Yaws is an endemic disease caused by Treponema pallidum subsp. pertenue (TPE) that primarily affects children in rural regions of the tropics. The endemic character of yaws infections and the expected exclusive reservoir of TPE in humans opened a new opportunity to start a yaws eradication campaign. We have developed a multi-locus sequence typing (MLST) scheme for TPE isolates combining the previously published (TP0548, TP0488) and new (TP0858) chromosomal loci, and we compared this typing scheme to the two previously published MLST schemes. We applied this scheme to TPE-containing clinical isolates obtained during a mass drug administration study performed in the Namatanai District of Papua New Guinea between June 2018 and December 2019. Of 1081 samples collected, 302 (28.5%) tested positive for TPE DNA, from which 255 (84.4%) were fully typed. The TPE PCR-positivity in swab samples was higher in younger patients, patients with single ulcers, first ulcer episodes, and with ulcer duration less than six months. Non-treponemal serological test positivity correlated better with PCR positivity compared to treponema-specific serological tests. The MLST revealed a low level of genetic diversity among infecting TPE isolates, represented by just three distinct genotypes (JE11, SE22, and TE13). Two previously used typing schemes revealed similar typing resolutions. Two new alleles (one in TP0858 and one in TP0136) were shown to arise by intragenomic recombination/deletion events. Compared to samples genotyped as JE11, the minor genotypes (TE13 and SE22) were more frequently detected in samples from patients with two or more ulcers and patients with higher values of specific TP serological tests. Moreover, the A2058G mutation in the 23S rRNA genes of three JE11 isolates was found, resulting in azithromycin resistance.

• MeSH
• Child MeSH
• Yaws * epidemiology   MeSH
• Genotype MeSH
• Humans MeSH
• Multilocus Sequence Typing MeSH
• Mutation MeSH
• Treponema pallidum * genetics   MeSH
• Treponema genetics   MeSH
• Ulcer MeSH
• Check Tag
• Child MeSH
• Humans MeSH
• Publication type
• Journal Article MeSH
• Geographicals
• Papua New Guinea epidemiology   MeSH

Treponema pallidum subsp. endemicum (TEN) is the causative agent of endemic syphilis (bejel). Until now, only a single TEN strain, Bosnia A, has been completely sequenced. The only other laboratory TEN strain available, Iraq B, was isolated in Iraq in 1951 by researchers from the US Centers for Disease Control and Prevention. In this study, the complete genome of the Iraq B strain was amplified as overlapping PCR products and sequenced using the pooled segment genome sequencing method and Illumina sequencing. Total average genome sequencing coverage reached 3469×, with a total genome size of 1,137,653 bp. Compared to the genome sequence of Bosnia A, a set of 37 single nucleotide differences, 4 indels, 2 differences in the number of tandem repetitions, and 18 differences in the length of homopolymeric regions were found in the Iraq B genome. Moreover, the tprF and tprG genes that were previously found deleted in the genome of the TEN Bosnia A strain (spanning 2.3 kb in length) were present in a subpopulation of TEN Iraq B and Bosnia A microbes, and their sequence was highly similar to those found in T. p. subsp. pertenue strains, which cause the disease yaws. The genome sequence of TEN Iraq B revealed close genetic relatedness between both available bejel-causing laboratory strains (i.e., Iraq B and Bosnia A) and also genetic variability within the bejel treponemes comparable to that found within yaws- or syphilis-causing strains. In addition, genetic relatedness to TPE strains was demonstrated by the sequence of the tprF and tprG genes found in subpopulations of both TEN Iraq B and Bosnia A. The loss of the tprF and tprG genes in most TEN microbes suggest that TEN genomes have been evolving via the loss of genomic regions, a phenomenon previously found among the treponemes causing both syphilis and rabbit syphilis.

• MeSH
• Genes, Bacterial MeSH
• Yaws microbiology   MeSH
• Phylogeny MeSH
• Genome, Bacterial MeSH
• Treponemal Infections microbiology   MeSH
• Bacterial Outer Membrane Proteins genetics   MeSH
• Whole Genome Sequencing MeSH
• Syphilis microbiology   MeSH
• Treponema pallidum genetics   MeSH
• Treponema genetics   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Geographicals
• Bosnia and Herzegovina MeSH
• Names of Substances
• Bacterial Outer Membrane Proteins MeSH

BACKGROUND: Treponema pallidum subspecies pallidum (TPA) and subsp. endemicum (TEN) are the causative agents of syphilis and bejel, respectively. TEN shows similar clinical manifestations and is morphologically and serologically indistinguishable from TPA. Recently, bejel was found outside of its assumed endemic areas. Using molecular typing we aimed to discover bejel and characterize circulating TPA types among syphilis cases with Surinamese, Antillean and Dutch ethnicity in Amsterdam. METHODS: DNA was extracted from 137 ulcer swabs, which tested positive in the in-house diagnostic PCR targeting the polA gene. Samples were collected between 2006 and 2018 from Surinamese, Antillean and Dutch patients attending the Amsterdam STI clinic. Multilocus sequence typing was performed by partial sequence analysis of the tp0136, tp0548 and tp0705 genes. In addition, the 23S rRNA loci were analyzed for A2058G and A2059G macrolide resistance mutations. RESULTS: We found 17 distinct allelic profiles in 103/137 (75%) fully typed samples, which were all TPA and none TEN. Of the strains, 82.5% were SS14-like and 17.5% Nichols-like. The prevalence of Nichols-like strains found in this study is relatively high compared to nearby countries. The most prevalent types were 1.3.1 (42%) and 1.1.1 (19%), in concordance with similar TPA typing studies. The majority of the TPA types found were unique per country. New allelic types (7) and profiles (10) were found. The successfully sequenced 23S rRNA loci from 123/137 (90%) samples showed the presence of 79% A2058G and 2% A2059G mutations. CONCLUSIONS: No TEN was found in the samples from different ethnicities residing in Amsterdam, the Netherlands, so no misdiagnoses occurred. Bejel has thus not (yet) spread as a sexually transmitted disease in the Netherlands. The strain diversity found in this study reflects the local male STI clinic population which is a diverse, mixed group.

• MeSH
• Alleles MeSH
• Genes, Bacterial * MeSH
• Adult MeSH
• Ethnicity statistics & numerical data   MeSH
• Humans MeSH
• Syphilis epidemiology   ethnology   microbiology   MeSH
• DNA Barcoding, Taxonomic MeSH
• Treponema pallidum classification   genetics   pathogenicity   MeSH
• Check Tag
• Adult MeSH
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Geographicals
• Netherlands Antilles MeSH
• Netherlands MeSH
• Suriname MeSH

In our most recent study, we found that in Tanzania infection with Treponema pallidum (TP) subsp. pertenue (TPE) is present in four different monkey species. In order to gain information on the diversity and epidemiological spread of the infection in Tanzanian nonhuman primates (NHP), we identified two suitable candidate genes for multi-locus sequence typing (MLST). We demonstrate the functionality of the MLST system in invasively and non-invasively collected samples. While we were not able to demonstrate frequent interspecies transmission of TPE in Tanzanian monkeys, our results show a clustering of TPE strains according to geography and not host species, which is suggestive for rare transmission events between different NHP species. In addition to the geographic stability, we describe the relative temporal stability of the strains infecting NHPs and identified multi-strain infection. Differences between TPE strains of NHP and human origin are highlighted. Our results show that antibiotic resistance does not occur in Tanzanian TPE strains of NHP origin.

• MeSH
• Chlorocebus aethiops microbiology   MeSH
• Cercopithecus microbiology   MeSH
• Species Specificity MeSH
• Feces microbiology   MeSH
• Phylogeny MeSH
• Genetic Variation MeSH
• Genetic Association Studies MeSH
• Gorilla gorilla microbiology   MeSH
• Host Specificity * MeSH
• Treponemal Infections epidemiology   microbiology   transmission   veterinary   MeSH
• Polymorphism, Single Nucleotide MeSH
• Multilocus Sequence Typing MeSH
• Ape Diseases epidemiology   microbiology   transmission   MeSH
• Monkey Diseases epidemiology   microbiology   transmission   MeSH
• Papio anubis microbiology   MeSH
• Papio cynocephalus microbiology   MeSH
• Treponema classification   genetics   isolation & purification   MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Geographicals
• Congo epidemiology   MeSH
• Tanzania epidemiology   MeSH

BACKGROUND: Pathogenic treponemes related to Treponema pallidum are both human (causing syphilis, yaws, bejel) and animal pathogens (infections of primates, venereal spirochetosis in rabbits). A set of 11 treponemal genome sequences including those of five Treponema pallidum ssp. pallidum (TPA) strains (Nichols, DAL-1, Mexico A, SS14, Chicago), four T. p. ssp. pertenue (TPE) strains (CDC-2, Gauthier, Samoa D, Fribourg-Blanc), one T. p. ssp. endemicum (TEN) strain (Bosnia A) and one strain (Cuniculi A) of Treponema paraluisleporidarum ecovar Cuniculus (TPeC) were tested for the presence of positively selected genes. METHODOLOGY/PRINCIPAL FINDINGS: A total of 1068 orthologous genes annotated in all 11 genomes were tested for the presence of positively selected genes using both site and branch-site models with CODEML (PAML package). Subsequent analyses with sequences obtained from 62 treponemal draft genomes were used for the identification of positively selected amino acid positions. Synthetic biotinylated peptides were designed to cover positively selected protein regions and these peptides were tested for reactivity with the patient's syphilis sera. Altogether, 22 positively selected genes were identified in the TP genomes and TPA sets of positively selected genes differed from TPE genes. While genetic variability among TPA strains was predominantly present in a number of genetic loci, genetic variability within TPE and TEN strains was distributed more equally along the chromosome. Several syphilitic sera were shown to react with some peptides derived from the protein sequences evolving under positive selection. CONCLUSIONS/SIGNIFICANCE: The syphilis-, yaws-, and bejel-causing strains differed relative to sets of positively selected genes. Most of the positively selected chromosomal loci were identified among the TPA treponemes. The local accumulation of genetic variability suggests that the diversification of TPA strains took place predominantly in a limited number of genomic regions compared to the more dispersed genetic diversity differentiating TPE and TEN strains. The identification of positively selected sites in tpr genes and genes encoding outer membrane proteins suggests their role during infection of human and animal hosts. The driving force for adaptive evolution at these loci thus appears to be the host immune response as supported by observed reactivity of syphilitic sera with some peptides derived from protein sequences showing adaptive evolution.

• MeSH
• Genes, Bacterial * MeSH
• Adaptation, Biological * MeSH
• Adult MeSH
• Genomics MeSH
• Genotype * MeSH
• Humans MeSH
• Young Adult MeSH
• Selection, Genetic MeSH
• Syphilis microbiology   pathology   MeSH
• Treponema pallidum classification   genetics   isolation & purification   MeSH
• Check Tag
• Adult MeSH
• Humans MeSH
• Young Adult MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

A recently introduced Multilocus Sequence Typing scheme for Treponema pallidum subsp. pallidum was applied to clinical samples collected from 2004 to 2017 from the two largest cities (Prague and Brno) in the Czech Republic. Altogether, a total of 675 samples were tested in this study and 281 of them were found PCR-positive for treponemal DNA and typeable. Most of the typed samples (n = 281) were swabs from primary or secondary syphilis lesions (n = 231), and only a minority were whole blood or tissue samples (n = 50). Swab samples from patients with rapid plasma regain (RPR) values of 1-1024 were more frequently PCR-positive (84.6%) compared to samples from patients with non-reactive RPR test (46.5%; p-value = 0.0001). Out of 281 typeable samples, 136 were fully-typed at all TP0136, TP0548, and TP0705 loci. Among the fully and partially typed samples, 25 different allelic profiles were identified. Altogether, eight novel allelic variants were found among fully (n = 5) and partially (n = 3) typed samples. The distribution of TPA allelic profiles identified in the Czech Republic from 2004 to 2017 revealed a dynamic character with allelic profiles disappearing and emerging over time. While the number of samples with the A2058G mutation was seen to increase (86.7% in 2016/2017), the number of samples harboring the A2059G mutation was found to have decreased over time (3.3% in 2016/2017). In addition, we found several allelic profile associations with macrolide resistance or susceptibility, the gender of patients, as well as patient residence.

• MeSH
• Alleles MeSH
• Anti-Bacterial Agents pharmacology   MeSH
• Drug Resistance, Bacterial genetics   MeSH
• DNA, Bacterial genetics   MeSH
• Adult MeSH
• Genotype MeSH
• Humans MeSH
• Young Adult MeSH
• Multilocus Sequence Typing * MeSH
• RNA, Ribosomal, 23S genetics   MeSH
• Syphilis genetics   microbiology   pathology   MeSH
• Treponema pallidum genetics   pathogenicity   MeSH
• Check Tag
• Adult MeSH
• Humans MeSH
• Young Adult MeSH
• Male MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Geographicals
• Czech Republic epidemiology   MeSH
• Names of Substances
• Anti-Bacterial Agents MeSH
• DNA, Bacterial MeSH
• RNA, Ribosomal, 23S MeSH

Treponema pallidum subsp. pallidum is the causative agent of syphilis, a sexually transmitted disease with worldwide prevalence. Several different molecular typing schemes are currently available for this pathogen. To enable population biology studies of the syphilis agent and for epidemiological surveillance at the global scale, a harmonized typing tool needs to be introduced. Recently, we published a new multi-locus sequence typing (MLST) with the potential to significantly enhance the epidemiological data in several aspects (e.g., distinguishing genetically different clades of syphilis, subtyping inside these clades, and finally, distinguishing different subspecies of non-cultivable pathogenic treponemes). In this short report, we introduce the PubMLST database for treponemal DNA data storage and for assignments of allelic profiles and sequencing types. Moreover, we have summarized epidemiological data of all treponemal strains (n = 358) with available DNA sequences in typing loci and found several association between genetic groups and characteristics of patients. This study proposes the establishment of a single MLST of T. p. pallidum and encourages researchers and public health communities to use this PubMLST database as a universal tool for molecular typing studies of the syphilis pathogen.

• Keywords
• Molecular typing, PubMLST, Treponema pallidum subsp. pallidum,
• Publication type
• Journal Article MeSH

BACKGROUND: Treponema pallidum subsp. pertenue (TPE) is the causative agent of yaws, a multistage disease endemic in tropical regions in Africa, Asia, Oceania, and South America. To date, seven TPE strains have been completely sequenced and analyzed including five TPE strains of human origin (CDC-2, CDC 2575, Gauthier, Ghana-051, and Samoa D) and two TPE strains isolated from the baboons (Fribourg-Blanc and LMNP-1). This study revealed the complete genome sequences of two TPE strains, Kampung Dalan K363 and Sei Geringging K403, isolated in 1990 from villages in the Pariaman region of Sumatra, Indonesia and compared these genome sequences with other known TPE genomes. METHODOLOGY/PRINCIPAL FINDINGS: The genomes were determined using the pooled segment genome sequencing method combined with the Illumina sequencing platform resulting in an average coverage depth of 1,021x and 644x for the TPE Kampung Dalan K363 and TPE Sei Geringging K403 genomes, respectively. Both Indonesian TPE strains were genetically related to each other and were more distantly related to other, previously characterized TPE strains. The modular character of several genes, including TP0136 and TP0858 gene orthologs, was identified by analysis of the corresponding sequences. To systematically detect genes potentially having a modular genetic structure, we performed a whole genome analysis-of-occurrence of direct or inverted repeats of 17 or more nucleotides in length. Besides in tpr genes, a frequent presence of repeats was found in the genetic regions spanning TP0126-TP0136, TP0856-TP0858, and TP0896 genes. CONCLUSIONS/SIGNIFICANCE: Comparisons of genome sequences of TPE Kampung Dalan K363 and Sei Geringging K403 with other TPE strains revealed a modular structure of several genomic loci including the TP0136, TP0856, and TP0858 genes. Diversification of TPE genomes appears to be facilitated by intra-strain genome recombination events.

• MeSH
• Genome, Bacterial * MeSH
• Humans MeSH
• Gene Order MeSH
• Recombination, Genetic MeSH
• Repetitive Sequences, Nucleic Acid MeSH
• Sequence Analysis, DNA * MeSH
• Treponema pallidum genetics   MeSH
• Computational Biology MeSH
• High-Throughput Nucleotide Sequencing MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Geographicals
• Indonesia MeSH

Syphilis is an important public health problem and an increasing incidence has been noted in recent years. Characterization of strain diversity through molecular data plays a critical role in the epidemiological understanding of this re-emergence. We here propose a new high-resolution multilocus sequence typing (MLST) scheme for Treponema pallidum subsp. pallidum (TPA). We analyzed 30 complete and draft TPA genomes obtained directly from clinical samples or from rabbit propagated strains to identify suitable typing loci and tested the new scheme on 120 clinical samples collected in Switzerland and France. Our analyses yielded three loci with high discriminatory power: TP0136, TP0548, and TP0705. Together with analysis of the 23S rRNA gene mutations for macrolide resistance, we propose these loci as MLST for TPA. Among clinical samples, 23 allelic profiles as well as a high percentage (80% samples) of macrolide resistance were revealed. The new MLST has higher discriminatory power compared to previous typing schemes, enabling distinction of TPA from other treponemal bacteria, distinction between the two main TPA clades (Nichols and SS14), and differentiation of strains within these clades.

• MeSH
• Alleles MeSH
• Anti-Bacterial Agents pharmacology   MeSH
• DNA, Bacterial genetics   MeSH
• Phylogeny MeSH
• Genome, Bacterial MeSH
• Genotype MeSH
• Globus Pallidus MeSH
• Polymorphism, Single Nucleotide MeSH
• Macrolides pharmacology   MeSH
• Multilocus Sequence Typing methods   MeSH
• RNA, Ribosomal, 23S genetics   MeSH
• Sequence Analysis, DNA methods   MeSH
• Syphilis epidemiology   MeSH
• Treponema pallidum genetics   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Geographicals
• France epidemiology   MeSH
• Switzerland epidemiology   MeSH
• Names of Substances
• Anti-Bacterial Agents MeSH
• DNA, Bacterial MeSH
• Macrolides MeSH
• RNA, Ribosomal, 23S MeSH

Treponema pallidum subsp. pallidum, the causative agent of sexually transmitted syphilis, detected in clinical samples from France, was subjected to molecular typing using the recently developed Multilocus Sequence Typing system. The samples (n = 133) used in this study were collected from 2010-2016 from patients with diagnosed primary or secondary syphilis attending outpatient centers or hospitals in several locations in France. Altogether, 18 different allelic profiles were found among the fully typed samples (n = 112). There were five allelic variants identified for TP0136, 12 for TP0548, and eight for TP0705. Out of the identified alleles, one, seven, and three novel alleles were identified in TP0136, TP0548, and TP0705, respectively. Partial allelic profiles were obtained from 6 samples. The majority of samples (n = 110) belonged to the SS14-like cluster of TPA isolates while 7 clustered with Nichols-like isolates. Patients infected with Nichols-like samples were more often older (p = 0.041) and more often diagnosed with secondary syphilis (p = 0.033) compared to patients infected with SS14-like samples. In addition, macrolide resistance caused by the A2058G mutation was found to be associated with allelic profile 1.3.1 or with strains belonging to the 1.3.1 lineage (p<0.001). The genetic diversity among TPA strains infecting the European population was surprisingly high, which suggests that additional studies are needed to reveal the full genetic diversity of TPA pathogens infecting humans.

• MeSH
• Alleles MeSH
• Biodiversity MeSH
• Child MeSH
• Adult MeSH
• Infant MeSH
• Middle Aged MeSH
• Humans MeSH
• Adolescent MeSH
• Young Adult MeSH
• Multilocus Sequence Typing MeSH
• Infant, Newborn MeSH
• Child, Preschool MeSH
• Aged MeSH
• Syphilis epidemiology   microbiology   MeSH
• Bacterial Typing Techniques MeSH
• Treponema pallidum genetics   isolation & purification   MeSH
• Check Tag
• Child MeSH
• Adult MeSH
• Infant MeSH
• Middle Aged MeSH
• Humans MeSH
• Adolescent MeSH
• Young Adult MeSH
• Male MeSH
• Infant, Newborn MeSH
• Child, Preschool MeSH
• Aged MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Geographicals
• France epidemiology   MeSH