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.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Treponema pallidum subsp. endemicum (TEN) is the causative agent of endemic syphilis (bejel). An unusual human TEN 11q/j isolate was obtained from a syphilis-like primary genital lesion from a ...patient that returned to France from Pakistan.
The TEN 11q/j isolate was characterized using nested PCR followed by Sanger sequencing and/or direct Illumina sequencing. Altogether, 44 chromosomal regions were analyzed. Overall, the 11q/j isolate clustered with TEN strains Bosnia A and Iraq B as expected from previous TEN classification of the 11q/j isolate. However, the 11q/j sequence in a 505 bp-long region at the TP0488 locus was similar to Treponema pallidum subsp. pallidum (TPA) strains, but not to TEN Bosnia A and Iraq B sequences, suggesting a recombination event at this locus. Similarly, the 11q/j sequence in a 613 bp-long region at the TP0548 locus was similar to Treponema pallidum subsp. pertenue (TPE) strains, but not to TEN sequences.
A detailed analysis of two recombinant loci found in the 11q/j clinical isolate revealed that the recombination event occurred just once, in the TP0488, with the donor sequence originating from a TPA strain. Since TEN Bosnia A and Iraq B were found to contain TPA-like sequences at the TP0548 locus, the recombination at TP0548 took place in a treponeme that was an ancestor to both TEN Bosnia A and Iraq B. The sequence of 11q/j isolate in TP0548 represents an ancestral TEN sequence that is similar to yaws-causing treponemes. In addition to the importance of the 11q/j isolate for reconstruction of the TEN phylogeny, this case emphasizes the possible role of TEN strains in development of syphilis-like lesions.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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.
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.
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.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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%.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Centers for Disease Control and Prevention and sequencingbased treponeme typing was used to analyze 72 blood samples, collected from human immunodeficiency virus and syphilis co-infected patients ...during 2014 to 2015 in Antwerp, Belgium. Twenty-nine (40.3%) isolates were polymerase chain reaction positive for Treponema pallidum, and all tested were macrolide-resistant. Four genotypes were identified by sequencing-based typing including two new genotypes, U4NR8 and SU9R8, whereas enhanced Centers for Disease Control and Prevention typing revealed 7 subtypes.
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.
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.
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.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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