Efficacy of linezolid on Treponema pallidum, the syphilis agent: A preclinical study

Abstract

Background: Penicillin G, the current standard treatment for syphilis, has important drawbacks, but virtually no preclinical or clinical studies have been performed to identify viable alternatives. We tested, both in vitro and in vivo, three marketed antibiotics with adequate pharmacological properties to treat syphilis.

Methods: We used an in vitro culturing system of T. pallidum to perform drug susceptibility testing and 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 experimental treatment (linezolid, moxifloxacin, clofazimine) or a control arm (benzathine penicillin G [BPG], untreated). The primary outcome was treatment efficacy defined as the time to lesion healing measured from the date of treatment start. Secondary outcomes were absence of treponemes or treponemal mRNA in injection sites, absence of seroconversion, and cerebrospinal fluid (CSF) abnormalities and negative rabbit infectivity tests (RIT).

Findings: Linezolid showed in vitro bactericidal activity at concentrations of 0.5 µg/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 2.05-7.17; 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 innovation program (Grant agreement No. 850450).

Keywords: Linezolid; Preclinical; Repurposing; Syphilis; Treponema pallidum; Yaws.

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 quantification 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, Clofazimine, Moxifloxacin, and Penicillin G, respectively.

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 injection of T. pallidum (Nichols stain) at eight sites on their shaved backs. A total of 10⁶T. 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 clofazimine-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.

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 message 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 rabbit 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.