The incidence of syphilis, a sexually transmitted disease caused by the Treponema pallidum subsp. pallidum (TPA), has been surging globally despite effective antibiotic therapy. A new strategy for syphilis control is the development of a multi-component syphilis vaccine with global efficacy, which requires the identification of surface-exposed candidate vaccinogens and the determination of their antigenic diversity within circulating TPA strains. To improve the quality of sequences from repetitive and paralogous regions of the TPA genome, we have developed a sequencing scheme that allows amplification and long-read sequencing of 25 targets encoding TPA proteins including 15 outer membrane proteins. We tested this approach on a set of 21 clinical TPA strains, mostly of European origin preselected by MLST typing. A total of 462 (88%) of 525 amplicons were sequenced. Of 58 new alleles identified in comparison to the SS14 and Nichols TPA reference strains, the majority encoded new protein sequences (n = 55; 94.8%). The 55 variant protein sequences were encoded by 99 individual TPA loci, where single amino acid replacements occurred most frequently (n = 50), followed by replacements of two to three amino acids (n = 35) and differences comprising four or more residues (n = 14); the latter included six intra-strain recombination events. Most differences were localized to predicted surface-exposed regions, consistent with adaptive evolution of bacterial determinants that function at the host-pathogen interface. Clinical strains having the same allelic profiles from different localities differed in several loci, suggesting that geographical origin significantly contributes to genetic diversity of circulating strains.IMPORTANCEOur findings underscore the importance of analyzing TPA clinical samples isolated from diverse geographical regions in order to understand TPA OMP variability.

• Keywords
• MinION sequencing, OMPeome, Treponema pallidum, genetic epidemiology, long-read sequencing, outer membrane proteins, syphilis,
• MeSH
• Alleles MeSH
• DNA, Bacterial genetics   MeSH
• Genetic Variation * MeSH
• Humans MeSH
• Multilocus Sequence Typing MeSH
• Bacterial Outer Membrane Proteins * genetics   MeSH
• Sequence Analysis, DNA MeSH
• Syphilis * microbiology   MeSH
• Treponema pallidum * genetics   classification   isolation & purification   MeSH
• Treponema MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• DNA, Bacterial MeSH
• Bacterial Outer Membrane Proteins * MeSH

Previous in vitro long-term cultivation studies of Treponema pallidum subsp. pallidum (T. pallidum) strains have indicated potential differences in the length of generation times among individual strains. In the present study, we have determined in vitro growth rates of seven T. pallidum strains including three from the Nichols-like cluster (DAL-1, Madras, and Haiti B) and four from the SS14-like cluster (Mexico A, SS14, Grady, and Philadelphia 1). Despite the observed considerable variability, the results of standard subcultures identified growth differences between the two clusters during the long-term cultivation. Furthermore, in vitro monocultures with defined inoculum revealed differences among individual strains. During three week-long binary co-cultivations of seven strains (n = 21), different growth rates were confirmed for individual strains (p < 0.001) using PCR amplicon sequencing of genomic regions differentiating treponemal-pairs. The order of strains by decreasing growth rate in vitro was DAL-1, Madras, Mexico A, Haiti B, SS14, Grady, and Philadelphia 1. While the generation time of strain DAL-1 was 32.97 h, the slowest strain, Philadelphia 1, had generation time 43.5 h. These experiments revealed significant physiological differences between the T. pallidum strains, which may also be involved in the variable presentation of syphilis symptoms observed in previous decades.

• MeSH
• Humans MeSH
• Syphilis * microbiology   MeSH
• Treponema pallidum * growth & development   genetics   classification   isolation & purification   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH

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.

• Keywords
• MLST, T. pallidum, epidemiology, typing,
• Publication type
• Journal Article MeSH
• Preprint MeSH

BACKGROUND: For many years, syphilis treatment was considered straightforward due to the universal susceptibility of Treponema pallidum subsp. pallidum (TPA) to penicillin antibiotics. METHODS: Penicillin-binding protein genes from a ceftriaxone treatment failure T. pallidum isolate were assessed, and the introduction of identified mutations into two laboratory strains via natural competence was aimed for, followed by in vitro analysis of antibiotic susceptibility of the recombinants. RESULTS: TPA from the ceftriaxone treatment failure case contained A1873G and G2122A mutations in the TP0705 gene. Introduction of the A1873G mutation into laboratory strains DAL-1 and SS14 resulted in partial resistance to ceftriaxone and penicillin G in vitro. Furthermore, in silico analyses revealed that the majority of contemporary TPA SS14-like strains harbors this mutation and are thus partially resistant to ceftriaxone and penicillin G. CONCLUSIONS: This finding indicates that TPA strains accumulate mutations that increase their resistance to β-lactam antibiotics. Alternative approaches for controlling syphilis will be needed, including the development of the syphilis vaccine.

Penicillin antibiotics have been used to treat syphilis since the 1950s. Resistance to antibiotics is a growing concern. We investigated cases where antibiotics had failed to treat infection and found two mutations in a specific gene that could be responsible. Introduction of one of these mutations into two laboratory T. pallidum strains (the bacteria that cause syphilis) resulted in partial resistance to both ceftriaxone and penicillin antibiotics. Moreover, analysis of existing data revealed the presence of this mutation in numerous circulating T. pallidum strains, suggesting widespread partial resistance may already exist and increasing concerns about the future emergence of fully resistant syphilis strains.

• Publication type
• Journal Article MeSH

BACKGROUND: Treponema pallidum subspecies pertenue (TPE) is the causative agent of human and nonhuman primate (NHP) yaws infection. The discovery of yaws bacterium in wild populations of NHPs opened the question of transmission mechanisms within NHPs, and this work aims to take a closer look at the transmission of the disease. METHODOLOGY/PRINCIPAL FINDINGS: Our study determined eleven whole TPE genomes from NHP isolates collected from three national parks in Tanzania: Lake Manyara National Park (NP), Serengeti NP, and Ruaha NP. The bacteria were isolated from four species of NHPs: Chlorocebus pygerythrus (vervet monkey), Cercopithecus mitis (blue monkey), Papio anubis (olive baboon), and Papio cynocephalus (yellow baboon). Combined with previously generated genomes of TPE originating from NHPs in Tanzania (n = 11), 22 whole-genome TPE sequences have now been analyzed. Out of 231 possible combinations of genome-to-genome comparisons, five revealed an unexpectedly high degree of genetic similarity in samples collected from different NHP species, consistent with inter-species transmission of TPE among NHPs. We estimated a substitution rate of TPE of NHP origin, ranging between 1.77 × 10-7 and 3.43 × 10-7 per genomic site per year. CONCLUSIONS/SIGNIFICANCE: The model estimations predicted that the inter-species transmission happened recently, within decades, roughly in an order of magnitude shorter time compared to time needed for the natural diversification of all tested TPE of Tanzanian NHP origin. Moreover, the geographical separation of the sampling sites (NPs) does not preclude TPE transmission between and within NHP species.

• MeSH
• Yaws * transmission   microbiology   veterinary   epidemiology   MeSH
• Phylogeny MeSH
• Genome, Bacterial * MeSH
• Primates * microbiology   MeSH
• Whole Genome Sequencing MeSH
• Treponema pallidum * genetics   isolation & purification   classification   MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Geographicals
• Tanzania epidemiology   MeSH

BACKGROUND: The increase in syphilis rates worldwide necessitates development of a vaccine with global efficacy. We aimed to explore Treponema pallidum subspecies pallidum (TPA) molecular epidemiology essential for vaccine research by analysing clinical data and specimens from early syphilis patients using whole-genome sequencing (WGS) and publicly available WGS data. METHODS: In this multicentre, cross-sectional, molecular epidemiology study, we enrolled patients with primary, secondary, or early latent syphilis from clinics in China, Colombia, Malawi, and the USA between Nov 28, 2019, and May 27, 2022. Participants aged 18 years or older with laboratory confirmation of syphilis by direct detection methods or serological testing, or both, were included. Patients were excluded from enrolment if they were unwilling or unable to give informed consent, did not understand the study purpose or nature of their participation, or received antibiotics active against syphilis in the past 30 days. TPA detection and WGS were conducted on lesion swabs, skin biopsies, skin scrapings, whole blood, or rabbit-passaged isolates. We compared our WGS data to publicly available genomes and analysed TPA populations to identify mutations associated with lineage and geography. FINDINGS: We screened 2802 patients and enrolled 233 participants, of whom 77 (33%) had primary syphilis, 154 (66%) had secondary syphilis, and two (1%) had early latent syphilis. The median age of participants was 28 years (IQR 22-35); 154 (66%) participants were cisgender men, 77 (33%) were cisgender women, and two (1%) were transgender women. Of the cisgender men, 66 (43%) identified as gay, bisexual, or other sexuality. Among all participants, 56 (24%) had HIV co-infection. WGS data from 113 participants showed a predominance of SS14-lineage strains with geographical clustering. Phylogenomic analyses confirmed that Nichols-lineage strains were more genetically diverse than SS14-lineage strains and clustered into more distinct subclades. Differences in single nucleotide variants (SNVs) were evident by TPA lineage and geography. Mapping of highly differentiated SNVs to three-dimensional protein models showed population-specific substitutions, some in outer membrane proteins (OMPs) of interest. INTERPRETATION: Our study substantiates the global diversity of TPA strains. Additional analyses to explore TPA OMP variability within strains is vital for vaccine development and understanding syphilis pathogenesis on a population level. FUNDING: US National Institutes of Health National Institute for Allergy and Infectious Disease, the Bill & Melinda Gates Foundation, Connecticut Children's, and the Czech Republic National Institute of Virology and Bacteriology.

• MeSH
• Bacterial Vaccines immunology   administration & dosage   MeSH
• Adult MeSH
• Phylogeny MeSH
• Genetic Variation genetics   MeSH
• Genome, Bacterial MeSH
• Genomics MeSH
• Middle Aged MeSH
• Humans MeSH
• Young Adult MeSH
• Molecular Epidemiology * MeSH
• Cross-Sectional Studies MeSH
• Whole Genome Sequencing * MeSH
• Syphilis * epidemiology   microbiology   MeSH
• Treponema pallidum * genetics   immunology   MeSH
• Treponema MeSH
• Check Tag
• Adult MeSH
• Middle Aged MeSH
• Humans MeSH
• Young Adult MeSH
• Male MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Multicenter Study MeSH
• Geographicals
• United States epidemiology   MeSH
• Names of Substances
• Bacterial Vaccines MeSH

The treponemes infecting lagomorphs include Treponema paraluisleporidarum ecovar Cuniculus (TPeC) and ecovar Lepus (TPeL), infecting rabbits and hares, respectively. In this study, we described the first complete genome sequence of TPeL, isolate V3603-13, from an infected mountain hare (Lepus timidus) in Sweden. In addition, we determined 99.0% of the genome sequence of isolate V246-08 (also from an infected mountain hare, Sweden) and 31.7% of the genome sequence of isolate Z27 A77/78 (from a European hare, Lepus europeaus, The Netherlands). The TPeL V3603-13 genome had considerable gene synteny with the TPeC Cuniculi A genome and with the human pathogen T. pallidum, which causes syphilis (ssp. pallidum, TPA), yaws (ssp. pertenue, TPE) and endemic syphilis (ssp. endemicum, TEN). Compared to the TPeC Cuniculi A genome, TPeL V3603-13 contained four insertions and 11 deletions longer than three nucleotides (ranging between 6 and2,932 nts). In addition, there were 25 additional indels, from one to three nucleotides long, altogether spanning 36 nts. The number of single nucleotide variants (SNVs) between TPeC Cuniculi A and TPeL V3603-13 were represented by 309 nucleotide differences. Major proteome coding differences between TPeL and TPeC were found in the tpr gene family, and (predicted) genes coding for outer membrane proteins, suggesting that these components are essential for host adaptation in lagomorph syphilis. The phylogeny revealed that the TPeL sample from the European brown hare was more distantly related to TPeC Cuniculi A than V3603-13 and V246-08.

• MeSH
• Phylogeny * MeSH
• Genome, Bacterial MeSH
• Rabbits MeSH
• Syphilis * microbiology   MeSH
• Treponema * genetics   isolation & purification   MeSH
• Hares * microbiology   MeSH
• Animals MeSH
• Check Tag
• Rabbits MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH

In this work, we determined that Treponema pallidum subsp. pallidum (TPA) DAL-1 (belonging to Nichols-like group of TPA strains) grew 1.53 (± 0.08) times faster compared to TPA Philadelphia 1 (SS14-like group) during in vitro cultivations. In longitudinal individual propagation in rabbit testes (n = 12, each TPA strain), infection with DAL-1 manifested clinical symptoms (induration, swelling, and erythema of testes) sooner than Philadelphia 1 infection, which resulted in a significantly shorter period of the experimental passages for DAL-1 (median = 15.0 and 23.5 days, respectively; p < 0.01). To minimize the confounding conditions during rabbit experiments, the growth characteristics of DAL-1 and Philadelphia 1 strains were determined during TPA co-infection of rabbit testes (n = 20, including controls). During two weeks of intratesticular co-infection, DAL-1 overgrew Philadelphia 1 in all twelve testes, regardless of inoculation ratio and dose (median of relative excess DAL-1 multiplication = 84.85×). Moreover, higher DAL-1 to Philadelphia 1 inoculum ratios appeared to increase differences in growth rates, suggesting direct competition between strains for available nutrients during co-infection. These experiments indicate important physiological differences between the two TPA strains and suggest growth differences between Nichols-like and SS14-like strains that are potentially linked to their virulence and pathogenicity.

• MeSH
• Rabbits MeSH
• Syphilis microbiology   pathology   MeSH
• Testis microbiology   metabolism   MeSH
• Treponema pallidum * MeSH
• Animals MeSH
• Check Tag
• Rabbits MeSH
• Male MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH

Syphilis is an ancient disease of humans and lagomorphs caused by two distinct but genetically closely related bacteria (>98% sequence identity based on the whole genome) of the genus Treponema. While human syphilis is well studied, little is known about the disease in the lagomorph host. Yet, comparative studies are needed to understand mechanisms in host-pathogen coevolution in treponematoses. Importantly, Treponema paraluisleporidarum-infected hare populations provide ample opportunity to study the syphilis-causing pathogen in a naturally infected model population without antibiotic treatment, data that cannot be obtained from syphilis infection in humans. We provide data on genetic diversity and are able to highlight various types of repetitions in one of the two hypervariable regions at the tp0548 locus that have not been described in the human syphilis-causing sister bacterium Treponema pallidum subsp. pallidum.

• Keywords
• European brown hare, Lepus, One Health, Oryctolagus, Treponema pallidum, rabbit, spirochetes, syphilis,
• MeSH
• Genetic Variation MeSH
• Lagomorpha * MeSH
• Humans MeSH
• Prevalence MeSH
• Syphilis * epidemiology   microbiology   MeSH
• Treponema pallidum MeSH
• Treponema genetics   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH

Globally, 94% of Treponema pallidum subsp. pallidum (TPA) clinical strains belong to the SS14-like group and 6% to the Nichols-like group, with a prevalence of macrolide resistance of 90%. Our goal was to determine whether local TPA strain distribution and macrolide resistance frequency have changed significantly since our last report, which revealed that Buenos Aires had a high frequency of Nichols-like strains (27%) and low levels of macrolide resistance (14%). Swab samples from patients with suspected syphilis were collected during 2015-2019 and loci TP0136, TP0548, TP0705 were sequenced in order to perform multilocus sequence typing. Strains were classified as Nichols-like or SS14-like. The presence of macrolide resistance-associated mutations was determined by examination of the 23S rDNA gene sequence. Of 46 typeable samples, 37% were classified as Nichols-like and 63% as SS14-like. Macrolide resistance prevalence was 45.7%. Seven allelic profiles were found, five were SS14-like and two were Nichols-like. The frequency of Nichols-like strains increased between studies (26.8% vs. 37%, p = 0.36). A dramatic increase was found in the frequency of macrolide resistant strains between studies (14.3% vs. 45.7%, p = 0.005). Our results are in agreement with international trends and underscore the need to pursue further TPA molecular typing studies in South America.

• MeSH
• Anti-Bacterial Agents pharmacology   MeSH
• Drug Resistance, Bacterial genetics   MeSH
• Treponemal Infections * MeSH
• Humans MeSH
• Macrolides pharmacology   MeSH
• Multilocus Sequence Typing MeSH
• DNA, Ribosomal MeSH
• Treponema pallidum * genetics   MeSH
• Treponema MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Geographicals
• Argentina epidemiology   MeSH
• Names of Substances
• Anti-Bacterial Agents MeSH
• Macrolides MeSH
• DNA, Ribosomal MeSH