| 1. |
- Harris, Simon R., et al.
(författare)
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Whole-genome analysis of diverse Chlamydia trachomatis strains identifies phylogenetic relationships masked by current clinical typing
- 2012
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Ingår i: Nature Genetics. - : Nature Publishing Group. - 1061-4036 .- 1546-1718. ; 44:4, s. 413-419
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Tidskriftsartikel (refereegranskat)abstract
- Chlamydia trachomatis is responsible for both trachoma and sexually transmitted infections, causing substantial morbidity and economic cost globally. Despite this, our knowledge of its population and evolutionary genetics is limited. Here we present a detailed phylogeny based on whole-genome sequencing of representative strains of C. trachomatis from both trachoma and lymphogranuloma venereum (LGV) biovars from temporally and geographically diverse sources. Our analysis shows that predicting phylogenetic structure using ompA, which is traditionally used to classify Chlamydia, is misleading because extensive recombination in this region masks any true relationships present. We show that in many instances, ompA is a chimera that can be exchanged in part or as a whole both within and between biovars. We also provide evidence for exchange of, and recombination within, the cryptic plasmid, which is another key diagnostic target. We used our phylogenetic framework to show how genetic exchange has manifested itself in ocular, urogenital and LGV C. trachomatis strains, including the epidemic LGV serotype L2b.
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| 2. |
- Seth-Smith, Helena M. B., et al.
(författare)
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Co-evolution of genomes and plasmids within Chlamydia trachomatis and the emergence in Sweden of a new variant strain
- 2009
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Ingår i: BMC Genomics. - : Springer Science and Business Media LLC. - 1471-2164. ; 10
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Tidskriftsartikel (refereegranskat)abstract
- Background: Chlamydia trachomatis is the most common cause of sexually transmitted infections globally and the leading cause of preventable blindness in the developing world. There are two biovariants of C trachomatis: 'trachoma', causing ocular and genital tract infections, and the invasive 'lymphogranuloma venereum' strains. Recently, a new variant of the genital tract C trachomatis emerged in Sweden. This variant escaped routine diagnostic tests because it carries a plasmid with a deletion. Failure to detect this strain has meant it has spread rapidly across the country provoking a worldwide alert. In addition to being a key diagnostic target, the plasmid has been linked to chlamydial virulence. Analysis of chlamydial plasmids and their cognate chromosomes was undertaken to provide insights into the evolutionary relationship between chromosome and plasmid. This is essential knowledge if the plasmid is to be continued to be relied on as a key diagnostic marker, and for an understanding of the evolution of Chlamydia trachomatis. Results: The genomes of two new C trachomatis strains were sequenced, together with plasmids from six C trachomatis isolates, including the new variant strain from Sweden. The plasmid from the new Swedish variant has a 377 bp deletion in the first predicted coding sequence, abolishing the site used for PCR detection, resulting in negative diagnosis. In addition, the variant plasmid has a 44 bp duplication downstream of the deletion. The region containing the second predicted coding sequence is the most highly conserved region of the plasmids investigated. Phylogenetic analysis of the plasmids and chromosomes are fully congruent. Moreover this analysis also shows that ocular and genital strains diverged from a common C trachomatis progenitor. Conclusion: The evolutionary pathways of the chlamydial genome and plasmid imply that inheritance of the plasmid is tightly linked with its cognate chromosome. These data suggest that the plasmid is not a highly mobile genetic element and does not transfer readily between isolates. Comparative analysis of the plasmid sequences has revealed the most conserved regions that should be used to design future plasmid based nucleic acid amplification tests, to avoid diagnostic failures.
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| 3. |
- Seth-Smith, Helena M. B., et al.
(författare)
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Whole-genome sequences of Chlamydia trachomatis directly from clinical samples without culture
- 2013
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Ingår i: Genome Research. - : Cold Spring Harbor Laboratory Press (CSHL). - 1088-9051 .- 1549-5469. ; 23:5, s. 855-866
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Tidskriftsartikel (refereegranskat)abstract
- The use of whole-genome sequencing as a tool for the study of infectious bacteria is of growing clinical interest. Chlamydia trachomatis is responsible for sexually transmitted infections and the blinding disease trachoma, which affect hundreds of millions of people worldwide. Recombination is widespread within the genome of C. trachomatis, thus whole-genome sequencing is necessary to understand the evolution, diversity, and epidemiology of this pathogen. Culture of C trachomatis has, until now, been a prerequisite to obtain DNA for whole-genome sequencing; however, as C trachomatis is an obligate intracellular pathogen, this procedure is technically demanding and time consuming. Discarded clinical samples represent a large resource for sequencing the genomes of pathogens, yet clinical swabs frequently contain very low levels of C trachomatis DNA and large amounts of contaminating microbial and human DNA. To determine whether it is possible to obtain whole-genome sequences from bacteria without the need for culture, we have devised an approach that combines immunomagnetic separation (IMS) for targeted bacterial enrichment with multiple displacement amplification (MDA) for whole-genome amplification. Using IMS-MDA ill conjunction with high-throughput multiplexed Illumina sequencing, we have produced the first whole bacterial genome sequences direct from clinical samples. We also show that this method can be used to generate genome data from nonviable archived samples. This method will prove a useful tool in answering questions relating to the biology of many difficult-to-culture or fastidious bacteria of clinical concern.
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| 4. |
- Labiran, Clare, et al.
(författare)
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Genotyping markers used for multi locus VNTR analysis with ompA (MLVA-ompA) and multi sequence typing (MST) retain stability in Chlamydia trachomatis
- 2012
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Ingår i: Frontiers in Cellular and Infection Microbiology. - : Frontiers Media SA. - 2235-2988. ; 2
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Tidskriftsartikel (refereegranskat)abstract
- We aimed to evaluate the stability of the Chlamydia trachomatis multi locus VNTR analysis (MLVA-ompA) and multi sequence typing (MST) systems through multiple passages in tissue culture. Firstly, we analyzed the stability of these markers through adaptation of C. trachomatis to tissue culture and secondly, we examined the stability of a four-locus MLVA-ompA and a five-locus MST system after multiple passages in tissue culture. Marker sequences were monitored through successive chlamydial developmental cycles to evaluate the stability of the individual DNA markers through many bacterial divisions and this, in turn, informed us of the usefulness of using such typing systems for short and long-term molecular epidemiology. Southampton genitourinary medicine (GUM) clinic isolates from endocervical swabs collected from C. trachomatis positive women were passaged through tissue culture. MLVA-ompA typing was applied to primary swab samples and to the same samples after C. trachomatis had been passaged through cell culture (eight passages). Sequence data from time-zero and passage-eight isolates were aligned with reference sequences to determine the stability of the markers. The Swedish new variant (nvCT) underwent 72 passages in cell culture and the markers of the two schemes were similarly analyzed. Analysis of genetic markers of the MLVA-ompA typing system before and after the isolates were introduced to tissue culture showed no change in the dominant sequence. The nvCT that had been passaged 72 times over the duration of a year also showed no variation in the dominant sequence for both the genotyping schemes. MLVA-ompA and MST markers are stable upon adaptation of C. trachomatis to tissue culture following isolation of strains from primary endocervical swab samples. These markers remain stable throughout multiple rounds of cell-division in tissue culture, concomitant with the incubation period and appearance of symptoms normally associated with host-infection. Both genotyping schemes are, therefore, suitable for epidemiology of C. trachomatis.
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| 5. |
- Unemo, Magnus, et al.
(författare)
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The Swedish new variant of Chlamydia trachomatis: genome sequence, morphology, cell tropism and phenotypic characterization
- 2010
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Ingår i: Microbiology. - : Microbiology Society. - 1465-2080 .- 1350-0872. ; 156, s. 1394-1404
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Tidskriftsartikel (refereegranskat)abstract
- Chlamydia trachomatis is a major cause of bacterial sexually transmitted infections worldwide. In 2006, a new variant of C. trachomatis (nvCT), carrying a 377 bp deletion within the plasmid, was reported in Sweden. This deletion included the targets used by the commercial diagnostic systems from Roche and Abbott. The nvCT is clonal (serovar/genovar E) and it spread rapidly in Sweden, undiagnosed by these systems. The degree of spread may also indicate an increased biological fitness of nvCT. The aims of this study were to describe the genome of nvCT, to compare the nvCT genome to all available C. trachomatis genome sequences and to investigate the biological properties of nvCT. An early nvCT isolate (Sweden2) was analysed by genome sequencing, growth kinetics, microscopy, cell tropism assay and antimicrobial susceptibility testing. It was compared with relevant C. trachomatis isolates, including a similar serovar E C. trachomatis wild-type strain that circulated in Sweden prior to the initially undetected expansion of nvCT. The nvCT genome does not contain any major genetic polymorphisms - the genes for central metabolism, development cycle and virulence are conserved - or phenotypic characteristics that indicate any altered biological fitness. This is supported by the observations that the nvCT and wild-type C. trachomatis infections are very similar in terms of epidemiological distribution, and that differences in clinical signs are only described, in one study, in women. In conclusion, the nvCT does not appear to have any altered biological fitness. Therefore, the rapid transmission of nvCT in Sweden was due to the strong diagnostic selective advantage and its introduction into a high-frequency transmitting population.
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| 6. |
- Wang, Yibing, et al.
(författare)
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Genetic Transformation of a Clinical (Genital Tract), Plasmid-Free Isolate of Chlamydia trachomatis: Engineering the Plasmid as a Cloning Vector
- 2013
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Ingår i: PLoS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 8:3
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Tidskriftsartikel (refereegranskat)abstract
- Our study had three objectives: to extend the plasmid-based transformation protocol to a clinical isolate of C. trachomatis belonging to the trachoma biovar, to provide "proof of principle'' that it is possible to "knock out'' selected plasmid genes (retaining a replication competent plasmid) and to investigate the plasticity of the plasmid. A recently developed, plasmid-based transformation protocol for LGV isolates of C. trachomatis was modified and a plasmid-free, genital tract C. trachomatis isolate from Sweden (SWFP-) was genetically transformed. Transformation of this non-LGV C. trachomatis host required a centrifugation step, but the absence of the natural plasmid removed the need for plaque purification of transformants. Transformants expressed GFP, were penicillin resistant and iodine stain positive for accumulated glycogen. The transforming plasmid did not recombine with the host chromosome. A derivative of pGFP::SW2 carrying a deletion of the plasmid CDS5 gene was engineered. CDS5 encodes pgp3, a protein secreted from the inclusion into the cell cytoplasm. This plasmid (pCDS5KO) was used to transform C. trachomatis SWFP-, and established that pgp3 is dispensable for plasmid function. The work shows it is possible to selectively delete segments of the chlamydial plasmid, and this is the first step towards a detailed molecular dissection of the role of the plasmid. The 3.6 kb beta-galactosidase cassette was inserted into the deletion site of CDS5 to produce plasmid placZ-CDS5KO. Transformants were penicillin resistant, expressed GFP and stained for glycogen. In addition, they expressed b-galactosidase showing that the lacZ cassette was functional in C. trachomatis. An assay was developed that allowed the visualisation of individual inclusions by X-gal staining. The ability to express active beta-galactosidase within chlamydial inclusions is an important advance as it allows simple, rapid assays to measure directly chlamydial infectivity without the need for plaquing, fluorescence or antibody staining.
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| 7. |
- Wang, Yibing, et al.
(författare)
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Transformation of a plasmid-free, genital tract isolate of Chlamydia trachomatis with a plasmid vector carrying a deletion in CDS6 revealed that this gene regulates inclusion phenotype
- 2013
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Ingår i: Pathogens and Disease. - 2049-632X. ; 67:2, s. 100-103
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Tidskriftsartikel (refereegranskat)abstract
- The development of a plasmid-based genetic transformation protocol for Chlamydia trachomatis provides the basis for the detailed investigation of the function of the chlamydial plasmid and its individual genes or coding sequences (CDS). In this study we constructed a plasmid vector with CDS6 deleted (pCDS6KO) from the original Escherichia coli/C. trachomatis shuttle vector pGFP::SW2. pCDS6KO was transformed into a clinical isolate of C. trachomatis from Sweden that is plasmid-free (C. trachomatis SWFP-). Penicillin-resistant transformants expressing the green fluorescent protein were selected. These transformants did not stain with iodine, indicating that this property is regulated by CDS6 or its gene product. In addition, mature inclusions of C. trachomatis SWFP-transformed by pCDS6KO displayed an identical morphological phenotype to the untransformed plasmid-free recipient host. In this phenotype the morphology of inclusions was altered with the chlamydiae lining the periphery of the inclusion leaving a 'hole' in the centre. These green fluorescent inclusions appear 'doughnut-shaped' with an empty centre when examined under blue light, giving rise to a characteristic 'black hole' phenotype. Our study demonstrates the power of the new genetic system for investigating chlamydial gene function using gene deletion technology.
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