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.

• Klíčová slova
• MinION sequencing, OMPeome, Treponema pallidum, genetic epidemiology, long-read sequencing, outer membrane proteins, syphilis,
• MeSH
• alely MeSH
• DNA bakterií genetika   MeSH
• genetická variace * MeSH
• lidé MeSH
• multilokusová sekvenční typizace MeSH
• proteiny vnější bakteriální membrány * genetika   MeSH
• sekvenční analýza DNA MeSH
• syfilis * mikrobiologie   MeSH
• Treponema pallidum * genetika   klasifikace   izolace a purifikace   MeSH
• Treponema MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• DNA bakterií MeSH
• proteiny vnější bakteriální membrány * 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.

• Klíčová slova
• MLST, T. pallidum, epidemiology, typing,
• Publikační typ
• časopisecké články MeSH
• preprinty 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
• frambézie * přenos   mikrobiologie   veterinární   epidemiologie   MeSH
• fylogeneze MeSH
• genom bakteriální * MeSH
• primáti * mikrobiologie   MeSH
• sekvenování celého genomu MeSH
• Treponema pallidum * genetika   izolace a purifikace   klasifikace   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Geografické názvy
• Tanzanie epidemiologie   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
• fylogeneze * MeSH
• genom bakteriální MeSH
• králíci MeSH
• syfilis * mikrobiologie   MeSH
• Treponema * genetika   izolace a purifikace   MeSH
• zajíci * mikrobiologie   MeSH
• zvířata MeSH
• Check Tag
• králíci MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Haemophilus ducreyi (HD) is an important cause of cutaneous ulcers in several endemic regions, including the Western Pacific Region, especially among children. An HD sequence typing on swab samples taken from 1,081 ulcers in the Namatanai district of Papua New Guinea, during the pilot study for treatment of yaws, has been performed using the Grant typing system. Of the 363 samples that tested positive for the 16S rDNA of HD, the dsrA sequences of 270 samples were determined. Altogether they revealed 8 HD strain types circulating in Namatanai, including seven strain types of Class I (I.3, I.4, I.5, I.9, I.10, I.11, I.12) and one strain of Class II (II.3); four Class I types (I.9, I.10, I.11, I.12) were novel. The southern region of Namatanai (Matalai Rural) was identified as the region with the lowest genotype diversity and with most infections caused by HD Class II. The middle and northern subdistricts were affected mainly by HD Class I. Analysis of patient characteristics revealed that Class II HD infections were more often represented by longer-lasting ulcers than Class I HD infections. An increase in the prevalence of the I.10 strain was found after azithromycin administration compared to the untreated population at baseline likely reflecting higher infectivity of HD Class I, and more specifically strain type I.10.

• MeSH
• antibakteriální látky * terapeutické užití   farmakologie   MeSH
• azithromycin * terapeutické užití   MeSH
• dítě MeSH
• DNA bakterií genetika   MeSH
• dospělí MeSH
• frambézie mikrobiologie   epidemiologie   farmakoterapie   MeSH
• fylogeneze MeSH
• genotyp * MeSH
• Haemophilus ducreyi * genetika   izolace a purifikace   účinky léků   MeSH
• lidé středního věku MeSH
• lidé MeSH
• měkký vřed * mikrobiologie   epidemiologie   farmakoterapie   MeSH
• mladiství MeSH
• mladý dospělý MeSH
• pilotní projekty MeSH
• předškolní dítě MeSH
• RNA ribozomální 16S genetika   MeSH
• sekvenční analýza DNA MeSH
• Check Tag
• dítě MeSH
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mladiství MeSH
• mladý dospělý MeSH
• mužské pohlaví MeSH
• předškolní dítě MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• Geografické názvy
• Papua Nová Guinea epidemiologie   MeSH
• Názvy látek
• antibakteriální látky * MeSH
• azithromycin * MeSH
• DNA bakterií MeSH
• RNA ribozomální 16S MeSH

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
• dítě MeSH
• frambézie * epidemiologie   MeSH
• genotyp MeSH
• lidé MeSH
• multilokusová sekvenční typizace MeSH
• mutace MeSH
• Treponema pallidum * genetika   MeSH
• Treponema genetika   MeSH
• vřed MeSH
• Check Tag
• dítě MeSH
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Geografické názvy
• Papua Nová Guinea epidemiologie   MeSH

BACKGROUND: Treponema pallidum subsp. pertenue (TPE) is the causative agent of human yaws. Yaws is currently reported in 13 endemic countries in Africa, southern Asia, and the Pacific region. During the mid-20th century, a first yaws eradication effort resulted in a global 95% drop in yaws prevalence. The lack of continued surveillance has led to the resurgence of yaws. The disease was believed to have no animal reservoirs, which supported the development of a currently ongoing second yaws eradication campaign. Concomitantly, genetic evidence started to show that TPE strains naturally infect nonhuman primates (NHPs) in sub-Saharan Africa. In our current study we tested hypothesis that NHP- and human-infecting TPE strains differ in the previously unknown parts of the genomes. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we determined complete (finished) genomes of ten TPE isolates that originated from NHPs and compared them to TPE whole-genome sequences from human yaws patients. We performed an in-depth analysis of TPE genomes to determine if any consistent genomic differences are present between TPE genomes of human and NHP origin. We were able to resolve previously undetermined TPE chromosomal regions (sequencing gaps) that prevented us from making a conclusion regarding the sequence identity of TPE genomes from NHPs and humans. The comparison among finished genome sequences revealed no consistent differences between human and NHP TPE genomes. CONCLUSION/SIGNIFICANCE: Our data show that NHPs are infected with strains that are not only similar to the strains infecting humans but are genomically indistinguishable from them. Although interspecies transmission in NHPs is a rare event and evidence for current spillover events is missing, the existence of the yaws bacterium in NHPs is demonstrated. While the low risk of spillover supports the current yaws treatment campaign, it is of importance to continue yaws surveillance in areas where NHPs are naturally infected with TPE even if yaws is successfully eliminated in humans.

• MeSH
• Bacteria MeSH
• frambézie * epidemiologie   MeSH
• lidé MeSH
• primáti MeSH
• Treponema genetika   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Bejel (endemic syphilis) is a neglected non-venereal disease caused by Treponema pallidum subsp. endemicum (TEN). Although it is mostly present in hot, dry climates, a few cases have been found outside of these areas. The aim of this work was the sequencing and analysis of TEN isolates obtained from "syphilis patients" in Cuba, which is not considered an endemic area for bejel. Genomes were obtained by pool segment genome sequencing or direct sequencing methods, and the bioinformatics analysis was performed according to an established pipeline. We obtained four genomes with 100%, 81.7%, 52.6%, and 21.1% breadth of coverage, respectively. The sequenced genomes revealed a non-clonal character, with nucleotide variability ranging between 0.2-10.3 nucleotide substitutions per 100 kbp among the TEN isolates. Nucleotide changes affected 27 genes, and the analysis of the completely sequenced genome also showed a recombination event between tprC and tprI, in TP0488 as well as in the intergenic region between TP0127-TP0129. Despite limitations in the quality of samples affecting breadth of sequencing coverage, the determined non-clonal character of the isolates suggests a persistent infection in the Cuban population rather than a single outbreak caused by imported case.

• MeSH
• epidemický výskyt choroby MeSH
• infekce bakteriemi rodu Treponema * epidemiologie   MeSH
• lidé MeSH
• nukleotidy MeSH
• syfilis * epidemiologie   MeSH
• Treponema pallidum genetika   MeSH
• Treponema MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• nukleotidy MeSH

The incidence of syphilis has risen worldwide in the last decade in spite of being an easily treated infection. The causative agent of this sexually transmitted disease is the bacterium Treponema pallidum subspecies pallidum (TPA), very closely related to subsp. pertenue (TPE) and endemicum (TEN), responsible for the human treponematoses yaws and bejel, respectively. Although much focus has been placed on the question of the spatial and temporary origins of TPA, the processes driving the evolution and epidemiological spread of TPA since its divergence from TPE and TEN are not well understood. Here, we investigate the effects of recombination and selection as forces of genetic diversity and differentiation acting during the evolution of T. pallidum subspecies. Using a custom-tailored procedure, named phylogenetic incongruence method, with 75 complete genome sequences, we found strong evidence for recombination among the T. pallidum subspecies, involving 12 genes and 21 events. In most cases, only one recombination event per gene was detected and all but one event corresponded to intersubspecies transfers, from TPE/TEN to TPA. We found a clear signal of natural selection acting on the recombinant genes, which is more intense in their recombinant regions. The phylogenetic location of the recombination events detected and the functional role of the genes with signals of positive selection suggest that these evolutionary processes had a key role in the evolution and recent expansion of the syphilis bacteria and significant implications for the selection of vaccine candidates and the design of a broadly protective syphilis vaccine.

• Klíčová slova
• genome analysis, phylogenetic congruence, recombination, selection, treponematoses,
• MeSH
• frambézie * mikrobiologie   MeSH
• fylogeneze MeSH
• infekce bakteriemi rodu Treponema * mikrobiologie   MeSH
• lidé MeSH
• syfilis * epidemiologie   mikrobiologie   MeSH
• Treponema pallidum genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH

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.

• MeSH
• lidé MeSH
• polymerázová řetězová reakce * MeSH
• retrospektivní studie MeSH
• sérologická diagnostika syfilis metody   MeSH
• syfilis krev   diagnóza   MeSH
• Treponema genetika   imunologie   izolace a purifikace   fyziologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH