Syphilis, caused by Treponema pallidum subsp. pallidum (TPA), remains an important public health problem with an increasing worldwide prevalence. Despite recent advances in in vitro cultivation, genetic variability of this pathogen during infection is poorly understood. Here, we present contemporary and geographically diverse complete treponemal genome sequences isolated directly from patients using a methyl-directed enrichment prior to sequencing. This approach reveals that approximately 50% of the genetic diversity found in TPA is driven by inter- and/or intra-strain recombination events, particularly in strains belonging to one of the defined genetic groups of syphilis treponemes: Nichols-like strains. Recombinant loci were found to encode putative outer-membrane proteins and the recombination variability was almost exclusively found in regions predicted to be at the host-pathogen interface. Genetic recombination has been considered to be a rare event in treponemes, yet our study unexpectedly showed that it occurs at a significant level and may have important impacts in the biology of this pathogen, especially as these events occur primarily in the outer membrane proteins. This study reveals the existence of strains with different repertoires of surface-exposed antigens circulating in the current human population, which should be taken into account during syphilis vaccine development.

• Keywords
• Treponema pallidum subsp. pallidum, culture-independent bacterial enrichment, direct whole genome sequencing, recombination-driven diversity, syphilis,
• Publication type
• Journal Article 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

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

OBJECTIVE: Treponema pallidum subsp. pallidum (TPA) is the causative agent of syphilis. Genetic analyses of TPA reference strains and human clinical isolates have revealed two genetically distinct groups of syphilis-causing treponemes, called Nichols-like and SS14-like groups. So far, no genetic intermediates, i.e. strains containing a mixed pattern of Nichols-like and SS14-like genomic sequences, have been identified. Recently, Sun et al. (Oncotarget 2016. https://doi.org/10.18632/oncotarget.10154 ) described a new "phylogenetic group" (called Lineage 2) among Chinese TPA strains. This lineage exhibited a "mosaic genomic structure" of Nichols-like and SS14-like lineages. RESULTS: We reanalyzed the primary sequencing data (Project Number PRJNA305961) from the Sun et al. publication with respect to the molecular basis of Lineage 2. While Sun et al. based the analysis on several selected genomic single nucleotide variants (SNVs) and a subset of highly variable but phylogenetically poorly informative genes, which may confound the phylogenetic analysis, our reanalysis primarily focused on a complete set of whole genomic SNVs. Based on our reanalysis, only two separate TPA clusters were identified: one consisted of Nichols-like TPA strains, the other was formed by the SS14-like TPA strains, including all Chinese strains.

• Keywords
• Genome sequencing, Phylogenetic analysis, Single nucleotide variant, Syphilis, Treponema pallidum,
• MeSH
• Phylogeny * MeSH
• Genome, Bacterial genetics   MeSH
• Polymorphism, Single Nucleotide genetics   MeSH
• Humans MeSH
• Sequence Analysis, DNA * MeSH
• Treponema pallidum genetics   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Geographicals
• China MeSH

A total of 54 clinical samples, including genital lesion swabs, whole blood and cerebrospinal fluid from patients diagnosed with syphilis were collected in 2006 and in 2013 in Buenos Aires, Argentina. Treponemal DNA was detected in 43 of the analyzed samples (79.6%) and further analyzed using Sequencing-based molecular typing (SBMT) and Enhanced CDC-typing (ECDCT). By SBMT, 10 different Treponema pallidum subsp. pallidum (TPA) genotypes were found, of which six were related to the TPA SS14 strain, and four to the TPA Nichols strain. The 23S rRNA gene was amplified in samples isolated from 42 patients, and in six of them (14.3%), either the A2058G (four patients, 9.5%) or the A2059G (two patients, 4.8%) mutations were found. In addition to Taiwan, Madagascar and Peru, Argentina is another country where the prevalence of Nichols-like isolates (26.8%) is greater than 10%.

• MeSH
• Anti-Bacterial Agents pharmacology   MeSH
• Drug Resistance, Bacterial genetics   MeSH
• DNA, Bacterial genetics   MeSH
• Adult MeSH
• Phylogeny MeSH
• Middle Aged MeSH
• Humans MeSH
• Macrolides pharmacology   MeSH
• Adolescent MeSH
• Young Adult MeSH
• Multilocus Sequence Typing MeSH
• DNA Mutational Analysis MeSH
• Base Sequence MeSH
• Aged MeSH
• Syphilis epidemiology   microbiology   MeSH
• Treponema pallidum genetics   isolation & purification   MeSH
• Cities MeSH
• Check Tag
• Adult MeSH
• Middle Aged MeSH
• Humans MeSH
• Adolescent MeSH
• Young Adult MeSH
• Male MeSH
• Aged MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Geographicals
• Argentina epidemiology   MeSH
• Cities MeSH
• Names of Substances
• Anti-Bacterial Agents MeSH
• DNA, Bacterial MeSH
• Macrolides MeSH