Microbial Genomics of Sexually Transmitted Infections

Whole-genome analysis has provided insights into the evolution of Chlamydia trachomatis and, recently, into circulating strains that cause lymphogranuloma venereum (LGV). A large LGV outbreak of a new ompA-genotype, L2b, was first reported in Europe in the early 2000s, primarily affecting men who have sex with men (MSM), and then expanded globally. More recent work shows that this outbreak is diversifying into variants of described ompA-genotypes, with the same L2b genomic backbone. This study extends the investigation of LGV cases to Argentina and Finland. In 2017, an LGV outbreak was described in Argentina characterized by distinct genomic features shown by both ompA-genotyping and Multi-Locus Sequence Typing (MLST) analysis. We have obtained whole-genome sequences from cultured isolates and clinical samples via SureSelect (Agilent) target enrichment. Based on ompA and phylogenetic analyses, we describe further diversity within the ompA-genotype L2b clade, illustrating the transmission dynamics in Argentina and Finland. A key finding is that of a novel clade of Argentinian samples, characterized by a proposed new ompA-genotype L4. Additionally, we present the genome sequence of a non-LGV strain associated with anorectal proctitis. These findings contribute to the investigation of LGV evolution, particularly with the presence of the novel L4 lineage, and provide insights into genomic diversity and transmission dynamics of C. trachomatis.

Neisseria meningitidis (Nm) is a bacterial pathogen responsible for invasive meningococcal disease. Though typically colonizing the nasopharynx, multiple outbreaks of meningococcal urethritis were first reported in 2015–2016; outbreaks originally presumed to be caused by Neisseria gonorrhoeae (Ng). Genomic analysis revealed that the Nm isolates causing these outbreaks were a distinct clade, and had integrated gonococcal DNA at multiple genomic sites, including the gonococcal denitrification apparatus aniA–norB, a partial gonococcal operon of five genes containing ispD, and the acetylglutamate kinase gene argB with the adjacent gonococcal locus NGO0843. The urethritis isolates had also deleted the group C capsule biosynthesis genes cssA/B/C and csc, resulting in loss of capsule. Collectively, these isolates form the N. meningitidis urethritis clade (NmUC). Genomic analysis of recent (2016–2022) NmUC isolates revealed that the genomic features have been maintained in the clade, implying that they are important for NmUC’s status as a urogenital pathogen. Furthermore, the analysis revealed the emergence of a sub-clade, designated NmUC-B, phylogenetically separated from the earlier NmUC-A. This sub-clade has integrated additional gonococcal alleles into the genome, including alleles associated with antimicrobial resistance. NmUC continues to adapt to a urethral niche and evolve as a urogenital pathogen.

Neisseria gonorrhoeae is a significant threat to global health with an estimated incidence of over 80 million cases each year and high levels of antimicrobial resistance. The gonococcal β-lactamase plasmid, pbla, carries the TEM β-lactamase, which requires only one or two amino acid changes to become an extended-spectrum β-lactamase (ESBL); this would render last resort treatments for gonorrhoea ineffective. Although pbla is not mobile, it can be transferred by the conjugative plasmid, pConj, found in N. gonorrhoeae . Seven variants of pbla have been described previously, but little is known about their frequency or distribution in the gonococcal population. We characterised sequences of pbla variants and devised a typing scheme, Ng_pblaST that allows their identification from whole genome short-read sequences. We implemented Ng_pblaST to assess the distribution of pbla variants in 15 532 gonococcal isolates. This demonstrated that only three pbla variants commonly circulate in gonococci, which together account for >99 % of sequences. The pbla variants carry different TEM alleles and are prevalent in distinct gonococcal lineages. Analysis of 2758 pbla-containing isolates revealed the co-occurrence of pbla with certain pConj types, indicating co-operativity between pbla and pConj variants in the spread of plasmid-mediated AMR in N. gonorrhoeae . Understanding the variation and distribution of pbla is essential for monitoring and predicting the spread of plasmid-mediated β-lactam resistance in N. gonorrhoeae .

The PorB porins are the major pore-forming proteins in the genus   Neisseria  . The trimeric PorB porins consist of 16 highly conserved transmembrane domains that form an amphipathic β-sheet connected by short periplasmic turns and eight extracellular hydrophilic loops. These loops are immunogenic and also play an important role in mediating antimicrobial influx. This study sought to (i) characterize the variations in Neisserial loop 3(355–438 bp) associated with intermediate resistance to penicillin/tetracycline and (ii) evaluate if there was evidence of horizontal gene transfer in any of the loops. We collated an integrated database consisting of 19 018   Neisseria   spp. genomes – 17 882   Neisseria gonorrhoeae  , 114   Neisseria meningitidis   and 1022 commensal   Neisseria   spp. To identify the porB alleles, a gene-by-gene approach (chewBBACA) was employed. To evaluate the presence of recombination events, the Recombination Detection Programme (RDP4) was used. In total, 3885 porB alleles were detected. Paralogues were identified in 17   Neisseria   isolates. Putative recombination was identified in loop regions. Intraspecies recombination among   N. gonorrhoeae   isolates and interspecies recombination between   N. meningitidis   and commensal   Neisseria   spp., and   N. gonorrhoeae   and   N. lactamica   were identified. Here, we present a large-scale study of 19 018   Neisseria   isolates to describe recombination and variation in the porB gene. Importantly, we found putative recombination in loop regions between the pathogenic and non-pathogenic   Neisseria   spp. These findings suggest the need for pheno- and genotypic surveillance of antimicrobial susceptibility in commensal   Neisseria   spp. to prevent the emergence of AMR in the pathogenic   Neisseria  . This article contains data hosted by Microreact.

The incidence of gonorrhoea is increasing at an alarming pace, and therapeutic options continue to narrow as a result of worsening drug resistance. Neisseria gonorrhoeae is naturally competent, allowing the organism to adapt rapidly to selection pressures including antibiotics. A sub-population of N. gonorrhoeae carries the Gonococcal Genetic Island (GGI), which encodes a type IV secretion system (T4SS) that secretes chromosomal DNA. Previous research has shown that the GGI increases transformation efficiency in vitro, but the extent to which it contributes to horizontal gene transfer (HGT) during infection is unknown. Here we analysed genomic data from clinical isolates of N. gonorrhoeae to better characterize GGI+ and GGI− sub-populations and to delineate patterns of variation at the locus itself. We found the element segregating at an intermediate frequency (61%), and it appears to act as a mobile genetic element with examples of gain, loss, exchange and intra-locus recombination within our sample. We further found evidence suggesting that GGI+ and GGI− sub-populations preferentially inhabit distinct niches with different opportunities for HGT. Previously, GGI+ isolates were reported to be associated with more severe clinical infections, and our results suggest this could be related to metal-ion trafficking and biofilm formation. The co-segregation of GGI+ and GGI− isolates despite mobility of the element suggests that both niches inhabited by N. gonorrhoeae remain important to its overall persistence as has been demonstrated previously for cervical- and urethral-adapted sub-populations. These data emphasize the complex population structure of N. gonorrhoeae and its capacity to adapt to diverse niches.

In Australia, gonococcal isolates are monitored for antimicrobial susceptibilities. In Western Australia (WA), gonorrhoea notification rates increased by 63 % between 2013 and 2016, with the steepest increase occurring between 2015 and 2016, before stabilizing at this higher baseline between 2017 and 2020. This increased prevalence was associated with antimicrobial-susceptible (AMS) lineages. To understand the provenance of these isolates causing gonorrhoea in WA, whether they were introduced or expanded from endogenous lineages, 741 isolates were collected in 2017 and characterized by both iPLEX typing and whole genome sequencing (WGS). Antibiograms and genocoding of the isolates revealed that AMS isolates were most prevalent in the remote regions, while the urban/rural regions were characterized by antimicrobial-resistant (AMR) isolates. iPLEX typing identified 78 iPLEX genotypes (WA-1 to WA-78) of which 20 accounted for over 88 % of isolates. WA-10 was the most frequently identified genotype in the urban/rural regions whilst WA-29 was the most frequently identified genotype in the remote regions. Genotypes WA-38, WA-52 and WA-13 accounted for 81 % (n=36/44) of the azithromycin-resistant N. gonorrhoeae (AziR) isolates. A representative isolate of each iPLEX genotype and AMR biotype was whole genome sequenced and analysed using MLST, NG-MAST and NG-STAR, and the novel core genome clustering Ng_cgc_400 typing scheme. Five predominant Bayesian population groups (termed BPG-1 to 5) were identified in the study collection. BPG-1 and BPG-2 were associated with AMS isolates from the remote regions. BPG-1 and BPG-2 were shown to be unique to the remote regions based on a minimum spanning tree against 4000 international isolates. AMS isolates in urban/rural regions were dominated by international lineages. AziR and Cef DS (decreased susceptibility to ceftriaxone) was concentrated in three urban/rural genomic groups (BPG-3, 4 and 5). Azithromycin minimum inhibitory concentrations (0.5–16 mg l−1) correlated with the accumulation of mtrR mutations or/and the fraction of 23S rRNA C2611T mutated copies. The majority of isolates in BPG-3, 4 and 5 could be correlated with known AMR lineages circulating globally and nationally. In conclusion, the surge in AMS isolates in WA in 2017 was due to importation of international AMS lineages into urban/rural regions, whilst the local AMS lineages persisted largely in the remote regions. Bridging between the urban/rural and remote regions was relatively rare, but continued surveillance is required to prevent ingress of AMR strains/lineages into the remote regions of WA.

Genomic data contribute invaluable information to the epidemiological investigation of pathogens of public health importance. However, whole-genome sequencing (WGS) of bacteria typically relies on culture, which represents a major hurdle for generating such data for a wide range of species for which culture is challenging. In this study, we assessed the use of culture-free target-enrichment sequencing as a method for generating genomic data for two bacterial species: (1) Bacillus anthracis, which causes anthrax in both people and animals and whose culture requires high-level containment facilities; and (2) Mycoplasma amphoriforme , a fastidious emerging human respiratory pathogen. We obtained high-quality genomic data for both species directly from clinical samples, with sufficient coverage (>15×) for confident variant calling over at least 80% of the baited genomes for over two thirds of the samples tested. Higher qPCR cycle threshold (Ct) values (indicative of lower pathogen concentrations in the samples), pooling libraries prior to capture, and lower captured library concentration were all statistically associated with lower capture efficiency. The Ct value had the highest predictive value, explaining 52 % of the variation in capture efficiency. Samples with Ct values ≤30 were over six times more likely to achieve the threshold coverage than those with a Ct > 30. We conclude that target-enrichment sequencing provides a valuable alternative to standard WGS following bacterial culture and creates opportunities for an improved understanding of the epidemiology and evolution of many clinically important pathogens for which culture is challenging.

Lymphogranuloma venereum (LGV), the invasive infection of the sexually transmissible infection (STI) Chlamydia trachomatis , is caused by strains from the LGV biovar, most commonly represented by ompA-genotypes L2b and L2. We investigated the diversity in LGV samples across an international collection over seven years using typing and genome sequencing. LGV-positive samples (n=321) from eight countries collected between 2011 and 2017 (Spain n=97, Netherlands n=67, Switzerland n=64, Australia n=53, Sweden n=37, Hungary n=31, Czechia n=30, Slovenia n=10) were genotyped for pmpH and ompA variants. All were found to contain the 9 bp insertion in the pmpH gene, previously associated with ompA-genotype L2b. However, analysis of the ompA gene shows ompA-genotype L2b (n=83), ompA-genotype L2 (n=180) and several variants of these (n=52; 12 variant types), as well as other/mixed ompA-genotypes (n=6). To elucidate the genomic diversity, whole genome sequencing (WGS) was performed from selected samples using SureSelect target enrichment, resulting in 42 genomes, covering a diversity of ompA-genotypes and representing most of the countries sampled. A phylogeny of these data clearly shows that these ompA-genotypes derive from an ompA-genotype L2b ancestor, carrying up to eight SNPs per isolate. SNPs within ompA are overrepresented among genomic changes in these samples, each of which results in an amino acid change in the variable domains of OmpA (major outer membrane protein, MOMP). A reversion to ompA-genotype L2 with the L2b genomic backbone is commonly seen. The wide diversity of ompA-genotypes found in these recent LGV samples indicates that this gene is under immunological selection. Our results suggest that the ompA-genotype L2b genomic backbone is the dominant strain circulating and evolving particularly in men who have sex with men (MSM) populations.

Many non-human primate species in sub-Saharan Africa are infected with Treponema pallidum subsp. pertenue , the bacterium causing yaws in humans. In humans, yaws is often characterized by lesions of the extremities and face, while T. pallidum subsp. pallidum causes venereal syphilis and is typically characterized by primary lesions on the genital, anal or oral mucosae. It remains unclear whether other T. pallidum subspecies found in humans also occur in non-human primates and how the genomic diversity of non-human primate T. pallidum subsp. pertenue lineages is distributed across hosts and space. We observed orofacial and genital lesions in sooty mangabeys (Cercocebus atys) in Taï National Park, Côte d’Ivoire and collected swabs and biopsies from symptomatic animals. We also collected non-human primate bones from 8 species in Taï National Park and 16 species from 11 other sites across sub-Saharan Africa. Samples were screened for T. pallidum DNA using polymerase chain reactions (PCRs) and we used in-solution hybridization capture to sequence T. pallidum genomes. We generated three nearly complete T. pallidum genomes from biopsies and swabs and detected treponemal DNA in bones of six non-human primate species in five countries, allowing us to reconstruct three partial genomes. Phylogenomic analyses revealed that both orofacial and genital lesions in sooty mangabeys from Taï National Park were caused by T. pallidum subsp. pertenue . We showed that T. pallidum subsp. pertenue has infected non-human primates in Taï National Park for at least 28 years and has been present in two non-human primate species that had not been described as T. pallidum subsp. pertenue hosts in this ecosystem, western chimpanzees (Pan troglodytes verus) and western red colobus (Piliocolobus badius), complementing clinical evidence that started accumulating in Taï National Park in 2014. More broadly, simian T. pallidum subsp. pertenue strains did not form monophyletic clades based on host species or the symptoms caused, but rather clustered based on geography. Geographical clustering of T. pallidum subsp. pertenue genomes might be compatible with cross-species transmission of T. pallidum subsp. pertenue within ecosystems or environmental exposure, leading to the acquisition of closely related strains. Finally, we found no evidence for mutations that confer antimicrobial resistance.

Chlamydia trachomatis is the most prevalent sexually transmitted bacterium worldwide and the causative agent of trachoma. Its strains are classified according to their ompA genotypes, which are strongly linked to differential tissue tropism and disease outcomes [ocular disease, urogenital disease and lymphogranuloma venereum (LGV)]. While the genome-based species phylogenetic tree presents four main clades correlating with tropism/prevalence, namely ocular, LGV, urogenital T1 (more prevalent genotypes) and urogenital T2 (less prevalent genotypes), inter-clade exchange of ompA is considered a rare phenomenon probably mediating marked tropism alterations. An LGV epidemic, associated with the clonal expansion of the L2b genotype, has emerged in the last few decades, raising concerns particularly due to its atypical clinical presentation (ulcerative proctitis) and circulation among men who have sex with men (MSM). Here, we report an LGV outbreak, mostly affecting human immunodeficiency virus-positive MSM engaging in high-risk sexual practices, caused by an L2b strain with a rather unique non-LGV ompA signature that precluded the laboratory notification of this outbreak as LGV. C. trachomatis whole-genome capture and sequencing directly from clinical samples was applied to deeply characterize the genomic backbone of this novel LGV outbreak-causing clone. It revealed a chimeric genome structure due to the genetic transfer of ompA and four neighbouring genes from a serovar D/Da strain, likely possessing the genomic backbone associated with the more prevalent urogenital genotypes (T1 clade), to an LGV (L2b) strain. The hybrid L2b/D-Da strain presents the adhesin and immunodominant antigen MOMP (major outer membrane protein) (encoded by ompA) with an epitope repertoire typical of non-invasive genital strains, while keeping the genome-dispersed virulence fingerprint of a classical LGV strain. As previously reported for inter-clade ompA exchange among non-LGV clades, this novel C. trachomatis genomic mosaic involving a contemporary epidemiologically and clinically relevant LGV strain may have implications on its transmission, tissue tropism and pathogenic capabilities. The emergence of variants with epidemic and pathogenic potential highlights the need for more focused surveillance strategies to capture C. trachomatis evolution in action.