Patterns of within-host spread of Chlamydia trachomatis between vagina, endocervix and rectum revealed by comparative genomic analysis

Abstract

Introduction: Chlamydia trachomatis, a gram-negative obligate intracellular bacterium, commonly causes sexually transmitted infections (STIs). Little is known about C. trachomatis transmission within the host, which is important for understanding disease epidemiology and progression.

Methods: We used RNA-bait enrichment and whole-genome sequencing to compare rectal, vaginal and endocervical samples collected at the same time from 26 study participants who attended Fijian Ministry of Health and Medical Services clinics and tested positive for C. trachomatis at each anatomic site.

Results: The 78 C. trachomatis genomes from participants resolved into two major clades of the C. trachomatis phylogeny (the "prevalent urogenital and anorectal" clade and "non-prevalent urogenital and anorectal" clade). For 21 participants, genome sequences were almost identical in each anatomic site. For the other five participants, two distinct C. trachomatis strains were present in different sites; in two cases, the vaginal sample was a mixture of strains.

Discussion: The absence of large numbers of fixed SNPs between C. trachomatis genomes within many of the participants could indicate recent acquisition of infection prior to the clinic visit without sufficient time to accumulate significant genetic variation in different body sites. This model suggests that many C. trachomatis infections may be resolved relatively quickly in the Fijian population, possibly reflecting common prescription or over-the-counter antibiotics usage.

Keywords: Chlamydia trachomatis; chlamydiae; sexually transmitted diseases; single nucleotide polymorphisms; single variable polymorphisms.

FIGURE 1

Relative load of C. trachomatis in the vagina and rectum estimated by qPCR. The non-transformed ratio of the C. trachomatis ompA genome copy number to the beta-actin genome copy number is shown (see section “Materials and methods”). C, endocervix; R, rectum; V, vagina. The lines connect the C. trachomatis load value for the vagina to the load value for the rectum for the same woman.

FIGURE 2

Global phylogeny with clade designations. The global phylogeny of high-quality C. trachomatis Fiji genomes plus selected complete C. trachomatis reference and clinical genomes representing global diversity from the National Center for Biotechnology Information (NCBI). Sample names are < ompA genotype > - < participant ID > - < body site code, where C = endocervix, R = rectum and V = vagina >. The round tips are colored by the 4 clade designations [LGV, ocular, prevalent- urogenital and anorectal (P-UA), non-prevalent urogenital and anorectal (NP-UA)]. The first column to the right of the tree denotes the ompA genotype with code at the lower right; the second column represents the source of the genomes from NCBI or the Fijian samples.

FIGURE 3

Distribution of shared SNPs by anatomic site in the 21 Group A participants. Venn diagram shows the number of participants with fixed SNPs (or fixed in one site with intermediate frequency in the other site in brackets) compared to the reference genome. All 21 participants had shared fixed SNPs in three body sites compared to the reference (center of the Venn diagram). More extensive breakdown of numbers of SNPs by participants are shown in Supplementary Table 3.

FIGURE 4

Patterns of SNP and SNV frequency across anatomic sites for representative Group A participant #1201. (A–C) Percent reference scores versus position on reference genome for “clonal SNPs” (CSSs) by body site. The set of 5,520 CSSs were chosen to differentiate NP-UA and P-UA genetic backgrounds. Each point shows the percentage of reads that mapped with the reference allele at each CSS position. The strains from #1201 are from the NP-UA clade and therefore, at most, CSSs are close to 100% match to the reference (D) allele, which is also in the NP-UA clade. The gaps in the distribution of CSSs across the chromosome are where there were regions of low variation or high recombination. (D) Box plot of distribution of% reference for clonal SNPs by body site. The minority of the CSSs with alternative alleles (<10% of reference genome) were likely the product of recombination events that have occurred since the divergence of the strains. Notably there is an intermediate frequency of SNVs.

FIGURE 5

Patterns of SNP and SNV frequency across anatomic sites for Group B participant #32. (A–C) Percent reference scores versus position on reference genome for “clonal SNPs” (CSSs) by body site. (D) Box plot of distribution of % reference for clonal SNPs by body site. The endocervical and rectal strains were in the P-UA clade and therefore the majority of the CSSs had an alternative allele (<10% reference genome). The vaginal genome showed intermediate allele frequency across the chromosome, which was evidence of mixture between P-UA and NP-UA strains.

FIGURE 6

Patterns of SNP and SNV frequency across anatomic sites for Group B participant #1176. (A–C) Percent reference scores versus position on reference genome for “clonal SNPs” (CSSs) by body site. (D) Box plot of distribution of % reference for clonal SNPs by body site. The patterns in this participant are similar to Figure 5 in showing evidence for the vaginal strain being a mixture of P-UA and NP-UA strains.