. An absolute quantifica- tion protocol using an external standard was used to quantify the tp0574 gene copy number at the time of sample harvest. Bar height is the mean of 18 data points for each antibiotic concentration tested, and error bars show the standard error. Asterisks indicates significance (p  0.05) calculated using one-way ANOVA in comparison to the No AB (No antibiotic) data. Carrier indicate wells containing antibiotic carrier only (water or DMSO). Panels a-d are Linezolid, Clofazi- mine, Moxifloxacin, and Penicillin G, respectively. mg/mL, although doses from 0.25 to 1.0 mg/mL were associated with were significantly associated with a shorter time to healing com- a significantly lower growth than untreated wells (Fig. 1b). Moxiflox- pared with untreated rabbits (Table S3, Figure S2): HR for healing acin only showed capacity to significantly reduce growth compared was 5.94 (95% CI 2.92  12.08) and 3.84 (2.05  7.17) for BPG with untreated wells at 2.0 mg/mL (Fig. 1c), whereas penicillin G sig- and linezolid, respectively (p < 0.001 for both molecules). This nificantly inhibited growth at concentrations of 0.003 mg/mL effect was observed for neither moxifloxacin (HR 1.19 [95% CI (approximately 0.06 U/mL) or higher (Fig. 1d). 0.97  1.46]; p = 0.102) nor clofazimine (0.90 [0.78  1.05]; After re-seeding treated treponemes into an antibiotic-free 96- p = 0.198). The corresponding analysis based on the expanded well plate, treponemes from wells without antibiotic grew but those definition of lesion (i.e., including erythema) showed a similar that had been exposed to either penicillin G or linezolid at any con- trend, with HRs of 10.60 (95% CI 3.75  29.94) and 3.60 (2.03  centration did not, indicating a bactericidal effect (Fig. S1). Growth 6.41) for BPG and linezolid, respectively (p < 0.001 for both mol- after re-seeding was observed in all treponemes that had been ecules), and 1.05 (0.89  1.24; p = 0.563) and 0.97 (0.79  1.19; treated with moxifloxacin and those treated with clofazimine at a p = 0.796) for moxifloxacin and clofazimine, respectively. concentration of 0.125 mg/mL or lower. Dark-field microscopy assessment showed absence of trepo- nemes in lesion aspirates from animals treated with BPG at all Treatment effects on the animal model time-points; treponemes could be found in animals in the line- zolid group at day 3 post-treatment start, but not after this time After intradermal infection with the treponemal inoculum of 15 point (Fig. 3a). The quantification of the tp0574 mRNA of T. pal- rabbits, all animals developed clinical signs of a lesion in each of their lidum in lesion biopsies, used as a surrogate of the burden of via- eight challenge sites; pre-treatment needle aspirates on day 7 ble treponemal cells, showed no evidence of treponemal growth showed the presence of treponemes. The mean lesion diameter of at any time point in animals treated with BPG. In animals treated rabbits treated with BPG and linezolid decreased from one and two with linezolid, treponemal growth was only identified at day 3 days after treatment start, respectively (Fig. 2a); no induration could post-treatment start (Fig. 3b). Amplification of tp0574 was be measured in the linezolid group at day 12 (or later) post-treatment detected in untreated animals and those treated with moxifloxa- initiation. Macroscopically, there were no differences between BPG- cin and clofazimine at all time points. and linezolid-treated animals at day 15 post-treatment (Fig. 2b). Lesions of untreated rabbits and those treated with moxifloxacin and Serology and CSF involvement clofazimine progressively increased along the week following treat- ment start. In clofazimine-treated animals, the mean lesion diameter Serologic nontreponemal VDRL tests, performed weekly for nine was significantly smaller than that of untreated controls between weeks after treatment start, showed no seroconversion of rabbits days 1726 post-infection (Fig. 2a). Moxifloxacin-treated animals treated with linezolid and BPG at any time point (Fig. 4). All rabbits ulcerated and presented a lesion size similar to untreated controls from the remaining groups (untreated controls, and moxifloxacin- over the study period. and clofazimine-treated animals) became VDRL-positive between In the primary analysis for time to lesion healing using a weeks 2 and 3 post-inoculation. Similarly, linezolid- and BPG-treated restrictive definition of the lesion (i.e., indurated or ulcerated, animals did not become positive for the FTA-ABS test throughout the with a measurable diameter), treatments with BPG and linezolid whole 9-week observation period, while sera from untreated A.M. Haynes et al. / EBioMedicine 65 (2021) 103281 5 Fig. 2. Monitoring of cutaneous lesion development in experimentally infected rabbits. Sample size was 120 lesional sites distributed in five treatment arms, each arm having three rabbits with eight injection sites each. Animals were infected by intradermal injec- tion of T. pallidum (Nichols stain) at eight sites on their shaved backs. A total of 106 T. pallidum viable cells were inoculated at each challenge site. (a) Dots represent the mean diameter (mm) of 21 indurated/ulcerated lesions (3 rabbits x 8 lesions sites) and error bars represent the standard error. The lesion diameter in clo- fazimine-treated animals was significantly lower (p < 0.05) compared to the untreated controls between day 17-27 post-infection. (b) Pictures of the rabbit right-side backs taken at day 23 post-infection (day 16 post-treatment initiation) in treated and control animals. One rabbit per group is shown. Black dots were used to mark the skin about 2 cm below the injection site. Subpanel a, b, d, e are pictures taken from linezolid-, BPG-, moxifloxacin-, and clofazimine- treated animals, respectively, while subpanel c is a picture from one of the untreated controls. The left side of the rabbit backs is not pictured because it was used to obtain lesion biopsies for mRNA quantification studies. controls, clofazimine- and moxifloxacin-treated animals matched the Discussion level of reactivity of the provided control sera within week 3 or 4 post-infection. In this study, we sequentially used in vitro and in vivo approaches The cellular and biochemical analysis of the CSF revealed a signifi- for repurposing marketed antibiotics with the potential for being a cantly higher protein content in the CSF of untreated animals than therapeutic alternative to penicillin G to treat syphilis. We sought out linezolid- or BPG-treated ones (Figure S3). Rabbits treated with clofa- molecules that could be administered orally, preferentially as a zimine and moxifloxacin had lower concentration of proteins in the short-course regimen (e.g., 5 days) to improve treatment acceptance CSF than controls but higher than those treated with linezolid or BPG and compliance, had no reported shortage or allergic reactions, and (Fig. S3 a). No differences in nucleated cell count or CSF glucose con- had reported at least in vitro activity against pathogenic spirochetes. centration were seen across all groups (Fig. S3 b-c), and CSF-VDRL Of the three selected molecules currently marketed that met the tests were negative in all animals. established criteria, only linezolid showed a significant in vitro and in In the RIT, only the recipient of the lymph nodes from the vivo efficacy. Remarkably, the bactericidal effect of linezolid on T. pal- untreated control group developed orchitis and became VDRL posi- lidum cultures and its efficacy to cure treponemal lesions in model tive (1:32) approximately a month following inoculation. None of the animals was similar to that of penicillin G. Furthermore, analyses of recipients from BPG treated and linezolid treated groups developed the lymph nodes (through RIT) and CSF of treated animals revealed orchitis or seroconverted. RIT was deemed unnecessary, and hence the ability of linezolid to prevent treponemal dissemination to these not performed, using lymph nodes from clofazimine- and moxifloxa- tissues. To our knowledge, no antibiotics of the oxazolidinone class, cin-treated animals because these antibiotics were shown to be inef- of which linezolid is the lead compound, have been tested against T. fective against T. pallidum based on lesion development, serology, pallidum in animal models or clinical studies of syphilis. Our in-silico and treponemal burden data. investigation ruled out the potential of T. pallidum harbouring efflux 6 A.M. Haynes et al. / EBioMedicine 65 (2021) 103281 Fig. 3. Assessment of treponemal burden within lesions of experimentally infected rabbits. Sample size was 120 lesional sites distributed in five treatment arms, each arm having three rabbits with eight injection sites each. Lesion aspirates were collected from all sites on days 3, 8, 12, and 16 post-treatment initiation. (a) Treponemal burdens in lesions from treated and control rabbits post-infection challenge were measured by dark-field microscopy (DFM) of lesion aspirates. Bar height is the mean of 24 data points (day 3 post-TI), 21 data points (day 8 post-TI), 18 data points (day 12 post-TI), and 16 data point (day 16 post-TI) for each antibiotic tested; error bars show the standard error. The number of data points decreased over time because once an injection site is biopsied, it is no longer sampled for DFM. (b) Treponemal burden measured by qPCR targeting T. pallidum tp0574 gene of lesion biopsies. Message quantification was performed using a qPCR approach targeting the mes- sage for the treponemal 47 kDa lipoprotein (encoded by the tp0574 gene) that normalizes the tp0574 signal to the message for the rabbit hypoxanthine-guanine phosphoribosyl transferase housekeeping gene (rHPRT). Bar height is the mean of 3 data points for each antibiotic tested, and error bars show the standard error. One biopsy was taken for each rab- bit at each time point (three rabbits each arm). Fig. 4. VDRL titers in experimentally infected rabbits. Sample size was 15 rabbits distributed in five treatment arms, each arm having three rabbits. Mean (§ standard error [SE]) serum VDRL titers in linezolid-, BPG-, moxifloxacin-, and clofazimine-treated rabbits, as well as in untreated controls. The data represent 3 rabbits per treatment group, and mean serum VDRL titres are shown through 10 weeks post- infection, and at least 8 weeks after initiation of treatment. For calculating mean § SE, titres were converted to log2 with nonreactive=0; R(1:2)= 1, and R(1:4) = 2, and so on. Antilog of mean § SE of titres are shown in this figure. A.M. Haynes et al. / EBioMedicine 65 (2021) 103281 7 pump genes typically associated with antibiotic resistance in gram- syphilis in human clinical trials. Low-cost generic linezolid was added negative bacteria. We cannot exclude that, like reported for other to the WHO list of prequalified medicinal products in 2018. The cur- bacteria [47], T. pallidum might develop resistance to linezolid rent price range of linezolid is 44.2  75.0 USD (ex-works price) for through selection of mutations in the 23S rRNA gene. To evaluate the 100 tablets blister; therefore, the cost range of a once-daily 5-day likelihood of this event, we cultivated T. pallidum SS14 strain for 10 regimen is 2.2  3.75 USD. Linezolid, as short-course chemotherapy, weeks in sub-therapeutic concentration of linezolid (~30 ng/mL). At is overall safe and generally very well tolerated. A drawback is that it the end of the experiment, we demonstrated that the linezolid mini- is in US-FDA pregnancy category C, meaning there have been no ade- mum inhibitory concentration (MIC) was not changed in these trepo- quate studies of its safety when used in pregnant women. Our pre- nemes, suggesting that no selection of a resistant strain occurred. clinical efficacy studies provide strong evidence on clinical promise Future work will aim at characterizing these treponemes at the of linezolid to treat syphilis. If shown efficacious in treating neurosy- molecular level and will provide a comprehensive overview of any philis and ocular syphilis it might overcome the need for 10-to- genomic and transcriptomics changes that might have occurred dur- 14 days hospital admission for intravenous aqueous crystalline peni- ing propagation in low linezolid concentration. We found that line- cillin G every 4 hours. zolid had MIC values of 0.5 mg/mL for T. pallidum cells. Assuming that the 24 h Area Under the Curve (AUC24) of linezolid is 155 mg £ h/mL Contributors and that efficacy is driven by AUC24/MIC >5080, we estimate that a single-dose can achieve effective drug concentration in the human L.G. and O.M. conceived of the study. C.N., C.P.M and O.M. did the infected tissue over 24 h; therefore, a once-a-day (QD) dosing is literature search, drug selection, and dose calculations. L.G, A.M.H., expected to be effective. Our in-silico analysis suggested that T. pal- and E. R. performed the experimental procedures, organized the data lidum might also be susceptible to the fluoroquinolone moxifloxacin. and contributed to data analysis. M.V.M., M.U.C., and LQ performed However, in vitro and in vivo experiments showed a very limited statistical analyses. All authors contributed to manuscript prepara- capacity of the molecule to inhibit treponemal growth. Fluroquino- tion and critically reviewed and approved the manuscript before sub- lones target two bacterial proteins, the gyrase (GyrA) and topoisom- mission. erase IV, with the latter not being encoded by T. pallidum genome [50]. Hence, the selection of mutations with substantial resistance Acknowledgements phenotypes might occur readily in this pathogen. Whether muta- tional alteration in target enzymes is the reason for resistance in trep- The authors wish to acknowledge the help received by Dr. Steve onema should be further investigated, particularly given that new- Norris and Dr. Diane Edmondson (University of Texas Health Center generation fluoroquinolones have shown good inhibitory effects on at Houston) to set up the in vitro cultivation system for T. pallidum. cultures of the other pathogenic spirochetes like Borrelia sp. [51]. We also wish to thank Dr. Marc Noguera (IrsiCaixa, Barcelona, Spain) Our study has some caveats intrinsically related to conducting for helpful discussion on the topic of this manuscript. We wish to research in syphilis. First, drug susceptibility testing in syphilis is thank Dr. Gerard Carot-Sans, PhD, for providing medical writing sup- remarkably challenged by the difficulties of propagating the infec- port during the revisions of the subsequent drafts of the manuscript. tious agent T. pallidum in laboratory cultures. In this regard, our adap- This project has received funding from the European Research tation of the cultivation method described by Edmonson et al., [28] Council (ERC) under the European Union’s Horizon 2020 research which was tailored for testing antibiotic efficacy against T. pallidum, and innovation program (Grant agreement No. 850450; Grant holder entails significant progress in the field. Owing to the inability to use ). Oriol Mitja traditional methods for estimating the MIC, we developed a qPCR- based assay in which tp0574 gene was used to identify treponemal Data sharing statement growth. The consistency of the DFM and qPCR findings, obtained after incubation in the presence of antibiotics and after re-seeding for an The data that support the findings of this study are available from additional week-long incubation in antibiotic-free media, allowed us the following repository at Harvard Dataverse: https://dataverse.har to confirm robustly which antibiotic concentrations were treponemi- vard.edu/dataset.xhtml?persistentId=doi:10.7910/DVN/6HE7IU cidal. The in vitro results were also in line with subsequent findings in model animals. Second, preclinical studies on model animals are typically limited in statistical power because of the small sample size Declaration of Competing Interests (in our case, three rabbits per group with eight challenges site each). Furthermore, quantitative methods for measuring the clinical pro- The authors have nothing to disclose. gression of treponemal lesions are scant. Lesion measurement was done using a calliper with an accuracy of about §0.02 mm. Precision Supplementary materials errors in measurement would affect equally both treatment arms because of randomization and blinding; hence, the difference Supplementary material associated with this article can be found between treatment arms is unlikely to be affected. In addition to the in the online version at doi:10.1016/j.ebiom.2021.103281. time-to-healing of the lesions, assessed by naked-eye examination, we analysed the treponemal burden by treponema cell count on DFM References and transcription levels of the tp0574 gene by qPCR. Like in the in vitro approach, the findings from the different approaches for [1] Gerbase AC, Rowley JT, Mertens TE. Global epidemiology of sexually transmitted diseases. Lancet 1998:351. measuring lesion progression were consistent regarding the ability [2] WHO. Report on global sexually transmitted infection surveillance 2015. 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