The global resurgence of treponematoses, particularly syphilis, poses a growing public health challenge. Despite recent advances in sequencing technologies, obtaining complete Treponema pallidum genome sequences for epidemiological studies remains time-consuming and challenging due to the difficulty related to procuring clinical samples with sufficient treponemal burden to fulfil the sequencing requirements. There is an urgent need for rapid, cost-effective and accessible typing methods suitable for laboratories with Sanger sequencing resources. Based on the analysis of 121 T. pallidum genomes from geographically diverse regions, we selected seven highly variable genes to form the basis of this new typing system. These seven genes show high discrimination capacity, identifying many allelic profiles among T. pallidum isolates. Importantly, the scheme employs a single-step PCR protocol for the amplification and sequencing of all seven targets enabling straightforward implementation in standard laboratory settings. The MLST was validated using a diverse set of T. pallidum clinical samples from across the globe. A significant proportion of the tested samples showed macrolide resistance, emphasizing the need for epidemiological surveillance. Utilizing this new tool, we have analyzed the genetic variation within and between populations of T. pallidum, considering the geographical origin of the samples. Population structure analysis revealed distinct genetic clusters, underlining complex transmission dynamics of T. pallidum, shaped by local epidemiological factors. The MLST scheme is publicly accessible through the PubMLST database, encouraging widespread adoption in standard laboratories due to this database being user-friendly, intuitive, and fast to implement. The novel MLST scheme offers a promising tool to advance the study of the molecular epidemiology of T. pallidum, facilitate tracking transmission, and establish a global surveillance network with the overall goal of strengthening public health interventions for syphilis control.

Catholic University of Valencia San Vicente Mártir Valencia Spain

Centers for Disease Control and Prevention Atlanta GA USA

CEU Cardenal Herrera University of Castellón Castellón Spain

CIBER in Epidemiology and Public Health Madrid Spain

Department of Biology Faculty of Medicine Masaryk University Brno Czech Republic

Department of Environmental Sciences University of Basel Basel Switzerland

Department of Medicine Division of Allergy and Infectious Diseases University of Washington Seattle WA USA

Department of Neurology University of Washington School of Medicine Seattle WA USA

Dermatology Service Consorcio Hospital General Universitario Valencia Spain

Division of Microbiology Osaka Institute of Public Health Osaka Japan

Institute for Bioinformatics and Medical Informatics University of Tübingen Tübingen Germany

Institute of Evolutionary Medicine University of Zurich Zurich Switzerland

ISGlobal Barcelona Spain

Joint Research Unit Infection and Public Health FISABIO Univ Valencia Institute for Integrative Systems Biology Valencia Spain

Microbiology Service Consorcio Hospital General Universitario Valencia Spain

National Reference Laboratory of Treponemes and Special Pathogens Tropical Medicine Institute Pedro Kourí Havana Cuba

School of Translational Medicine Monash University Melbourne Victoria Australia

University Hospital Zurich University of Zurich Zurich Switzerland

Zurich Institute of Forensic Medicine University of Zurich Zurich Switzerland

See more in PubMed

Noda AA, Grillová L, Lienhard R, Blanco O, Rodríguez I, Šmajs D. Bejel in Cuba: molecular identification of PubMed

Shinohara K, Furubayashi K, Kojima Y, Mori H, Komano J, Kawahata T. Clinical perspectives of PubMed

Grange PA, Allix-Beguec C, Chanal J, Benhaddou N, Gerhardt P, Morini J-P, et al. Molecular subtyping of PubMed

World Health Organization. Global progress report on HIV, viral hepatitis and sexually transmitted infections, 2021. 2021.

Health Organization W. Summary report of a consultation on the eradication of yaws, 5-7 March 2012, Morges, Switzerland. 2012. Available: https://apps.who.int/iris/bitstream/handle/10665/75528/WHO?sequence=1

Mitjà O, Godornes C, Houinei W, Kapa A, Paru R, Abel H, et al. Re-emergence of yaws after single mass azithromycin treatment followed by targeted treatment: a longitudinal study. Lancet. 2018;391: 1599–1607. PubMed PMC

Dofitas BL, Kalim SP, Toledo CB, Richardus JH. Yaws in the Philippines: first reported cases since the 1970s. Infect Dis Poverty. 2020;9: 1. PubMed PMC

Elo A, Dégboé B, Barogui Y, Gomido IC, Wadagni A, d’Almeida C, et al. Resurgence of yaws in Benin: Four confirmed cases in the district of Z, Southern Benin. Journal of public health and epidemiology. 2019;11: 201–208.

Timothy JWS, Beale MA, Rogers E, Zaizay Z, Halliday KE, Mulbah T, et al. Epidemiologic and Genomic Reidentification of Yaws, Liberia. Emerg Infect Dis. 2021;27: 1123–1132. PubMed PMC

Mitjà O, Šmajs D, Bassat Q. Advances in the diagnosis of endemic treponematoses: yaws, bejel, and pinta. PLoS Negl Trop Dis. 2013;7: e2283. PubMed PMC

Pillay A, Liu H, Chen CY, Holloway B, Sturm WA, Steiner B, et al. Molecular Subtyping of PubMed DOI

Grillová L, Bawa T, Mikalová L, Gayet-Ageron A, Nieselt K, Strouhal M, et al. Molecular characterization of PubMed PMC

Lieberman NAP, Lin MJ, Xie H, Shrestha L, Nguyen T, Huang M-L, et al. PubMed PMC

Vrbová E, Pospíšilová P, Dastychová E, Kojanová M, Kreidlová M, Rob F, et al. Majority of PubMed PMC

Marra CM, Sahi SK, Tantalo LC, Godornes C, Reid T, Behets F, et al. Enhanced molecular typing of treponema pallidum: geographical distribution of strain types and association with neurosyphilis. J Infect Dis. 2010;202: 1380–1388. PubMed PMC

Godornes C, Giacani L, Barry AE, Mitja O, Lukehart SA. Development of a Multilocus Sequence Typing (MLST) scheme for PubMed PMC

Katz SS, Chi K-H, Nachamkin E, Danavall D, Taleo F, Kool JL, et al. Molecular strain typing of the yaws pathogen, PubMed PMC

Thurlow CM, Joseph SJ, Ganova-Raeva L, Katz SS, Pereira L, Chen C, et al. Selective Whole-Genome Amplification as a Tool to Enrich Specimens with Low PubMed PMC

Beale MA, Marks M, Cole MJ, Lee M-K, Pitt R, Ruis C, et al. Global phylogeny of PubMed PMC

Edmondson DG, Delay BD, Kowis LE, Norris SJ. Parameters affecting continuous in vitro culture of PubMed PMC

Pereira LE, Katz SS, Sun Y, Mills P, Taylor W, Atkins P, et al. Successful isolation of PubMed PMC

Maiden MC, Bygraves JA, Feil E, Morelli G, Russell JE, Urwin R, et al. Multilocus sequence typing: a portable approach to the identification of clones within populations of pathogenic microorganisms. Proc Natl Acad Sci U S A. 1998;95: 3140–3145. PubMed PMC

Enright MC, Day NP, Davies CE, Peacock SJ, Spratt BG. Multilocus sequence typing for characterization of methicillin-resistant and methicillin-susceptible clones of PubMed PMC

Dingle KE, Colles FM, Wareing DR, Ure R, Fox AJ, Bolton FE, et al. Multilocus sequence typing system for PubMed PMC

Enright MC, Spratt BG. A multilocus sequence typing scheme for PubMed

Katz KA, Pillay A, Ahrens K, Kohn RP, Hermanstyne K, Bernstein KT, et al. Molecular Epidemiology of Syphilis—San Francisco, 2004-2007. Sexually Transmitted Diseases. 2010. pp. 660–663. doi: 10.1097/olq.0b013e3181e1a77a PubMed DOI

Mikalová L, Pospíšilová P, Woznicová V, Kuklová I, Zákoucká H, Smajs D. Comparison of CDC and sequence-based molecular typing of syphilis treponemes: PubMed PMC

Grillova L, Jolley K, Šmajs D, Picardeau M. A public database for the new MLST scheme for PubMed PMC

Flasarová M, Smajs D, Matejková P, Woznicová V, Heroldová-Dvoráková M, Votava M. [Molecular detection and subtyping of PubMed

Woznicová V, Smajs D, Wechsler D, Matĕjková P, Flasarová M. Detection of PubMed PMC

Chuma IS, Roos C, Atickem A, Bohm T, Anthony Collins D, Grillová L, et al. Strain diversity of PubMed PMC

Medappa M, Pospíšilová P, Madruga MPM, John LN, Beiras CG, Grillová L, et al. Low genetic diversity of PubMed PMC

Peltzer A, Jäger G, Herbig A, Seitz A, Kniep C, Krause J, et al. EAGER: efficient ancient genome reconstruction. Genome Biol. 2016;17: 60. PubMed PMC

Li C. A Burrows-Wheeler Transform Based Method for DNA Sequence Comparison. Computational Biology and Bioinformatics. 2014. p. 33. doi: 10.11648/j.cbb.20140203.11 DOI

Okonechnikov K, Conesa A, García-Alcalde F. Qualimap 2: advanced multi-sample quality control for high-throughput sequencing data. Bioinformatics. 2016;32: 292–294. PubMed PMC

McKenna A, Hanna M, Banks E, Sivachenko A, Cibulskis K, Kernytsky A, et al. The Genome Analysis Toolkit: a MapReduce framework for analyzing next-generation DNA sequencing data. Genome Res. 2010;20: 1297–1303. PubMed PMC

Arora N, Schuenemann VJ, Jäger G, Peltzer A, Seitz A, Herbig A, et al. Origin of modern syphilis and emergence of a pandemic PubMed

Minh BQ, Schmidt HA, Chernomor O, Schrempf D, Woodhams MD, von Haeseler A, et al. IQ-TREE 2: New Models and Efficient Methods for Phylogenetic Inference in the Genomic Era. Mol Biol Evol. 2020;37: 1530–1534. PubMed PMC

Strimmer K, von Haeseler A. Likelihood-mapping: a simple method to visualize phylogenetic content of a sequence alignment. Proc Natl Acad Sci U S A. 1997;94: 6815–6819. PubMed PMC

Lukehart SA, Godornes C, Molini BJ, Sonnett P, Hopkins S, Mulcahy F, et al. Macrolide resistance in Treponema pallidum in the United States and Ireland. N Engl J Med. 2004;351: 154–158. PubMed

Staden R, Judge DP, Bonfield JK. Managing Sequencing Projects in the GAP4 Environment. Introduction to Bioinformatics. pp. 327–344. doi: 10.1385/1-59259-335-6:327 DOI

Larsson A. AliView: a fast and lightweight alignment viewer and editor for large datasets. Bioinformatics. 2014;30: 3276–3278. PubMed PMC

Matejkova P, Flasarova M, Zakoucka H, Borek M, Kremenova S, Arenberger P, et al. Macrolide treatment failure in a case of secondary syphilis: a novel A2059G mutation in the 23S rRNA gene of PubMed

Molini BJ, Tantalo LC, Sahi SK, Rodriguez VI, Brandt SL, Fernandez MC, et al. Macrolide Resistance in PubMed PMC

Katoh K, Asimenos G, Toh H. Multiple alignment of DNA sequences with MAFFT. Methods Mol Biol. 2009;537: 39–64. PubMed

Fu L, Niu B, Zhu Z, Wu S, Li W. CD-HIT: accelerated for clustering the next-generation sequencing data. Bioinformatics. 2012;28: 3150–3152. PubMed PMC

Pla-Díaz M, Akgül G, Molak M, du Plessis L, Panagiotopoulou H, Doan K, et al. Insights into PubMed PMC

Nei M. F-statistics and analysis of gene diversity in subdivided populations. Ann Hum Genet. 1977;41: 225–233. PubMed

Rozas J, Ferrer-Mata A, Sánchez-DelBarrio JC, Guirao-Rico S, Librado P, Ramos-Onsins SE, et al. DnaSP 6: DNA Sequence Polymorphism Analysis of Large Data Sets. Mol Biol Evol. 2017;34: 3299–3302. PubMed

Jolley KA, Bray JE, Maiden MCJ. Open-access bacterial population genomics: BIGSdb software, the PubMLST.org website and their applications. Wellcome Open Res. 2018;3: 124. PubMed PMC

Taouk ML, Taiaroa G, Pasricha S, Herman S, Chow EPF, Azzatto F, et al. Characterisation of PubMed DOI

Vrbová E, Noda AA, Grillová L, Rodríguez I, Forsyth A, Oppelt J, et al. Whole genome sequences of PubMed PMC

Seña AC, Matoga MM, Yang L, Lopez-Medina E, Aghakhanian F, Chen JS, et al. Clinical and genomic diversity of PubMed PMC

Beale MA, Thorn L, Cole MJ, Pitt R, Charles H, Ewens M, et al. Genomic epidemiology of syphilis in England: a population-based study. Lancet Microbe. 2023;4: e770–e780. PubMed PMC

Pla-Díaz M, Sánchez-Busó L, Giacani L, Šmajs D, Bosshard PP, Bagheri HC, et al. Evolutionary Processes in the Emergence and Recent Spread of the Syphilis Agent, PubMed DOI PMC

Grillová L, Oppelt J, Mikalová L, Nováková M, Giacani L, Niesnerová A, et al. Directly Sequenced Genomes of Contemporary Strains of Syphilis Reveal Recombination-Driven Diversity in Genes Encoding Predicted Surface-Exposed Antigens. Front Microbiol. 2019;10: 1691. PubMed PMC

Strouhal M, Mikalová L, Haviernik J, Knauf S, Bruisten S, Noordhoek GT, et al. Complete genome sequences of two strains of PubMed PMC

Beale MA, Marks M, Sahi SK, Tantalo LC, Nori AV, French P, et al. Genomic epidemiology of syphilis reveals independent emergence of macrolide resistance across multiple circulating lineages. Nat Commun. 2019;10: 1–9. PubMed PMC

Obraztsova O, Shpilevaya MV, Katunin G, Obukhov A, Shagabieva YZ, Solomka V. Prevalence of the A2058G mutation in 23S rRNA gene, which determines DOI

Morando N, Vrbová E, Melgar A, Rabinovich RD, Šmajs D, Pando MA. High frequency of Nichols-like strains and increased levels of macrolide resistance in PubMed DOI PMC

Wang X, Abliz P, Deng S. Molecular Characteristics of Macrolide Resistance in from Patients with Latent Syphilis in Xinjiang, China. Infect Drug Resist. 2023;16: 1231–1236. PubMed PMC

Lieberman NAP, Reid TB, Cannon CA, Nunley BE, Berzkalns A, Cohen SE, et al. Near-Universal Resistance to Macrolides of PubMed PMC

Janier M, Hegyi V, Dupin N, Unemo M, Tiplica GS, Potočnik M, et al. 2014 European guideline on the management of syphilis. J Eur Acad Dermatol Venereol. 2014;28: 1581–1593. PubMed