Bright New Resources for Syphilis Research: Genetically Encoded Fluorescent Tags for Treponema pallidum and Sf1Ep Cells.

The recently discovered methodologies to cultivate and genetically manipulate Treponema pallidum subsp. pallidum ( T. pallidum ) have significantly helped syphilis research, allowing the in vitro evaluation of antibiotic efficacy, performance of controlled studies to assess differential treponemal gene expression, and generation of loss-of-function mutants to evaluate the contribution of specific genetic loci to T. pallidum virulence. Building on this progress, we engineered the T. pallidum SS14 strain to express a red-shifted Green Fluorescent Protein (GFP) and Sf1Ep cells to express mCherry and blue fluorescent protein (BFP) for enhanced visualization. These new resources improve microscopy- and cell sorting-based applications for T. pallidum , better capturing the physical interaction between the host and pathogen, among other possibilities. Continued efforts to develop and share new tools and resources are required to help our overall knowledge of T. pallidum biology and syphilis pathogenesis reach that of other bacterial pathogens, including spirochetes. Graphical abstract: By employing genetic engineering, T. pallidum was modified to express GFP, and Sf1Ep cells to express mCherry on the cytoplasmic membrane and BFP in the nucleus. These new resources for syphilis research will facilitate experimental designs to better define the complex interplay between T. pallidum and the host during infection.

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.

High prevalence and genetic diversity of Treponema paraluisleporidarum isolates in European lagomorphs.

IMPORTANCE: 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.

Core genome sequencing and genotyping of Leptospira interrogans in clinical samples by target capture sequencing.

BACKGROUND: The life-threatening pathogen Leptospira interrogans is the most common agent of leptospirosis, an emerging zoonotic disease. However, little is known about the strains that are currently circulating worldwide due to the fastidious nature of the bacteria and the difficulty to isolate cultures. In addition, the paucity of bacteria in blood and other clinical samples has proven to be a considerable challenge for directly genotyping the agent of leptospirosis directly from patient material. Our understanding of the genetic diversity of strains during human infection is therefore limited. METHODS: Here, we carried out hybridization capture followed by Illumina sequencing of the core genome directly from 20 clinical samples that were PCR positive for pathogenic Leptospira to elucidate the genetic diversity of currently circulating Leptospira strains in mainland France. RESULTS: Capture with RNA probes covering the L. interrogans core genome resulted in a 72 to 13,000-fold increase in pathogen reads relative to standard sequencing without capture. Variant analysis of the genomes sequenced from the biological samples using 273 Leptospira reference genomes was then carried out to determine the genotype of the infecting strain. For samples with sufficient coverage (19/20 samples with coverage > 8×), we could unambiguously identify L. interrogans serovars Icterohaemorrhagiae and Copenhageni (14 samples), L. kirschneri serovar Grippotyphosa (4 samples), and L. interrogans serovar Pyrogenes (1 sample) as the infecting strains. CONCLUSIONS: We obtained high-quality genomic data with suitable coverage for confident core genome genotyping of the agent of leptospirosis for most of our clinical samples. The recovery of the genome of the serovars Icterohaemorrhagiae and Copenhageni directly from multiple clinical samples revealed low adaptive diversification of the core genes during human infection. The ability to generate culture-free genomic data opens new opportunities for better understanding of the epidemiology of this fastidious pathogen and pathogenesis of this neglected disease.

Whole genome sequences of Treponema pallidum subsp. endemicum isolated from Cuban patients: The non-clonal character of isolates suggests a persistent human infection rather than a single outbreak.

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.

Genetic diversity of Leptospira isolates in Lao PDR and genome analysis of an outbreak strain.

BACKGROUND: Although Southeast Asia is one of the most leptospirosis afflicted regions, little is known about the diversity and molecular epidemiology of the causative agents of this widespread and emerging zoonotic disease. METHODOLOGY/PRINCIPAL FINDINGS: We used whole genome sequencing to examine genetic variation in 75 Leptospira strains isolated from patients in the Lao PDR (Laos) between 2006 and 2017. Eleven serogroups from 4 Leptospira species and 43 cgMLST-defined clonal groups (CGs) were identified. The most prevalent CG was CG272 (n = 18, 26.8%), composed of L. interrogans serogroup Autumnalis isolates. This genotype was recovered throughout the 12-year period and was associated with deaths, and with a large outbreak in neighbouring Thailand. Genome analysis reveals that the CG272 strains form a highly clonal group of strains that have, for yet unknown reasons, recently spread in Laos and Thailand. Additionally, accessory genes clearly discriminate CG272 strains from the other Leptospira strains. CONCLUSIONS/SIGNIFICANCE: The present study reveals a high diversity of Leptospira genotypes in Laos, thus extending our current knowledge of the pan- and core-genomes of these life-threatening pathogens. Our results demonstrate that the CG272 strains belong to a unique clonal group, which probably evolved through clonal expansion following niche adaptation. Additional epidemiological studies are required to better evaluate the spread of this genotype in Southeast Asia. To further investigate the key factors driving the virulence and spread of these pathogens, more intense genomic surveillance is needed, combining detailed clinical and epidemiological data.

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.

Circulating genotypes of Leptospira in French Polynesia : An 9-year molecular epidemiology surveillance follow-up study.

BACKGROUND: Leptospirosis is a widespread zoonosis with global impact, particularly among vulnerable populations in resource-poor settings in tropical countries. Rodents have been considered to be the main reservoir of the disease; however, a wide variety of mammals can act as hosts as well. Here we examine the genetic diversity of Leptospira strains from biological samples of patients and animals in French Polynesia (FP) from 2011 to 2019. METHODOLOGY/PRINCIPAL FINDINGS: From 2011 to 2019, we have collected 444 blood samples from patients diagnosed as having leptospirosis. The limited volume of clinical material and low amount of leptospiral DNA in blood samples led us to develop a nested PCR targeting the secY locus that enabled us to amplify and sequence 244 samples (55%). In addition, 20 Leptospira strains recovered from the blood of patients from 2002 to 2011 were sequenced and fully characterized at the serogroup level and used as reference strains for the association of different phylogenetic branches with respective serogroups. The secY sequences were compared with publicly available sequences from patients and animal reservoirs in FP (n = 79). We identified rats as the main source of infection for L. borgpetersenii serogroup Ballum and L. interrogans serogroup Icterohaemorrhagiae, dogs as the main source of infection for L. interrogans serogroup Australis, and farm pigs as the main source of infection for L. interrogans serogroups Pomona or Canicola. L. interrogans was associated with the most severe infections with 10 and 5 fatal cases due to serogroups Icterohaemorrhagiae and Australis, respectively. Mortality was significantly associated with older age (p-value < 0.001). CONCLUSIONS/SIGNIFICANCE: We described the population dynamics of leptospires circulating among patients in FP, including two patients who were reinfected with unrelated Leptospira genotypes, and clarified the local role of the animal reservoirs in the transmission route of leptospirosis to humans. Routine Leptospira genotyping directly on biological samples should allow the epidemiological follow-up of circulating strains and assess the impact of control interventions on disease transmission.

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.

Core Genome Multi-locus Sequence Typing Analyses of Leptospira spp. Using the Bacterial Isolate Genome Sequence Database.

With the advent of whole-genome sequencing (WGS), comparative analysis has led to the use of core genome MLST (cgMLST) schemes for the high-resolution reproducible typing of bacterial isolates. In cgMLST, hundreds of loci are used for gene-by-gene comparisons of assembled genomes for studying the genetic diversity of clinically important pathogens. Combination of the cgMLST data and metadata of the isolates is useful for epidemiological investigations.Here we present a cgMLST scheme for the high-resolution typing of isolates from the whole Leptospira genus, enabling identification at the level of species, clades, clonal groups, and sequence types. We show several examples how the cgMLST Leptospira database, which is a publicly available web-based database, can be used for the analyses of WGS data of Leptospira isolates. This effort was undertaken in order to facilitate international collaborations and support the global surveillance of leptospirosis.

A first insight into the genomic diversity of Leptospira strains isolated from patients in Cuba.

Leptospirosis is a neglected disease causing severe infections in humans and animals. Due in part to misdiagnosis, this infectious disease results in nearly 60,000 deaths per year around the globe. This study represents the first effort to describe the diversity of pathogenic Leptospira in Cuba based on whole-genome sequencing. We have collected nineteen whole-blood samples from patients that were diagnosed as having leptospirosis between 2008 and 2012 in Cuba. In addition, we have enhanced our sample set by three historical strains that were used for the development of a human vaccine in 1990s. The Leptospira strains were grown and serotyped by the microscopic agglutination test, and the draft genomes were generated by NGS (Illumina). Subsequently, the core genomes were analyzed and compared to the genetic data available from other Caribbean islands and countries in Central America. Core genome Multi-locus Sequence Typing (cgMLST) revealed four different core genome clonal groups (cgCGs), with the highest number of samples belonging to L. interrogans, followed by L. borgpetersenii and L. kirschneri. All cgCGs that were found in Cuba have been also identified from multiple origins across the globe, except in neighbor countries and Central America. Serotyping divided the samples into the serogroups Canicola, Ballum and Pomona. The most frequent cgCGs, cgCG28, associated with serogroup Canicola, and cgCG15, associated with serogroup Ballum, have also been identified from samples isolated from dogs, rodents, and pigs; suggesting that these hosts represent the major source of human infection in Cuba. The vaccine strains did not significantly differ from the recent patient isolates. However, the increasing prevalence of samples belonging to the serogroup Ballum combined with the fact that the available vaccine in Cuba represents inactivated Leptospira belonging to serogroups other than Ballum, should be a valuable information for the National and Regional Leptospirosis Control Programs.

Strain diversity of Treponema pallidum subsp. pertenue suggests rare interspecies transmission in African nonhuman primates.

In our most recent study, we found that in Tanzania infection with Treponema pallidum (TP) subsp. pertenue (TPE) is present in four different monkey species. In order to gain information on the diversity and epidemiological spread of the infection in Tanzanian nonhuman primates (NHP), we identified two suitable candidate genes for multi-locus sequence typing (MLST). We demonstrate the functionality of the MLST system in invasively and non-invasively collected samples. While we were not able to demonstrate frequent interspecies transmission of TPE in Tanzanian monkeys, our results show a clustering of TPE strains according to geography and not host species, which is suggestive for rare transmission events between different NHP species. In addition to the geographic stability, we describe the relative temporal stability of the strains infecting NHPs and identified multi-strain infection. Differences between TPE strains of NHP and human origin are highlighted. Our results show that antibiotic resistance does not occur in Tanzanian TPE strains of NHP origin.

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.

A public database for the new MLST scheme for Treponema pallidum subsp. pallidum: surveillance and epidemiology of the causative agent of syphilis.

Treponema pallidum subsp. pallidum is the causative agent of syphilis, a sexually transmitted disease with worldwide prevalence. Several different molecular typing schemes are currently available for this pathogen. To enable population biology studies of the syphilis agent and for epidemiological surveillance at the global scale, a harmonized typing tool needs to be introduced. Recently, we published a new multi-locus sequence typing (MLST) with the potential to significantly enhance the epidemiological data in several aspects (e.g., distinguishing genetically different clades of syphilis, subtyping inside these clades, and finally, distinguishing different subspecies of non-cultivable pathogenic treponemes). In this short report, we introduce the PubMLST database for treponemal DNA data storage and for assignments of allelic profiles and sequencing types. Moreover, we have summarized epidemiological data of all treponemal strains (n = 358) with available DNA sequences in typing loci and found several association between genetic groups and characteristics of patients. This study proposes the establishment of a single MLST of T. p. pallidum and encourages researchers and public health communities to use this PubMLST database as a universal tool for molecular typing studies of the syphilis pathogen.

Multilocus Sequence Typing of Treponema pallidum subsp. pallidum in Cuba From 2012 to 2017.

BACKGROUND: The increased prevalence of syphilis in Cuba prompted us to map the circulating Treponema pallidum subsp. pallidum allelic profiles in this geographic region. METHODS: Samples were collected from 2012 to 2017, from 83 male patients with ulcers or skin lesions, and were examined using multilocus sequence typing. Additionally, we analyzed the 23S rDNA and 16S rDNA regions for the presence of possible mutations leading to macrolide and tetracycline resistance. RESULTS: Among 94% of fully typed strains, we found 7 different allelic profiles, of which 4 had not been previously described. More than 87% of patients were infected with the T. pallidum SS14-like group and only 8.2% with T. pallidum Nichols-like group. As in other countries, the 1.3.1 allelic profile (ie, SS14-like) was the most common. In addition, 1 of the newly described allelic profiles represents T. pallidum strains that arose by recombination events between members of different T. pallidum subgroups. More than 90% of patients were infected with treponemes harboring the A2058G mutation. However, we found no potential tetracycline-resistant T. pallidum mutations. CONCLUSIONS: Our results suggest that, in Cuba, tetracycline antibiotics could be used to treat syphilis in penicillin-allergic patients instead of macrolides.

Sequencing of Treponema pallidum subsp. pallidum from isolate UZ1974 using Anti-Treponemal Antibodies Enrichment: First complete whole genome sequence obtained directly from human clinical material.

Treponema pallidum subsp. pallidum (TPA) is the infectious agent of syphilis, a disease that infects more than 5 million people annually. Since TPA is an uncultivable bacterium, most of the information on TPA genetics comes from genome sequencing and molecular typing studies. This study presents the first complete TPA genome (without sequencing gaps) of clinical isolate (UZ1974), which was obtained directly from clinical material, without multiplication in rabbits. Whole genome sequencing was performed using a newly developed Anti-Treponemal Antibody Enrichment technique combined with previously reported Pooled Segment Genome Sequencing. We identified the UW074B genome, isolated from a sample previously propagated in rabbits, to be the closest relative of the UZ1974 genome and calculated the TPA mutation rate as 2.8 x 10(-10) per site per generation.