1887

Abstract

A culture-independent genome sequencing approach was developed and used to examine genomic variability in -positive specimens that were collected from patients in the Seattle, WA, USA, area. The procedure is based on an immunomagnetic separation approach with chlamydial LPS-specific mAbs, followed by DNA purification and total DNA amplification, and subsequent Illumina-based sequence analysis. Quality of genome sequencing was independent of the total number of inclusion-forming units determined for the sample and the amount of non-chlamydial DNA in the Illumina libraries. A geographically and temporally linked clade of isolates was identified with evidence of several different regions of recombination and variable sequence types, suggesting that recombination is common within outbreaks. Culture-independent sequence analysis revealed a linkage pattern at two nucleotide positions that was unique to the genomes of isolates from patients, but not in recombinants generated . These data demonstrated that culture-independent sequence analysis can be used to rapidly and inexpensively collect genome data from patients infected by , and that this approach can be used to examine genomic variation within this species.

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2013-10-01
2020-01-20
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References

  1. Borges V., Ferreira R., Nunes A., Sousa-Uva M., Abreu M., Borrego M. J., Gomes J. P..( 2013;). Effect of long-term laboratory propagation on Chlamydia trachomatis genome dynamics. Infect Genet Evol17:23–32 [CrossRef][PubMed]
    [Google Scholar]
  2. Bruen T. C., Philippe H., Bryant D..( 2006;). A simple and robust statistical test for detecting the presence of recombination. Genetics172:2665–2681 [CrossRef][PubMed]
    [Google Scholar]
  3. Centers for Disease Control and Prevention( 2011;). CDC Grand Rounds: Chlamydia prevention: challenges and strategies for reducing disease burden and sequelae. MMWR Morb Mortal Wkly Rep60:370–373[PubMed]
    [Google Scholar]
  4. Dean D., Bruno W. J., Wan R., Gomes J. P., Devignot S., Mehari T., de Vries H. J., Morré S. A., Myers G..& other authors ( 2009;). Predicting phenotype and emerging strains among Chlamydia trachomatis infections. Emerg Infect Dis15:1385–1394 [CrossRef][PubMed]
    [Google Scholar]
  5. Dugan J., Rockey D. D., Jones L., Andersen A. A..( 2004;). Tetracycline resistance in Chlamydia suis mediated by genomic islands inserted into the chlamydial inv-like gene. Antimicrob Agents Chemother48:3989–3995 [CrossRef][PubMed]
    [Google Scholar]
  6. Eckert L. O., Suchland R. J., Hawes S. E., Stamm W. E..( 2000;). Quantitative Chlamydia trachomatis cultures: correlation of chlamydial inclusion-forming units with serovar, age, sex, and race. J Infect Dis182:540–544 [CrossRef][PubMed]
    [Google Scholar]
  7. Gomes J. P., Bruno W. J., Borrego M. J., Dean D..( 2004;). Recombination in the genome of Chlamydia trachomatis involving the polymorphic membrane protein C gene relative to ompA and evidence for horizontal gene transfer. J Bacteriol186:4295–4306 [CrossRef][PubMed]
    [Google Scholar]
  8. Gomes J. P., Bruno W. J., Nunes A., Santos N., Florindo C., Borrego M. J., Dean D..( 2007;). Evolution of Chlamydia trachomatis diversity occurs by widespread interstrain recombination involving hotspots. Genome Res17:50–60 [CrossRef][PubMed]
    [Google Scholar]
  9. Harris S. R., Clarke I. N., Seth-Smith H. M., Solomon A. W., Cutcliffe L. T., Marsh P., Skilton R. J., Holland M. J., Mabey D..& other authors ( 2012;). Whole-genome analysis of diverse Chlamydia trachomatis strains identifies phylogenetic relationships masked by current clinical typing. Nat Genet44:413–419, S1 [CrossRef][PubMed]
    [Google Scholar]
  10. Jeck W. R., Reinhardt J. A., Baltrus D. A., Hickenbotham M. T., Magrini V., Mardis E. R., Dangl J. L., Jones C. D..( 2007;). Extending assembly of short DNA sequences to handle error. Bioinformatics23:2942–2944 [CrossRef][PubMed]
    [Google Scholar]
  11. Jeffrey B. M., Suchland R. J., Quinn K. L., Davidson J. R., Stamm W. E., Rockey D. D..( 2010;). Genome sequencing of recent clinical Chlamydia trachomatis strains identifies loci associated with tissue tropism and regions of apparent recombination. Infect Immun78:2544–2553 [CrossRef][PubMed]
    [Google Scholar]
  12. Jeffrey B. M., Suchland R. J., Eriksen S. G., Sandoz K. M., Rockey D. D..( 2013;). Genomic and phenotypic characterization of in vitro-generated Chlamydia trachomatis recombinants. BMC Microbiol13:142 [CrossRef][PubMed]
    [Google Scholar]
  13. Joseph S. J., Didelot X., Gandhi K., Dean D., Read T. D..( 2011;). Interplay of recombination and selection in the genomes of Chlamydia trachomatis. Biol Direct6:28 [CrossRef][PubMed]
    [Google Scholar]
  14. Katoh K., Misawa K., Kuma K., Miyata T..( 2002;). mafft: a novel method for rapid multiple sequence alignment based on fast Fourier transform. Nucleic Acids Res30:3059–3066 [CrossRef][PubMed]
    [Google Scholar]
  15. Katoh K., Asimenos G., Toh H..( 2009;). Multiple alignment of DNA sequences with mafft. Methods Mol Biol537:39–64 [CrossRef][PubMed]
    [Google Scholar]
  16. Li H., Ruan J., Durbin R..( 2008;). Mapping short DNA sequencing reads and calling variants using mapping quality scores. Genome Res18:1851–1858 [CrossRef][PubMed]
    [Google Scholar]
  17. R Development Core Team( 2008;). R: A Language and Environment for Statistical Computing Vienna: R Foundation for Statistical Computing;
    [Google Scholar]
  18. Seth-Smith H. M., Harris S. R., Skilton R. J., Radebe F. M., Golparian D., Shipitsyna E., Duy P. T., Scott P., Cutcliffe L. T..& other authors ( 2013;). Whole-genome sequences of Chlamydia trachomatis directly from clinical samples without culture. Genome Res23:855–866 [CrossRef][PubMed]
    [Google Scholar]
  19. Somboonna N., Wan R., Ojcius D. M., Pettengill M. A., Joseph S. J., Chang A., Hsu R., Read T. D., Dean D..( 2011;). Hypervirulent Chlamydia trachomatis clinical strain is a recombinant between lymphogranuloma venereum (L(2)) and D lineages. MBio2:e00045-11 [CrossRef][PubMed]
    [Google Scholar]
  20. Srinivasan T., Bruno W. J., Wan R., Yen A., Duong J., Dean D..( 2012;). In vitro recombinants of antibiotic-resistant Chlamydia trachomatis strains have statistically more breakpoints than clinical recombinants for the same sequenced loci and exhibit selection at unexpected loci. J Bacteriol194:617–626 [CrossRef][PubMed]
    [Google Scholar]
  21. Stephens R. S., Kalman S., Lammel C., Fan J., Marathe R., Aravind L., Mitchell W., Olinger L., Tatusov R. L..& other authors ( 1998;). Genome sequence of an obligate intracellular pathogen of humans: Chlamydia trachomatis. Science282:754–759 [CrossRef][PubMed]
    [Google Scholar]
  22. Sturdevant G. L., Kari L., Gardner D. J., Olivares-Zavaleta N., Randall L. B., Whitmire W. M., Carlson J. H., Goheen M. M., Selleck E. M..& other authors ( 2010;). Frameshift mutations in a single novel virulence factor alter the in vivo pathogenicity of Chlamydia trachomatis for the female murine genital tract. Infect Immun78:3660–3668 [CrossRef][PubMed]
    [Google Scholar]
  23. Suchland R. J., Stamm W. E..( 1991;). Simplified microtiter cell culture method for rapid immunotyping of Chlamydia trachomatis. J Clin Microbiol29:1333–1338[PubMed]
    [Google Scholar]
  24. Suchland R. J., Rockey D. D., Bannantine J. P., Stamm W. E..( 2000;). Isolates of Chlamydia trachomatis that occupy nonfusogenic inclusions lack IncA, a protein localized to the inclusion membrane. Infect Immun68:360–367 [CrossRef][PubMed]
    [Google Scholar]
  25. Suchland R. J., Eckert L. O., Hawes S. E., Stamm W. E..( 2003;). Longitudinal assessment of infecting serovars of Chlamydia trachomatis in Seattle public health clinics: 1988–1996. Sex Transm Dis30:357–361 [CrossRef][PubMed]
    [Google Scholar]
  26. Suchland R. J., Jeffrey B. M., Xia M., Bhatia A., Chu H. G., Rockey D. D., Stamm W. E..( 2008;). Identification of concomitant infection with Chlamydia trachomatis IncA-negative mutant and wild-type strains by genomic, transcriptional, and biological characterizations. Infect Immun76:5438–5446 [CrossRef][PubMed]
    [Google Scholar]
  27. Wang Y., Kahane S., Cutcliffe L. T., Skilton R. J., Lambden P. R., Clarke I. N..( 2011;). Development of a transformation system for Chlamydia trachomatis: restoration of glycogen biosynthesis by acquisition of a plasmid shuttle vector. PLoS Pathog7:e1002258 [CrossRef][PubMed]
    [Google Scholar]
  28. Zhang Y. X., Stewart S. J., Caldwell H. D..( 1989;). Protective monoclonal antibodies to Chlamydia trachomatis serovar- and serogroup-specific major outer membrane protein determinants. Infect Immun57:636–638[PubMed]
    [Google Scholar]
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