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

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
• lidé MeSH
• syfilis * mikrobiologie   MeSH
• Treponema pallidum * růst a vývoj   genetika   klasifikace   izolace a purifikace   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články 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.

• Publikační typ
• časopisecké články 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

Syphilis is a multistage sexually transmitted disease caused by Treponema pallidum ssp. pallidum. In the Czech Republic, there are around 700-800 new syphilis cases annually, continuously increasing since 2012. This study analyzed a total of 1228 samples from 2004 to 2022. Of the PCR-positive typeable samples (n = 415), 68.7% were fully-typed (FT), and 31.3% were partially-typed. Most of the identified isolates belonged to the SS14-clade and only 6.3% were the Nichols-like cluster. While in the beginning of sample collection isolates have been macrolide-susceptible, recent isolates are completely resistant to macrolides. Among the FT samples, 34 different allelic profiles (APs) were found. Most of the profiles (n = 27) appeared just once in the Czech population, while seven profiles were detected more than twice. The most frequent APs belonged to two separate groups of SS14-like isolates, including group of 1.3.1 (ST 1) and 1.26.1 (ST 25) profiles, and the second group containing 1.1.8 (ST 3), 1.1.1 (ST 2), and 1.1.3 (ST 11) (representing 57.5%, and 25.3% of all detected APs, respectively). Both groups consistently differed in 6 nucleotide positions in five genes (TP0150, TP0324, TP0515, TP0548, and TP0691) coding amino-acid replacements suggesting that one or more of these differences could be involved in the higher success of the first group.

• MeSH
• alely * MeSH
• dospělí MeSH
• genotyp MeSH
• lidé středního věku MeSH
• lidé MeSH
• makrolidy farmakologie   MeSH
• multilokusová sekvenční typizace * MeSH
• syfilis * mikrobiologie   epidemiologie   genetika   MeSH
• Treponema pallidum * genetika   izolace a purifikace   MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• Geografické názvy
• Česká republika MeSH
• Názvy látek
• makrolidy 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
• králíci MeSH
• syfilis mikrobiologie   patologie   MeSH
• testis mikrobiologie   metabolismus   MeSH
• Treponema pallidum * MeSH
• zvířata MeSH
• Check Tag
• králíci MeSH
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články 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.

• Klíčová slova
• European brown hare, Lepus, One Health, Oryctolagus, Treponema pallidum, rabbit, spirochetes, syphilis,
• MeSH
• genetická variace MeSH
• Lagomorpha * MeSH
• lidé MeSH
• prevalence MeSH
• syfilis * epidemiologie   mikrobiologie   MeSH
• Treponema pallidum MeSH
• Treponema genetika   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

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.

• Klíčová slova
• Oryctolagus cuniculus, dermatitis, in vivo propagation, penicillin, rabbit, sexually transmitted diseases, syphilis,
• Publikační typ
• časopisecké články 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
• bakteriální geny MeSH
• frambézie mikrobiologie   MeSH
• fylogeneze MeSH
• genom bakteriální MeSH
• infekce bakteriemi rodu Treponema mikrobiologie   MeSH
• proteiny vnější bakteriální membrány genetika   MeSH
• sekvenování celého genomu MeSH
• syfilis mikrobiologie   MeSH
• Treponema pallidum genetika   MeSH
• Treponema genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Geografické názvy
• Bosna a Hercegovina MeSH
• Názvy látek
• proteiny vnější bakteriální membrány MeSH