Chlamydia, gonorrhoea and syphilis are the three most common bacterial sexually transmitted infections (STIs) worldwide , caused by Chlamydia trachomatis, Neisseria gonorrhoeae and Treponema pallidum subsp. pallidum (T. p. pallidum), respectively. Of these infections, chlamydia is the most commonly diagnosed in the UK, USA and globally, while there has been a recent rise in the global incidence of gonorrhoea, and in syphilis cases in the UK and USA [1–3] (Table 1 [1–9]). This review focusses on how genomics can inform our understanding of these pathogens evolutionarily, clinically and epidemiologically.
Chlamydia and gonorrhoea share some aspects of their disease outcomes, as both are able to cause pelvic inflammatory disease and epithelial scarring which can lead to infertility, although they can also be asymptomatic. However, N. gonorrhoeae infections tend to be more acute, with symptoms including purulent discharge and acute local inflammation; in rare cases when the bacterium disseminates through the host, rashes and septic arthritis can occur. An invasive biovar of C. trachomatis causes lymphogranuloma venereum (LGV) which exists as a genital ulcer disease in many resource-poor locations and was seen recently as an outbreak amongst men who have sex with men (MSM) associated with proctitis. C. trachomatis is also the leading cause of infectious blindness (trachoma , which will not be discussed in detail here). Early signs of syphilis are also localized to the genital area, usually characterized by a chancre or sore, yet within a few weeks the disease can move to a secondary stage characterized by a rash on one or more areas of the body. Without treatment a tertiary stage can develop, even after a latent period of many years, resulting in damage to internal organs including the brain, nerves, and cardiovascular system, which can lead to a range of complications, neurological disorders and ultimately death. T. p. pallidum can also pass through the placenta, causing congenital syphilis leading to foetal developmental problems or stillbirth. All three bacteria have been implicated in preterm birth.
CELL BIOLOGY OF BACTERIAL SEXUALLY TRANSMITTED INFECTION PATHOGENS
C. trachomatis, N. gonorrhoeae and T. p. pallidum are all human-restricted pathogens, despite having closely related species that are less restricted in their host range. All are Gram-negative, yet belong to different bacterial phyla (Fig. 1 ) and infect the host in very different ways. C. trachomatis strains are obligate intracellular bacteria with a unique biphasic developmental cycle. Small, infectious elementary bodies are endocytosed into the host cell, sequestered into specialized inclusion vesicles where they differentiate into reticular bodies. This metabolically active form replicates until the inclusion fills the host cell, when the reticular bodies differentiate into elementary bodies which are released from the cell to continue the infection. N. gonorrhoeae are facultative intracellular bacteria, and T. p. pallidum are highly motile spirochaetes which can move between the cells to aid dissemination. All three pathogens are difficult to culture: C. trachomatis through its intracellular nature and requirement for tissue culture, N. gonorrhoeae through its fastidious nature and specific nutritional requirements , and T. p. pallidum which has not been successfully cultured in vitro and must be propagated in a rabbit host [13▪]. As these bacteria naturally only infect humans, suitable animal models of these diseases do not exist.
INFORMATION GAINED FROM GENOMIC STUDIES
Until recently, there were very few genomes of bacterial STIs available, in contrast to the number of other bacterial genomes sequenced [currently over 16 000 (http://www.genomesonline.org)]. This is despite their importance to public health: complications associated with chlamydia in the UK are estimated to cost £100 million annually (www.chlamydiascreening.nhs.uk) and some gonorrhoea strains have emerged that are resistant to all treatment antibiotics.
Genomics has helped to open a window into parts of the biology that were previously inaccessible. Particularly in the cases of C. trachomatis and T. p. pallidum, very little was known about the genetics of these organisms because of a lack of molecular tools and the inability to grow them outside cell culture or the host. All three of these STI pathogens have small genomes of 1.0–2.2 Mb, encoding only 889–2662 genes (Table 1). The genomes of C. trachomatis and T. p. pallidum appear to have undergone genome degradation, losing many of the genes required to grow independently of the host. This process is known as reductive evolution and is usually associated with specialization to a new, isolated niche with a narrow ecological spectrum. All three bacteria also contain large families of outer membrane proteins, which may be associated with immune evasion (Table 1) [4,8,9].
The first forays into C. trachomatis genomics generated reference genomes for the three disease-causing types (pathotypes): urogenital, trachoma and LGV [4,5,14]. Comparative analyses of these small genomes (1.0 Mb) found that all possess the same gene order along the chromosome (synteny). The high sequence conservation, with 846 genes in common and few or no true whole-gene differences , indicates that only subtle differences must determine the disease phenotype. Variable genes can provide clues to the differences between pathotypes: these include a cytotoxin gene within the region of the genome called the plasticity zone  which is largely degraded in all C. trachomatis genomes, but may retain some activity within urogenital strains [16,17], and genes encoding tryptophan synthase which are inactivated in trachoma strains . This enzyme converts indole, found in the genital tract, to tryptophan, which is otherwise degraded through an immune trigger [4,18,19]. Additionally, all strains possess an almost identical cryptic plasmid of 7.5 kb.
C. trachomatis genomics has recently moved towards large-scale, population-based sequencing projects, with 66 genomes now sequenced covering temporal and geographic diversity [6,20▪▪,21–24]. These studies confirm the high similarity between genomes, with only 7616 base changes or single-nucleotide polymorphisms (SNPs) separating urogenital and LGV strains (Table 1). Phylogenetic analysis of the data shows us that the LGV strains form a separate clade exhibiting low diversity, with the STI strains split into two clades separated by 5509 SNPs, and the ocular strains branching from one of these clades (Fig. 2) [20▪▪]. The most surprising finding in the analysis of these genomes was the extent of recombination apparent between strains of C. trachomatis [20▪▪]. Although full recombination machinery was identified within the first genome sequence , it was thought that the barriers for recombination were too high because of the intracellular nature of this pathogen. However, these studies have shown that recombination is rife, even between pathotypes associated with different anatomical sites, a finding which has major implications for our understanding of chlamydial biology and epidemiology.
Four genome sequences of N. gonorrhoeae (2.2 Mb) are available for analysis [7,25,26], the most divergent strains being separated by 7331 SNPs (Table 1); a further 14 genomes exist in draft form (http://www.broadinstitute.org/). N. gonorrhoeae is naturally competent and able to take up exogenous DNA  which can be incorporated and used to shuffle novel functions into its genome. Specialized 10–12 bp repeat elements called DNA uptake sequences (DUSs)  are present at high density (over 1900 copies) throughout the genome. Recipient bacteria actively recognize DUSs in free DNA, bind them and import the DNA, involving Type IV pili which are also used in bacterial motility . The canonical N. gonorrhoeae DUSs are found in closely related commensal and pathogenic Neisseria species including Neisseria meningitidis that also inhabit human mucosal surfaces, providing an ideal opportunity for genetic exchange . Indeed regions of apparent recombination have been identified in the genome of strain SM-3, associated with transfer of antibiotic resistance determinants . A range of plasmids are associated with the strains of N. gonorrhoeae, from the small cryptic plasmid of 4.2 kb to larger conjugative plasmids which promote their own transfer between strains and may carry determinants of antibiotic resistance .
Treponema pallidum subsp. pallidum
All treponemes, of which T. p. pallidum is one subspecies, have reduced, highly conserved genomes (1.1 Mb), despite significant differences in their host preference and clinical manifestations [31▪]. A comparison of the four sequenced T. p. pallidum genomes [9,32–35] shows that only 327 SNPs differentiate them (excluding hypervariable regions ). Much of the variation lies within the specific regions including the gene encoding the acidic repeat protein (arp) and the trpD and trpK genes , and there is also evidence of intrastrain gene conversion of variable (V) regions within trpK during infection . Key genes encoding the flagellar structural proteins enabling the characteristic spiralling motility have been identified, along with 13 genes involved in chemotaxis.
GENOMICS IN EPIDEMIOLOGY
Molecular epidemiological typing is often used for finer resolution of bacterial strain identity in epidemiological studies. For C. trachomatis and N. gonorrhoeae, there are several gene or repeat-based typing schemes [38,39,40▪]. If the aim is macroepidemiological, following long-term or global trends, multilocus sequence typing (MLST) schemes are often suitable [41–44], using a set of stable relatively conserved, slowly evolving and evolutionary more neutral housekeeping genes, distributed throughout the genome [40▪,45]. On the other hand, if the study is looking at community epidemics, local outbreaks or contact tracing over a short timescale, microepidemiological high-resolution schemes which focus on hypervariable regions of the genome are preferable [40▪,45]. These include multiple-locus variable number tandem repeat analysis (MLVA) and multilocus sequence analysis (MLSA) in C. trachomatis [38,46] and N. gonorrhoeae multiantigen sequence typing (NG-MAST) . The most common typing technique in T. p. pallidum  was developed using the number of repeats in arp and restriction fragment length polymorphism (RFLP) analysis of trp genes .
The most widely used typing scheme in Chlamydia is ompA genotyping, based on the gene encoding the major outer membrane protein (MOMP), with genotypes reflecting tropism: ocular A–C, urogenital D–K and LGV L1–L3. Global studies show that the most common urogenital genotypes are E, F and D, leading to the common notion that over the last few decades the overall populations of urogenital C. trachomatis genotypes has been relatively stable. Until recently, this nomenclature has been the only method available to predict relationships between isolates and to perform association studies. Such studies looking for associations between genotype and age, sex, number of sexual partners or clinical symptoms are divided as to whether there is a significant link [50–55].
In the light of whole-genome phylogenies (Fig. 2) and ongoing recombination, we can no longer infer strain relatedness from ompA genotype. The ompA gene has been found to undergo recombination, and the same ompA genotype can exist on the unrelated branches of the C. trachomatis phylogenetic tree [20▪▪]. This may explain why C. trachomatis genotypes appear epidemiologically static, as genomic flux is masked by the ompA genotype, and associations with disease or demographic cannot be determined. It also explains why all the existing gene-based C. trachomatis typing schemes can give conflicting results with the underlying whole-genome phylogeny and with each other (Fig. 3).
In contrast to other human pathogens [56–60], the use of whole-genome sequencing for the epidemiological investigation of bacterial STIs has hardly begun. What we have learnt so far has rephrased what we thought we knew about the prevalence and spread of C. trachomatis worldwide. If we are truly to understand both the local and global epidemiology of bacterial STIs, we must go back to the beginning. Using genomic methods, we can gain an understanding of the baseline variation within the pathogen population, providing us with information regarding how these bacteria evolve, acquire antibiotic resistance and cause outbreaks.
ADVANCES IN DIAGNOSIS
Diagnosis has historically involved microscopy (chlamydia, gonorrhoea and syphilis), growth (chlamydia and gonorrhoea) and serology (chlamydia and syphilis). For greater speed, throughput, sensitivity and specificity, nucleic acid amplification tests (NAATs) are currently the gold standard for chlamydia and gonorrhoea in well resourced settings.
Two recent cases emphasize how caution must be used in the choice of diagnostic targets. Previously, two of the main commercial C. trachomatis NAATs used a single target on the multicopy cryptic plasmid. However, a deletion of 377 bp covering the target site for both NAATs led to the emergence of a new strain which gave a false-negative diagnostic result and so went untreated, being passed between infected individuals without their knowledge, before being recognized in Sweden in 2006 . From genomic and phenotypic studies, it became apparent that the deletion of the 377 bp and consequent inactivation of a plasmid gene did not impact negatively on the growth or virulence of the strain [6,22], instead giving a huge advantage in being able to propagate under the diagnostic radar. The redesigned NAATs for C. trachomatis diagnosis from these manufacturers now use dual targets.
Diagnostic-driven evolution is clearly a phenomenon to be aware of. The porA gene in N. gonorrhoeae is a pseudogene used as a diagnostic target in several commercial and in-house NAATs, whereas the closely related N. meningitidis (Fig. 1) has an intact porA gene with a substantially different sequence, and commensal Neisseria strains have no porA gene. Four N. gonorrhoeae isolates, belonging to different clonal lineages, have been found in Australia, Scotland and Sweden which appear to have gained the porA gene from N. meningitidis, again becoming diagnostically ‘invisible’ [62,63,64▪▪]. These strains were identified through culture of the samples and use of additional diagnostic methods, but the extent of the spread of these clones remains unknown. In this case also, dual target NAATs would increase sensitivity and specificity.
These examples highlight a major public health issue, telling us that diagnostic targets must be carefully chosen and constantly monitored. On a population level, the lack of treatment following a negative NAAT provides a strong Darwinian selective pressure on the bacterial community by removing, through antibiotic treatment, only those isolates providing a positive test result. An understanding of how the genomes of these bacteria evolve is essential if we are to reduce the likelihood of future ‘diagnostic escapes’.
Treatment failure because of antibiotic resistance is a serious threat to human health for many bacterial pathogens. The increasing use of NAATs for STI diagnostics is a worrying trend; culture-based techniques are being eliminated leading to a dearth of live strains for resistance testing. This is compounded by a lack of routine test-of-cure, meaning it is impossible to distinguish treatment failure from reinfection. Although known genetic mechanisms of resistance can be tested through NAATs, novel or obscure resistance mechanisms are recalcitrant to this approach, and correlation with treatment outcome is not absolute.
N. gonorrhoeae has a history of acquiring or developing resistance to antibiotics, including all first-line treatment drugs such as sulphonamides, penicillins, tetracyclines, macrolides, fluoroquinolones and recently cephalosporins [65▪,66]. The resistance mechanisms may involve mutations in chromosomal targets, efflux systems or acquisition of novel genes by transformation or plasmid-mediated conjugation [65▪,67–69]. Most recently, chromosomal mosaic penA genes have been identified, conferring resistance to extended-spectrum cephalosporins including ceftriaxone and cefixime and threatening us with the development of an untreatable ‘superbug’ [65▪].
Syphilis has not yet developed resistance to its treatment antibiotic penicillin. However, two mutations conferring resistance to macrolides have been identified [70,71], possibly indirectly selected as a result of using azithromycin to treat other STIs and ‘genital discharge syndromes’ . Similarly, there is no evidence that C. trachomatis, treated in most cases with a single dose of azithromycin, has developed resistance, although resistance to several antibiotics has been reported in vitro [73▪▪,74,75]. For both chlamydia and syphilis, there are no internationally agreed standard methods to assess minimum inhibitory concentrations in vitro , and assessment of antibiotic resistance relies on the identification of treatment failures.
Genome sequence data is essential for understanding the biology, pathogenesis, virulence and epidemiology of these pathogens, particularly those whose study is intractable by other means. For known recombinogenic bacteria such as C. trachomatis and N. gonorrhoeae, accurate whole-genome sequences are the ultimate typing tool. Genome sequencing is becoming faster, cheaper, more high throughput and more available, meaning that genomic epidemiology can now be performed within real-time clinical situations [11,59,77–83]. Combined with a growing body of genomic data, it is hoped that recent advances in the genetic manipulation of C. trachomatis [73▪▪,84] will allow further investigations into chlamydia genetics, building on the output of comparative genomics.
The authors thank Magnus Unemo for useful discussions in the preparation of this manuscript. This work was supported by the Wellcome Trust (grant number 098051).
Conflicts of interest
There are no conflicts of interest.
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