Culture-independent sequence analysis of Chlamydia trachomatis in urogenital specimens identifies regions of recombination and in-patient sequence mutations
A culture-independent genome sequencing approach was developed and used to examine genomic variability in Chlamydia trachomatis-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 ompA 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 C. trachomatis recombinants generated in vitro. These data demonstrated that culture-independent sequence analysis can be used to rapidly and inexpensively collect genome data from patients infected by C. trachomatis, and that this approach can be used to examine genomic variation within this species.
BruenT. C., PhilippeH., BryantD.(2006). A simple and robust statistical test for detecting the presence of recombination. Genetics 172:2665–2681 [View Article][PubMed]
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 Rep 60:370–373[PubMed]
DuganJ., RockeyD. D., JonesL., AndersenA. A.(2004). Tetracycline resistance in Chlamydia suis mediated by genomic islands inserted into the chlamydial inv-like gene. Antimicrob Agents Chemother 48:3989–3995 [View Article][PubMed]
GomesJ. P., BrunoW. J., BorregoM. J., DeanD.(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 Bacteriol 186:4295–4306 [View Article][PubMed]
HarrisS. R., ClarkeI. N., Seth-SmithH. M., SolomonA. W., CutcliffeL. T., MarshP., SkiltonR. J., HollandM. J., MabeyD.& other authors (2012). Whole-genome analysis of diverse Chlamydia trachomatis strains identifies phylogenetic relationships masked by current clinical typing. Nat Genet 44:413–419, S1 [View Article][PubMed]
JeckW. R., ReinhardtJ. A., BaltrusD. A., HickenbothamM. T., MagriniV., MardisE. R., DanglJ. L., JonesC. D.(2007). Extending assembly of short DNA sequences to handle error. Bioinformatics 23:2942–2944 [View Article][PubMed]
JeffreyB. M., SuchlandR. J., QuinnK. L., DavidsonJ. R., StammW. E., RockeyD. D.(2010). Genome sequencing of recent clinical Chlamydia trachomatis strains identifies loci associated with tissue tropism and regions of apparent recombination. Infect Immun 78:2544–2553 [View Article][PubMed]
JeffreyB. M., SuchlandR. J., EriksenS. G., SandozK. M., RockeyD. D.(2013). Genomic and phenotypic characterization of in vitro-generated Chlamydia trachomatis recombinants. BMC Microbiol 13:142 [View Article][PubMed]
JosephS. J., DidelotX., GandhiK., DeanD., ReadT. D.(2011). Interplay of recombination and selection in the genomes of Chlamydia trachomatis. Biol Direct 6:28 [View Article][PubMed]
KatohK., MisawaK., KumaK., MiyataT.(2002).mafft: a novel method for rapid multiple sequence alignment based on fast Fourier transform. Nucleic Acids Res 30:3059–3066 [View Article][PubMed]
LiH., RuanJ., DurbinR.(2008). Mapping short DNA sequencing reads and calling variants using mapping quality scores. Genome Res 18:1851–1858 [View Article][PubMed]
Seth-SmithH. M., HarrisS. R., SkiltonR. J., RadebeF. M., GolparianD., ShipitsynaE., DuyP. T., ScottP., CutcliffeL. T.& other authors (2013). Whole-genome sequences of Chlamydia trachomatis directly from clinical samples without culture. Genome Res 23:855–866 [View Article][PubMed]
SomboonnaN., WanR., OjciusD. M., PettengillM. A., JosephS. J., ChangA., HsuR., ReadT. D., DeanD.(2011). Hypervirulent Chlamydia trachomatis clinical strain is a recombinant between lymphogranuloma venereum (L(2)) and D lineages. MBio 2:e00045-11 [View Article][PubMed]
SrinivasanT., BrunoW. J., WanR., YenA., DuongJ., DeanD.(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 Bacteriol 194:617–626 [View Article][PubMed]
SturdevantG. L., KariL., GardnerD. J., Olivares-ZavaletaN., RandallL. B., WhitmireW. M., CarlsonJ. H., GoheenM. M., SelleckE. 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 Immun 78:3660–3668 [View Article][PubMed]
SuchlandR. J., RockeyD. D., BannantineJ. P., StammW. E.(2000). Isolates of Chlamydia trachomatis that occupy nonfusogenic inclusions lack IncA, a protein localized to the inclusion membrane. Infect Immun 68:360–367 [View Article][PubMed]
SuchlandR. J., EckertL. O., HawesS. E., StammW. E.(2003). Longitudinal assessment of infecting serovars of Chlamydia trachomatis in Seattle public health clinics: 1988–1996. Sex Transm Dis 30:357–361 [View Article][PubMed]
SuchlandR. J., JeffreyB. M., XiaM., BhatiaA., ChuH. G., RockeyD. D., StammW. E.(2008). Identification of concomitant infection with Chlamydia trachomatis IncA-negative mutant and wild-type strains by genomic, transcriptional, and biological characterizations. Infect Immun 76:5438–5446 [View Article][PubMed]
WangY., KahaneS., CutcliffeL. T., SkiltonR. J., LambdenP. R., ClarkeI. N.(2011). Development of a transformation system for Chlamydia trachomatis: restoration of glycogen biosynthesis by acquisition of a plasmid shuttle vector. PLoS Pathog 7:e1002258 [View Article][PubMed]
Culture-independent sequence analysis of Chlamydia trachomatis in urogenital specimens identifies regions of recombination and in-patient sequence mutations