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  • 1.
    Harris, Simon R.
    et al.
    Pathogen Genomics, Wellcome Trust Sanger Institute, Hinxton, United Kingdom.
    Clarke, Ian N.
    Molecular Microbiology Group, Southampton General Hospital, University Medical School, Southampton, United Kingdom.
    Seth-Smith, Helena M. B.
    Pathogen Genomics, Wellcome Trust Sanger Institute, Hinxton, United Kingdom.
    Solomon, Anthony W.
    Department of Clinical Research, Faculty of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London, United Kingdom.
    Cutcliffe, Lesley T.
    Molecular Microbiology Group, Southampton General Hospital, University Medical School, Southampton, United Kingdom.
    Marsh, Peter
    Health Protection Agency, Public Health Laboratory Southampton, Southampton General Hospital, Southampton, United Kingdom.
    Skilton, Rachel J.
    Molecular Microbiology Group, Southampton General Hospital, University Medical School, Southampton, United Kingdom.
    Holland, Martin J.
    Department of Clinical Research, Faculty of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London, United Kingdom.
    Mabey, David
    Department of Clinical Research, Faculty of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London, United Kingdom.
    Peeling, Rosanna W.
    Department of Clinical Research, Faculty of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London, United Kingdom.
    Lewis, David A.
    Department of Clinical Research, Faculty of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London, United Kingdom; Sexually Transmitted Infections Reference Centre, National Health Laboratory Service, National Institute for Communicable Diseases, Johannesburg, South Africa; Department of Internal Medicine, Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, South Africa.
    Spratt, Brian G.
    Department of Infectious Disease Epidemiology, St. Mary's Hospital Campus, Imperial College, London, United Kingdom.
    Unemo, Magnus
    Örebro University Hospital. Department of Laboratory Medicine and Clinical Microbiology, National Reference Laboratory for Pathogenic Neisseria, Örebro University Hospital, Örebro, Sweden.
    Persson, Kenneth
    Department of Laboratory Medicine, Clinical Microbiology, Malmö University Hospital, Malmö, Sweden.
    Bjartling, Carina
    Department of Obstetrics and Gynecology, Institute of Clinical Sciences, Malmö University Hospital, Malmö, Sweden.
    Brunham, Robert
    British Columbia Centre for Disease Control, Vancouver BC, Canada.
    de Vries, Henry J. C.
    Department of Dermatology, Academic Medical Centre, University of Amsterdam, Amsterdam, Netherlands; Sexually Transmitted Infections Outpatient Clinic, Infectious Diseases Cluster, Public Health Service Amsterdam, Amsterdam, Netherlands; Centre for Infection and Immunity Amsterdam, Academic Medical Centre, University of Amsterdam, Amsterdam, The Netherlands.
    Morré, Servaas A.
    Department of Medical Microbiology of Infection Prevention, Laboratory of Immunogenetics, Vrije University Medical Center, Amsterdam, Netherlands; Department of Genetics and Cell Biology, Institute of Public Health Genomics, University of Maastricht, Maastricht, Netherlands.
    Speksnijder, Arjen
    Geneeskundige en Gezondheidsdienst (GGD), Amsterdam, The Netherlands.
    Bébéar, Cecile M.
    Unité Sous Contrat (USC) Mycoplasmal and Chlamydial Infections in Humans, French National Reference Center for Chlamydial Infections, Université de Bordeaux, Bordeaux, France; USC Mycoplasmal and Chlamydial Infections in Humans, French National Reference Center for Chlamydial Infections, Institut National de la Recherche Agronomique, Bordeaux, France.
    Clerc, Maite
    Unité Sous Contrat (USC) Mycoplasmal and Chlamydial Infections in Humans, French National Reference Center for Chlamydial Infections, Université de Bordeaux, Bordeaux, France; USC Mycoplasmal and Chlamydial Infections in Humans, French National Reference Center for Chlamydial Infections, Institut National de la Recherche Agronomique, Bordeaux, France.
    de Barbeyrac, Bertille
    Unité Sous Contrat (USC) Mycoplasmal and Chlamydial Infections in Humans, French National Reference Center for Chlamydial Infections, Université de Bordeaux, Bordeaux, France; USC Mycoplasmal and Chlamydial Infections in Humans, French National Reference Center for Chlamydial Infections, Institut National de la Recherche Agronomique, Bordeaux, France.
    Parkhill, Julian
    Pathogen Genomics, Wellcome Trust Sanger Institute, Hinxton, United Kingdom.
    Thomson, Nicholas R.
    Pathogen Genomics, Wellcome Trust Sanger Institute, Hinxton, United Kingdom.
    Whole-genome analysis of diverse Chlamydia trachomatis strains identifies phylogenetic relationships masked by current clinical typing2012In: Nature Genetics, ISSN 1061-4036, E-ISSN 1546-1718, Vol. 44, no 4, p. 413-419Article in journal (Refereed)
    Abstract [en]

    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.

  • 2. Seth-Smith, Helena M. B.
    et al.
    Harris, Simon R.
    Scott, Paul
    Parmar, Surendra
    Clin Microbiol & Publ Hlth Lab, Hlth Protect Agcy, Addenbrookes Hosp, Cambridge, England.
    Marsh, Peter
    Publ Hlth England Lab Southampton, Southampton Gen Hosp, Southampton, England.
    Unemo, Magnus
    Örebro University Hospital. Natl Reference Lab Pathogen Neisseria, World Hlth Org WHO Collaborating Ctr Gonorrhoea & other STIs, Örebro University Hospital, Örebro, Sweden.
    Clarke, Ian N.
    Fac Med, Mol Microbiol Grp, Southampton Gen Hosp, Univ Southampton, Southampton, England.
    Parkhill, Julian
    Thomson, Nicholas R.
    Generating whole bacterial genome sequences of low-abundance species from complex samples with IMS-MDA2013In: Nature Protocols, ISSN 1754-2189, E-ISSN 1750-2799, Vol. 8, no 12, p. 2404-2412Article in journal (Refereed)
    Abstract [en]

    The study of bacterial populations using whole-genome sequencing is of considerable scientific and clinical interest. However, obtaining bacterial genomic information is not always trivial: the target bacteria may be difficult to culture or uncultured, and they may be found within samples containing complex mixtures of other contaminating microbes and/or host cells, from which it is very difficult to derive robust sequencing data. Here we describe our procedure to generate sufficient DNA for whole-genome sequencing from clinical samples and without the need for culture, as successfully used on the difficult-to-culture, obligate intracellular pathogen Chlamydia trachomatis. Our protocol combines immunomagnetic separation (IMS) for targeted bacterial enrichment with multiple displacement amplification (MDA) for whole-genome amplification (WGA), which is followed by high-throughput sequencing. Compared with other techniques that might be used to generate such data, IMS-MDA is an inexpensive, low-technology and highly transferable process that provides amplified genomic DNA for sequencing from target bacteria in under 5 h, with little hands-on time.

  • 3.
    Seth-Smith, Helena M. B.
    et al.
    Wellcome Trust Sanger Inst, Hinxton, England.
    Harris, Simon R.
    Wellcome Trust Sanger Inst, Hinxton, England.
    Skilton, Rachel J.
    Fac Med, Mol Microbiol Grp, Southampton Gen Hosp, Univ Southampton, Southampton, England.
    Radebe, Frans M.
    Natl Inst Communicable Dis, Ctr HIV & Sexually Transmitted Infect, National Health Laboratory Service, Johannesburg, South Africa.
    Golparian, Daniel
    Natl Reference Lab Pathogen Neisseria, WHO Collaborating Ctr Gonorrhoea & Other STIs, Örebro University Hospital, Örebro, Sweden.
    Shipitsyna, Elena
    Lab Microbiol, DO Ott Res Inst Obstet & Gynaecol, St Petersburg, Russia.
    Duy, Pham Thanh
    Scott, Paul
    Wellcome Trust Sanger Inst, Hinxton, England.
    Cutcliffe, Lesley T.
    Fac Med, Mol Microbiol Grp, Southampton Gen Hosp, Univ Southampton, Southampton, England.
    O'Neill, Colette
    Fac Med, Mol Microbiol Grp, Southampton Gen Hosp, Univ Southampton, Southampton, England.
    Parmar, Surendra
    Hlth Protect Agcy, Clin Microbiol & Publ Hlth Lab, Addenbrookes Hosp, Cambridge, England.
    Pitt, Rachel
    Sexually Transmitted Bacteria Reference Lab, Health Protection Agency, London, England.
    Baker, Stephen
    Clin Res Unit, Wellcome Trust Major Overseas List, Hosp Trop Dis, Univ Oxford, Ho Chi Minh City, Vietnam.
    Ison, Catherine A.
    Sexually Transmitted Bacteria Reference Lab, Health Protection AgencyLondon NW9 5HT, England..
    Marsh, Peter
    Publ Hlth Lab Southampton, Health Protection Agency, Southampton Gen Hosp, Southampton, England.
    Jalal, Hamid
    Health Protection Agency, Clin Microbiol & Publ Hlth Lab, Addenbrookes Hosp, Cambridge, England.
    Lewis, David A.
    Natl Inst Communicable Dis, Ctr HIV & Sexually Transmitted Infect, National Health Laboratory ServiceJohannesburg, South Africa; Fac Hlth Sci, Dept Internal Med, Univ Witwatersrand, Johannesburg, South Africa.
    Unemo, Magnus
    Örebro University Hospital. Natl Reference Lab Pathogen Neisseria, WHO Collaborating Ctr Gonorrhoea & Other STIs, Örebro University Hospital, Örebro, Sweden.
    Clarke, Ian N.
    Fac Med, Mol Microbiol Grp, Southampton Gen Hosp, Univ Southampton, Southampton, England.
    Parkhill, Julian
    Wellcome Trust Sanger Inst, Hinxton, England.
    Thomson, Nicholas R.
    Wellcome Trust Sanger Inst, Hinxton, England.
    Whole-genome sequences of Chlamydia trachomatis directly from clinical samples without culture2013In: Genome Research, ISSN 1088-9051, E-ISSN 1549-5469, Vol. 23, no 5, p. 855-866Article in journal (Refereed)
    Abstract [en]

    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.

  • 4.
    Sigar, Ira M.
    et al.
    Dept Microbiol & Immunol, Chicago Coll Osteopath Med, Midwestern Univ, Downers Grove IL, USA.
    Schripsema, Justin H.
    Dept Microbiol & Immunol, Chicago Coll Osteopath Med, Midwestern Univ, Downers Grove IL, USA.
    Wang, Yibing
    Dept Clin & Expt Sci, Mol Microbiol Grp, Fac Med, Univ Southampton, Southampton, England.
    Clarke, Ian N.
    Dept Clin & Expt Sci, Mol Microbiol Grp, Fac Med, Univ Southampton, Southampton, England.
    Cutcliffe, Lesley T.
    Dept Clin & Expt Sci, Mol Microbiol Grp, Fac Med, Univ Southampton, Southampton, England.
    Seth-Smith, Helena M. B.
    Wellcome Trust Sanger Inst, Hinxton, England.
    Thomson, Nicholas R.
    Wellcome Trust Sanger Inst, Hinxton, England.
    Bjartling, Carina
    Dept Obstet & Gynecol, Malmö Univ Hosp, Malmö, Sweden.
    Unemo, Magnus
    Örebro University Hospital. Dept Lab Med, Örebro University Hospital, Örebro, Sweden.
    Persson, Kenneth
    Dept Lab Med, Malmö Univ Hosp, Malmö, Sweden.
    Ramsey, Kyle H.
    Dept Microbiol & Immunol, Chicago Coll Osteopath Med, Midwestern Univ, Downers Grove IL, USA.
    Plasmid deficiency in urogenital isolates of Chlamydia trachomatis reduces infectivity and virulence in a mouse model2014In: Pathogens and Disease, E-ISSN 2049-632X, Vol. 70, no 1, p. 61-69Article in journal (Refereed)
    Abstract [en]

    We hypothesized that the plasmid of urogenital isolates of Chlamydia trachomatis would modulate infectivity and virulence in a mouse model. To test this hypothesis, we infected female mice in the respiratory or urogenital tract with graded doses of a human urogenital isolate of C.trachomatis, serovar F, possessing the cognate plasmid. For comparison, we inoculated mice with a plasmid-free serovar F isolate. Following urogenital inoculation, the plasmid-free isolate displayed significantly reduced infectivity compared with the wild-type strain with the latter yielding a 17-fold lower infectious dose to yield 50% infection. When inoculated via the respiratory tract, the plasmid-free isolate exhibited reduced infectivity and virulence (as measured by weight change) when compared to the wild-type isolate. Further, differences in infectivity, but not in virulence were observed in a C.trachomatis, serovar E isolate with a deletion within the plasmid coding sequence 1 when compared to a serovar E isolate with no mutations in the plasmid. We conclude that plasmid loss reduces virulence and infectivity in this mouse model. These findings further support a role for the chlamydial plasmid in infectivity and virulence in vivo.

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