Successful isolation of Treponema pallidum strains from patients’ cryopreserved ulcer exudate using the rabbit model

Pereira, Lara; Katz, Samantha S.; Sun, Yongcheng; Mills, Patrick; Taylor, Willie; Atkins, Peter; Thurlow, Charles M.; Chi, Kai-Hua; Danavall, Damien; Cook, Nicholas; Ahmed, Tamanna; Philip, Susan; Cohen, Stéphanie; Workowski, Kimberly A.; Kersh, Ellen N.; Fakile, Yetunde; Chen, Cheng Y.; Pillay, Allan

doi:10.1371/journal.pone.0227769

Cited by 10 publications

(16 citation statements)

References 58 publications

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“…In silico variant analysis correlated with real-time PCR detection of the mutations associated with macrolide resistance in clinical isolate CDC-SF003. Further, phylogenetics revealed that this strain belonged to the SS14 lineage, which correlated with its enhanced CDC typing method (ECDCT) strain type, 4d9f, as previously reported (7). While this enrichment method yielded good results with isolates, most clinical specimens collected in this study had lower than 100 polA DNA copies/µl of T. pallidum leading us to consider an alternative method.…”

Section: Discussionsupporting

confidence: 84%

“…Molecular studies have relied primarily on T. pallidum strains propagated in rabbits or DNA amplified directly from clinical specimens, because T. pallidum cannot be grown on routine laboratory media. However, advances have been made with in vitro tissue culture and the propagation of T. pallidum in rabbits from cryopreserved genital lesion specimens, which may make routine culture directly from clinical specimens a possibility in the near future (6)(7). Despite these advances, the methods are still time-consuming and impractical for laboratory diagnosis and molecular epidemiological studies of syphilis.…”

Section: Introductionmentioning

confidence: 99%

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Selective whole genome amplification as a tool to enrich specimens with lowTreponema pallidumgenomic DNA copies for whole genome sequencing

Cm¹,

Joseph²,

et al. 2021

Preprint

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Downstream next generation sequencing of the syphilis spirochete Treponema pallidum subspecies pallidum (T. pallidum) is hindered by low bacterial loads and the overwhelming presence of background metagenomic DNA in clinical specimens. In this study, we investigated selective whole genome amplification (SWGA) utilizing Multiple Displacement Amplification (MDA) in conjunction with custom oligonucleotides with an increased specificity for the T. pallidum genome, and the capture and removal of CpG-methylated host DNA followed by MDA as enrichment methods to improve the yields of T. pallidum DNA in rabbit propagated isolates and lesion specimens from patients with primary and secondary syphilis. Sequencing was performed using the Illumina MiSeq v2 500 cycle or NovaSeq 6000 SP platform. These two enrichment methods led to 93-98% genome coverage at 5 reads/site in 5 clinical specimens from the United States and rabbit propagated isolates, containing >14 T. pallidum genomic copies/µl input for SWGA and >129 genomic copies/µl for CpG methylation capture with MDA. Variant analysis using sequencing data derived from SWGA-enriched specimens, showed that all 5 clinical strains had the A2058G mutation associated with azithromycin resistance. SWGA is a robust method that allows direct whole genome sequencing (WGS) of specimens containing very low numbers of T. pallidum, which have been challenging until now.ImportanceSyphilis is a sexually transmitted, disseminated acute and chronic infection caused by the bacterial pathogen Treponema pallidum subspecies pallidum. Primary syphilis typically presents as single or multiple mucocutaneous lesions, and if left untreated, can progress through multiple stages with varied clinical manifestations. Molecular studies rely on direct amplification of DNA sequences from clinical specimens; however, this can be impacted by inadequate samples due to disease progression or timing of patients seeking clinical care. While genotyping has provided important data on circulating strains over the past two decades, whole genome sequencing data is needed to better understand strain diversity, perform evolutionary tracing, and monitor antimicrobial resistance markers. The significance of our research is the development of a SWGA DNA enrichment method that expands the range of clinical specimens that can be directly sequenced to include samples with low numbers of T. pallidum.

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Section: Discussionsupporting

confidence: 84%

Section: Introductionmentioning

confidence: 99%

Selective whole genome amplification as a tool to enrich specimens with lowTreponema pallidumgenomic DNA copies for whole genome sequencing

Cm¹,

Joseph²,

et al. 2021

Preprint

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“…Rabbits are susceptible not only to rabbit (TPeC) but also to hare (TPeL) and human syphilis. Hence, laboratory rabbits played a fundamental role in propagation and diagnostics of human pathogenic treponemes for decades [e.g., (13,14)]. While recent advance in in vitro cultivation of TPA could lead to decreased need of animals for propagation, rabbit infection model will probably remain vital for a variety of biological experiments with treponemes (15).…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2021

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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.

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“…Surface-exposed lipoproteins of exclusively extracellular pathogen T. pallidum are essential for initial infection, dissemination to distal tissues, and chronic pathogenesis. Study of syphilis has been dependent on propagation of T. pallidum in rabbit testes and examination of orchitic and skin lesions in the rabbit model of infection (Morgan et al, 2002 ; Pereira et al, 2020 ). Recovery of these spirochetes often damages their fragile outer membrane making it difficult to unambiguously determine the localization of various proteins at the subcellular level of T. pallidum , preventing a consensus among researchers on whether a protein is located on the spirochete surface or periplasmic space.…”

Section: Introductionmentioning

confidence: 99%

Identification and Functional Assessment of the First Placental Adhesin of Treponema pallidum That May Play Critical Role in Congenital Syphilis

et al. 2020

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Syphilis is a global, re-emerging sexually transmitted infection and congenital syphilis remains a major cause of adverse pregnancy outcomes due to bacterial infection in developing nations with a high rate of fetus loss. The molecular mechanisms involved in pathogenesis of the causative agent, Treponema pallidum subsp. pallidum remain poorly understood due to the difficulties of working with this pathogen, including the inability to grow it in pure culture. To reduce the spread of syphilis, we must first increase our knowledge of the virulence factors of T. pallidum and their contribution to syphilis manifestations. Tp0954 was predicted to be a surface lipoprotein of T. pallidum. Therefore, we experimentally demonstrated that Tp0954 is indeed a surface protein and further investigated its role in mediating bacterial attachment to various mammalian host cells. We found that expression of Tp0954 in a poorly adherent, but physiologically related derivative strain of the Lyme disease causing spirochete Borrelia burgdorferi B314 strain promotes its binding to epithelial as well as non-epithelial cells including glioma and placental cell lines. We also found that Tp0954 expression facilitates binding of this strain to purified dermatan sulfate and heparin, and also that bacterial binding to mammalian cell lines is mediated by the presence of heparan sulfate and dermatan sulfate in the extracellular matrix of the specific cell lines. These results suggest that Tp0954 may be involved not only in initiating T. pallidum infection by colonizing skin epithelium, but it may also contribute to disseminated infection and colonization of distal tissues. Significantly, we found that Tp0954 promotes binding to the human placental choriocarcinoma BeWo cell line, which is of trophoblastic endocrine cell type, as well as human placental tissue sections, suggesting its role in placental colonization and possible contribution to transplacental transmission of T. pallidum. Altogether, these novel findings offer an important step toward unraveling syphilis pathogenesis, including placental colonization and T. pallidum vertical transmission from mother to fetus during pregnancy.

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Successful isolation of Treponema pallidum strains from patients’ cryopreserved ulcer exudate using the rabbit model

Cited by 10 publications

References 58 publications

Selective whole genome amplification as a tool to enrich specimens with lowTreponema pallidumgenomic DNA copies for whole genome sequencing

Selective whole genome amplification as a tool to enrich specimens with lowTreponema pallidumgenomic DNA copies for whole genome sequencing

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

Identification and Functional Assessment of the First Placental Adhesin of Treponema pallidum That May Play Critical Role in Congenital Syphilis

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