Low genetic diversity of Treponema pallidum ssp. pertenue (TPE) isolated from patients' ulcers in Namatanai District of Papua New Guinea: Local human population is infected by three TPE genotypes.

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.

Clinical and genomic diversity of Treponema pallidum subsp. pallidum: A global, multi-center study of early syphilis to inform vaccine research.

Background: The continuing increase in syphilis rates worldwide necessitates development of a vaccine with global efficacy. We conducted a multi-center, observational study to explore Treponema pallidum subsp. pallidum ( TPA ) molecular epidemiology essential for vaccine research by analyzing clinical data and specimens from early syphilis patients using whole-genome sequencing (WGS) and publicly available WGS data. Methods: We enrolled patients with primary (PS), secondary (SS) or early latent (ELS) syphilis from clinics in China, Colombia, Malawi and the United States between November 2019 - May 2022. Inclusion criteria included age ≥18 years, and syphilis confirmation by direct detection methods and/or serological testing. TPA detection and WGS were conducted on lesion swabs, skin biopsies/scrapings, whole blood, and/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 2,820 patients and enrolled 233 participants - 77 (33%) with PS, 154 (66%) with SS, and two (1%) with ELS. Median age of participants was 28; 66% were cis -gender male, of which 43% reported identifying as "gay", "bisexual", or "other sexuality". Among all participants, 56 (24%) had HIV co-infection. WGS data from 113 participants demonstrated a predominance of SS14-lineage strains with geographic clustering. Phylogenomic analysis confirmed that Nichols-lineage strains are more genetically diverse than SS14-lineage strains and cluster 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 demonstrated population-specific substitutions, some in outer membrane proteins (OMPs) of interest. Interpretation: Our study involving participants from four countries substantiates the global diversity of TPA strains. Additional analyses to explore TPA OMP variability within strains will be vital for vaccine development and improved understanding of syphilis pathogenesis on a population level. Funding: National Institutes of Health, Bill and Melinda Gates Foundation.

Trial of Three Rounds of Mass Azithromycin Administration for Yaws Eradication.

BACKGROUND: Treponema pallidum subspecies pertenue causes yaws. Strategies to better control, eliminate, and eradicate yaws are needed. METHODS: In an open-label, cluster-randomized, community-based trial conducted in a yaws-endemic area of Papua New Guinea, we randomly assigned 38 wards (i.e., clusters) to receive one round of mass administration of azithromycin followed by two rounds of target treatment of active cases (control group) or three rounds of mass administration of azithromycin (experimental group); round 1 was administered at baseline, round 2 at 6 months, and round 3 at 12 months. The coprimary end points were the prevalence of active cases of yaws, confirmed by polymerase-chain-reaction assay, in the entire trial population and the prevalence of latent yaws, confirmed by serologic testing, in a subgroup of asymptomatic children 1 to 15 years of age; prevalences were measured at 18 months, and the between-group differences were calculated. RESULTS: Of the 38 wards, 19 were randomly assigned to the control group (30,438 persons) and 19 to the experimental group (26,238 persons). A total of 24,848 doses of azithromycin were administered in the control group (22,033 were given to the participants at round 1 and 207 and 2608 were given to the participants with yaws-like lesions and their contacts, respectively, at rounds 2 and 3 [combined]), and 59,852 doses were administered in the experimental group. At 18 months, the prevalence of active yaws had decreased from 0.46% (102 of 22,033 persons) at baseline to 0.16% (47 of 29,954 persons) in the control group and from 0.43% (87 of 20,331 persons) at baseline to 0.04% (10 of 25,987 persons) in the experimental group (relative risk adjusted for clustering, 4.08; 95% confidence interval [CI], 1.90 to 8.76). The prevalence of other infectious ulcers decreased to a similar extent in the two treatment groups. The prevalence of latent yaws at 18 months was 6.54% (95% CI, 5.00 to 8.08) among 994 children in the control group and 3.28% (95% CI, 2.14 to 4.42) among 945 children in the experimental group (relative risk adjusted for clustering and age, 2.03; 95% CI, 1.12 to 3.70). Three cases of yaws with resistance to macrolides were found in the experimental group. CONCLUSIONS: The reduction in the community prevalence of yaws was greater with three rounds of mass administration of azithromycin at 6-month intervals than with one round of mass administration of azithromycin followed by two rounds of targeted treatment. Monitoring for the emergence and spread of antimicrobial resistance is needed. (Funded by Fundació "la Caixa" and others; ClinicalTrials.gov number, NCT03490123.).

Penicillin Treatment Failure in Rabbit Syphilis Due to the Persistence of Treponemes (Treponema paraluisleporidarum Ecovar Cuniculus) in the Focus of Infection.

Rabbit venereal spirochetosis, a disease caused by Treponema paraluisleporidarum ecovar Cuniculus (TPeC), affects both wild and pet rabbits, and is transmitted sexually and via direct contact among animals. Treatment of syphilis in pet rabbits requires administration of antibiotics, including penicillin G, chloramphenicol, or fluoroquinolones. The aim of this work was to elucidate the cause of penicillin treatment failure in rabbit syphilis in a pet rabbit treated in Brno, Czech Republic, and to assess the phylogenetic relatedness of the agent to previously characterized pathogenic treponemes. Following amputation of the infected digits, the second round of penicillin treatment using the same dosage and application route resulted in the disappearance of clinical symptoms within a period of two weeks. The bacterium was successfully isolated from the claws, propagated in three experimental rabbits, and the resulting TPeC strain was designated as Cz-2020. Analysis of four genetic loci revealed that the Cz-2020 strain was similar but also clearly distinct from the only TPeC strain, which had been characterized in detail to date, i.e., the Cuniculi A strain, which was isolated in North America. The strain Cz-2020 represents the first available viable TPeC strain of European origin. DNA sequences encoding five penicillin-binding proteins of the strain Cz-2020 were compared to those of Cuniculi A, which is known to be sensitive to penicillin. The sequences differed in six nucleotides resulting in single amino acid changes in Penicillin-binding protein 1, 2, and 3. Since the second round of treatment was successful, we conclude that the penicillin treatment failure in the first round resulted from the presence of infection foci in claws where treponemes persisted.

Analysis of Treponema pallidum Strains From China Using Improved Methods for Whole-Genome Sequencing From Primary Syphilis Chancres.

BACKGROUND: Whole-genome sequencing (WGS) of Treponema pallidum subspecies pallidum (TPA) has been constrained by the lack of in vitro cultivation methods for isolating spirochetes from patient samples. METHODS: We built upon recently developed enrichment methods to sequence TPA directly from primary syphilis chancre swabs collected in Guangzhou, China. RESULTS: By combining parallel, pooled whole-genome amplification with hybrid selection, we generated high-quality genomes from 4 of 8 chancre-swab samples and 2 of 2 rabbit-passaged isolates, all subjected to challenging storage conditions. CONCLUSIONS: This approach enabled the first WGS of Chinese samples without rabbit passage and provided insights into TPA genetic diversity in China.

The Arg753Gln Polymorphism of Toll-Like Receptor 2 Has a Lower Occurrence in Patients with Syphilis, Suggesting Its Protective Effect in Czech and Slovak Individuals.

Syphilis is a bacterial infection caused by Treponema pallidum subsp. pallidum Infection with T. pallidum subsp. pallidum and its dissemination lead to the synthesis of proinflammatory cytokines triggered by the interaction of bacterial lipoproteins with Toll-like receptor 2 (TLR2). TLR2 contains several nonsynonymous single-nucleotide polymorphisms that may impact the activation of its signaling cascade and alter the responsiveness to, or the course of, various infectious diseases, including those caused by pathogenic spirochetes. To investigate whether TLR2 polymorphism may influence susceptibility to syphilis, 221 healthy individuals with no history of syphilis (controls) and 137 patients diagnosed with syphilis (cases) were screened for the presence of the Arg753Gln polymorphism in the TLR2 gene (2258G→A; rs5743708). The Arg753Gln variant occurs at a significantly lower frequency in syphilis patients (4 of 137 [3%]) than in controls (24 of 221 [10.9%]). These data suggest that TLR2 Arg753Gln may protect from the development of syphilis due to reduced signaling.

A retrospective study on nested PCR detection of syphilis treponemes in clinical samples: PCR detection contributes to the diagnosis of syphilis in patients with seronegative and serodiscrepant results.

Syphilis, caused by Treponema pallidum ssp. pallidum (TPA), is a persisting global health problem. Although syphilis diagnostics relies mainly on serology, serological tests have some limitations, and it is recommended that the final diagnosis be supported by additional tests. The purpose of this study was to analyze the relationship between serology and PCR in syphilis diagnostics. From the year 2004 to May 2019, a total of 941 samples were taken from 833 patients suspected of having syphilis, in Czech Republic. In all these samples, both nested PCR detection of TPA and serology testing were performed. Of the 941 samples, 126 were seronegative, 651 were seropositive, and 164 were serodiscrepant. Among seronegative samples (n = 126), 11 were PCR-positive (8.7%). Among seropositive samples (n = 651; i.e., samples positive for both non-treponemal and treponemal serology tests), 368 samples were PCR-positive (56.5%). The remaining 164 serodiscrepant samples included RPR negative and treponemal serological test-positive samples (n = 154) and a set of 10 RPR-positive samples negative in treponemal serological tests. While the first group revealed 73 PCR-positive samples (47.4%), the second revealed 5 PCR positive samples (50.0%). PCR detection rates were highest in primary syphilis, with lower rates in the secondary and undetermined syphilis stages. As shown here, the nested PCR can improve diagnostics of syphilis, especially in seronegative patients and in patients with discrepant serology.

Complete Chromosomal Sequences of Two Borrelia miyamotoi Samples Obtained from Ixodes ricinus Eggs in Czechia.

Here, we present complete chromosome sequences of Borrelia miyamotoi samples CZ-F1E and CZ-F190E, which were obtained from Ixodes ricinus eggs from Czechia. The chromosome sequences, assembled from Illumina and Sanger sequencing data, had average coverage values of 647× and 3,216×, respectively. They belong to the European genotype, distinct from the Asian and American strains.

Directly Sequenced Genomes of Contemporary Strains of Syphilis Reveal Recombination-Driven Diversity in Genes Encoding Predicted Surface-Exposed Antigens.

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.

MLST typing of Treponema pallidum subsp. pallidum in the Czech Republic during 2004-2017: Clinical isolates belonged to 25 allelic profiles and harbored 8 novel allelic variants.

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.

Identification of positively selected genes in human pathogenic treponemes: Syphilis-, yaws-, and bejel-causing strains differ in sets of genes showing adaptive evolution.

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.

Multi-locus sequence typing of Treponema pallidum subsp. pallidum present in clinical samples from France: Infecting treponemes are genetically diverse and belong to 18 allelic profiles.

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.

Syphilis-causing strains belong to separate SS14-like or Nichols-like groups as defined by multilocus analysis of 19 Treponema pallidum strains.

Treponema pallidum strains are closely related at the genome level but cause distinct diseases. Subspecies pallidum (TPA) is the causative agent of syphilis, subspecies pertenue (TPE) causes yaws while subspecies endemicum (TEN) causes bejel (endemic syphilis). Compared to the majority of treponemal genomic regions, several chromosomal loci were found to be more diverse. To assess genetic variability in diverse genomic positions, we have selected (based on published genomic data) and sequenced five variable loci, TP0304, TP0346, TP0488, TP0515 and TP0558, in 19 reference Treponema pallidum strains including all T. pallidum subspecies (TPA, TPE and TEN). Results of this multilocus analysis divided syphilitic isolates into two groups: SS14-like and Nichols-like. The SS14-like group is comprised of SS14, Grady, Mexico A and Philadelphia 1 strains. The Nichols-like group consisted of strains Nichols, Bal 73-1, DAL-1, MN-3, Philadelphia 2, Haiti B and Madras. The TP0558 locus was selected for further studies because it clearly distinguished between the SS14- and Nichols-like groups and because the phylogenetic tree derived from the TP0558 locus showed the same clustering pattern as the tree constructed from whole genome sequences. In addition, TP0558 was shown as the only tested locus that evolved under negative selection within TPA strains. Sequencing of a short fragment (573bp) of the TP0558 locus in a set of 25 clinical isolates from 22 patients collected in the Czech Republic during 2012-2013 revealed that clinical isolates follow the SS14- and Nichols-like distribution.

Resequencing of Treponema pallidum ssp. pallidum strains Nichols and SS14: correction of sequencing errors resulted in increased separation of syphilis treponeme subclusters.

BACKGROUND: Treponema pallidum ssp. pallidum (TPA), the causative agent of syphilis, is a highly clonal bacterium showing minimal genetic variability in the genome sequence of individual strains. Nevertheless, genetically characterized syphilis strains can be clearly divided into two groups, Nichols-like strains and SS14-like strains. TPA Nichols and SS14 strains were completely sequenced in 1998 and 2008, respectively. Since publication of their complete genome sequences, a number of sequencing errors in each genome have been reported. Therefore, we have resequenced TPA Nichols and SS14 strains using next-generation sequencing techniques. METHODOLOGY/PRINCIPAL FINDINGS: The genomes of TPA strains Nichols and SS14 were resequenced using the 454 and Illumina sequencing methods that have a combined average coverage higher than 90x. In the TPA strain Nichols genome, 134 errors were identified (25 substitutions and 109 indels), and 102 of them affected protein sequences. In the TPA SS14 genome, a total of 191 errors were identified (85 substitutions and 106 indels) and 136 of them affected protein sequences. A set of new intrastrain heterogenic regions in the TPA SS14 genome were identified including the tprD gene, where both tprD and tprD2 alleles were found. The resequenced genomes of both TPA Nichols and SS14 strains clustered more closely with related strains (i.e. strains belonging to same syphilis treponeme subcluster). At the same time, groups of Nichols-like and SS14-like strains were found to be more distantly related. CONCLUSION/SIGNIFICANCE: We identified errors in 11.5% of all annotated genes and, after correction, we found a significant impact on the predicted proteomes of both Nichols and SS14 strains. Corrections of these errors resulted in protein elongations, truncations, fusions and indels in more than 11% of all annotated proteins. Moreover, it became more evident that syphilis is caused by treponemes belonging to two separate genetic subclusters.

Comparison of CDC and sequence-based molecular typing of syphilis treponemes: tpr and arp loci are variable in multiple samples from the same patient.

BACKGROUND: Molecular typing of syphilis-causing strains provides important epidemiologic data. We tested whether identified molecular subtypes were identical in PCR-positive parallel samples taken from the same patient at a same time. We also tested whether subtype prevalence differs in skin and blood samples. RESULTS: Eighteen syphilis positive patients (showing both positive serology and PCR), with two PCR-typeable parallel samples taken at the same time, were tested with both CDC (Centers for Disease Control and Prevention) and sequence-based typing. Samples taken from 9 of 18 patients were completely typed for TP0136, TP0548, 23S rDNA, arp, and tpr loci. The CDC typing revealed 11 distinct genotypes while the sequence-based typing identified 6 genotypes. When results from molecular typing of TP0136, TP0548, and 23S rDNA were analyzed in samples taken from the same patient, no discrepancies in the identified genotypes were found; however, there were discrepancies in 11 of 18 patients (61.1%) samples relative to the arp and tpr loci. In addition to the above described typing, 127 PCR-positive swabs and whole blood samples were tested for individual genotype frequencies. The repetition number for the arp gene was lower in whole blood (WB) samples compared to swab samples. Similarly, the most common tpr RFLP type "d" was found to have lower occurrence rates in WB samples while type "e" had an increased occurrence in these samples. CONCLUSIONS: Differences in the CDC subtypes identified in parallel samples indicated genetic instability of the arp and tpr loci and suggested limited applicability of the CDC typing system in epidemiological studies. Differences in treponemal genotypes detected in whole blood and swab samples suggested important differences between both compartments and/or differences in adherence of treponeme variants to human cells.

Whole genome sequence of the Treponema Fribourg-Blanc: unspecified simian isolate is highly similar to the yaws subspecies.

BACKGROUND: Unclassified simian strain Treponema Fribourg-Blanc was isolated in 1966 from baboons (Papio cynocephalus) in West Africa. This strain was morphologically indistinguishable from T. pallidum ssp. pallidum or ssp. pertenue strains, and it was shown to cause human infections. METHODOLOGY/PRINCIPAL FINDINGS: To precisely define genetic differences between Treponema Fribourg-Blanc (unclassified simian isolate, FB) and T. pallidum ssp. pertenue strains (TPE), a high quality sequence of the whole Fribourg-Blanc genome was determined with 454-pyrosequencing and Illumina sequencing platforms. Combined average coverage of both methods was greater than 500×. Restriction target sites (n = 1,773), identified in silico, of selected restriction enzymes within the Fribourg-Blanc genome were verified experimentally and no discrepancies were found. When compared to the other three sequenced TPE genomes (Samoa D, CDC-2, Gauthier), no major genome rearrangements were found. The Fribourg-Blanc genome clustered with other TPE strains (especially with the TPE CDC-2 strain), while T. pallidum ssp. pallidum strains clustered separately as well as the genome of T. paraluiscuniculi strain Cuniculi A. Within coding regions, 6 deletions, 5 insertions and 117 substitutions differentiated Fribourg-Blanc from other TPE genomes. CONCLUSIONS/SIGNIFICANCE: The Fribourg-Blanc genome showed similar genetic characteristics as other TPE strains. Therefore, we propose to rename the unclassified simian isolate to Treponema pallidum ssp. pertenue strain Fribourg-Blanc. Since the Fribourg-Blanc strain was shown to cause experimental infection in human hosts, non-human primates could serve as possible reservoirs of TPE strains. This could considerably complicate recent efforts to eradicate yaws. Genetic differences specific for Fribourg-Blanc could then contribute for identification of cases of animal-derived yaws infections.

Structure of rrn operons in pathogenic non-cultivable treponemes: sequence but not genomic position of intergenic spacers correlates with classification of Treponema pallidum and Treponema paraluiscuniculi strains.

This study examined the sequences of the two rRNA (rrn) operons of pathogenic non-cultivable treponemes, comprising 11 strains of T. pallidum ssp. pallidum (TPA), five strains of T. pallidum ssp. pertenue (TPE), two strains of T. pallidum ssp. endemicum (TEN), a simian Fribourg-Blanc strain and a rabbit T. paraluiscuniculi (TPc) strain. PCR was used to determine the type of 16S-23S ribosomal intergenic spacers in the rrn operons from 30 clinical samples belonging to five different genotypes. When compared with the TPA strains, TPc Cuniculi A strain had a 17 bp deletion, and the TPE, TEN and Fribourg-Blanc isolates had a deletion of 33 bp. Other than these deletions, only 17 heterogeneous sites were found within the entire region (excluding the 16S-23S intergenic spacer region encoding tRNA-Ile or tRNA-Ala). The pattern of nucleotide changes in the rrn operons corresponded to the classification of treponemal strains, whilst two different rrn spacer patterns (Ile/Ala and Ala/Ile) appeared to be distributed randomly across species/subspecies classification, time and geographical source of the treponemal strains. It is suggested that the random distribution of tRNA genes is caused by reciprocal translocation between repetitive sequences mediated by a recBCD-like system.

Whole genome sequence of Treponema pallidum ssp. pallidum, strain Mexico A, suggests recombination between yaws and syphilis strains.

BACKGROUND: Treponema pallidum ssp. pallidum (TPA), the causative agent of syphilis, and Treponema pallidum ssp. pertenue (TPE), the causative agent of yaws, are closely related spirochetes causing diseases with distinct clinical manifestations. The TPA Mexico A strain was isolated in 1953 from male, with primary syphilis, living in Mexico. Attempts to cultivate TPA Mexico A strain under in vitro conditions have revealed lower growth potential compared to other tested TPA strains. METHODOLOGY/PRINCIPAL FINDINGS: The complete genome sequence of the TPA Mexico A strain was determined using the Illumina sequencing technique. The genome sequence assembly was verified using the whole genome fingerprinting technique and the final sequence was annotated. The genome size of the Mexico A strain was determined to be 1,140,038 bp with 1,035 predicted ORFs. The Mexico A genome sequence was compared to the whole genome sequences of three TPA (Nichols, SS14 and Chicago) and three TPE (CDC-2, Samoa D and Gauthier) strains. No large rearrangements in the Mexico A genome were found and the identified nucleotide changes occurred most frequently in genes encoding putative virulence factors. Nevertheless, the genome of the Mexico A strain, revealed two genes (TPAMA_0326 (tp92) and TPAMA_0488 (mcp2-1)) which combine TPA- and TPE- specific nucleotide sequences. Both genes were found to be under positive selection within TPA strains and also between TPA and TPE strains. CONCLUSIONS/SIGNIFICANCE: The observed mosaic character of the TPAMA_0326 and TPAMA_0488 loci is likely a result of inter-strain recombination between TPA and TPE strains during simultaneous infection of a single host suggesting horizontal gene transfer between treponemal subspecies.

Sequencing-based molecular typing of treponema pallidum strains in the Czech Republic: all identified genotypes are related to the sequence of the SS14 strain.

A set of 415 clinical samples isolated from 294 patients suspected of having syphilis collected in the Czech Republic between 2004 and 2010 was tested for the presence of treponemal DNA. Standard serological tests showed that 197 patients were syphilis-seropositive and 97 patients were syphilis-seronegative. In each sample, PCR tests for polA (TP0105), tmpC (TP0319), TP0136, TP0548 and 23S rRNA genes were performed. Samples taken from 91 patients were PCR-positive. Molecular typing of treponemal DNA was based on the sequencing of TP0136, TP0548 and 23S rRNA genes. Treponemal DNA was typeable in samples taken from 64 PCR-positive patients and 9 different genotypes were found. The proportion of treponemal strains resistant to macrolide antibiotics was 37.3%. In the DNA samples taken from 39 patients, a parallel treponemal typing approved by Centers for Disease Control and Prevention was performed. The variants of arp and tpr genes appear to combine independently with sequence variants of TP0136, TP0548 and 23S rRNA genes.