SummaryBackgroundTraditional methods for molecular epidemiology of Neisseria gonorrhoeae are suboptimal. Whole-genome sequencing (WGS) offers ideal resolution to describe population dynamics and to predict and infer transmission of antimicrobial resistance, and can enhance infection control through linkage with epidemiological data. We used WGS, in conjunction with linked epidemiological and phenotypic data, to describe the gonococcal population in 20 European countries. We aimed to detail changes in phenotypic antimicrobial resistance levels (and the reasons for these changes) and strain distribution (with a focus on antimicrobial resistance strains in risk groups), and to predict antimicrobial resistance from WGS data.MethodsWe carried out an observational study, in which we sequenced isolates taken from patients with gonorrhoea from the European Gonococcal Antimicrobial Surveillance Programme in 20 countries from September to November, 2013. We also developed a web platform that we used for automated antimicrobial resistance prediction, molecular typing (N gonorrhoeae multi-antigen sequence typing [NG-MAST] and multilocus sequence typing), and phylogenetic clustering in conjunction with epidemiological and phenotypic data.FindingsThe multidrug-resistant NG-MAST genogroup G1407 was predominant and accounted for the most cephalosporin resistance, but the prevalence of this genogroup decreased from 248 (23%) of 1066 isolates in a previous study from 2009–10 to 174 (17%) of 1054 isolates in this survey in 2013. This genogroup previously showed an association with men who have sex with men, but changed to an association with heterosexual people (odds ratio=4·29). WGS provided substantially improved resolution and accuracy over NG-MAST and multilocus sequence typing, predicted antimicrobial resistance relatively well, and identified discrepant isolates, mixed infections or contaminants, and multidrug-resistant clades linked to risk groups.InterpretationTo our knowledge, we provide the first use of joint analysis of WGS and epidemiological data in an international programme for regional surveillance of sexually transmitted infections. WGS provided enhanced understanding of the distribution of antimicrobial resistance clones, including replacement with clones that were more susceptible to antimicrobials, in several risk groups nationally and regionally. We provide a framework for genomic surveillance of gonococci through standardised sampling, use of WGS, and a shared information architecture for interpretation and dissemination by use of open access software.FundingThe European Centre for Disease Prevention and Control, The Centre for Genomic Pathogen Surveillance, Örebro University Hospital, and Wellcome.

Chlamydia trachomatis is an obligate intracellular bacterium of major public health significance, infecting over one-tenth of the world's population and causing blindness and infertility in millions. Mounting evidence supports recombination as a key source of genetic diversity among free-living bacteria. Previous research shows that intracellular bacteria such as Chlamydiaceae may also undergo recombination but whether this plays a significant evolutionary role has not been determined. Here, we examine multiple loci dispersed throughout the chromosome to determine the extent and significance of recombination among 19 laboratory reference strains and 10 present-day ocular and urogenital clinical isolates using phylogenetic reconstructions, compatibility matrices, and statistically based recombination programs. Recombination is widespread; all clinical isolates are recombinant at multiple loci with no two belonging to the same clonal lineage. Several reference strains show nonconcordant phylogenies across loci; one strain is unambiguously identified as recombinantly derived from other reference strain lineages. Frequent recombination contrasts with a low level of point substitution; novel substitutions relative to reference strains occur less than one per kilobase. Hotspots for recombination are identified downstream from ompA, which encodes the major outer membrane protein. This widespread recombination, unexpected for an intracellular bacterium, explains why strain-typing using one or two genes, such as ompA, does not correlate with clinical phenotypes. Our results do not point to specific events that are responsible for different pathogenicities but, instead, suggest a new approach to dissect the genetic basis for clinical strain pathology with implications for evolution, host cell adaptation, and emergence of new chlamydial diseases.

Chlamydia trachomatis is an intracellular bacterium responsible for ocular, respiratory, and sexually transmitted diseases. The genome contains a nine-member polymorphic membrane protein (Pmp) family unique to members of the order Chlamydiales. Genomic and molecular analyses were performed for the entire pmp gene family for the 18 reference serological variants (serovars) and genovariant Ja to identify specific gene and protein regions that differentiate chlamydial disease groups. The mean genetic distance among all serovars varied from 0.1% for pmpA to 7.0% for pmpF. Lymphogranuloma venereum (LGV) serovars were the most closely related for the pmp genes and were also the most divergent, compared to ocular and non-LGV urogenital disease groups. Phylogenetic reconstructions showed that for six of nine pmp genes (not pmpA, pmpD, or pmpE), the serovars clustered based on tissue tropism. The most globally successful serovars, E and F, clustered distantly from the urogenital group for five pmp genes. These pmp genes may confer a biologic advantage that may facilitate infection and transmission for E and F. Surprisingly, serovar Da clustered with the ocular group from pmpE to pmpI, which are located together in the chromosome, providing statistically significant evidence for intergenomic recombination and acquisition of a genetic composition that could hypothetically expand the host cell range of serovar Da. We also identified distinct domains for pmpE, pmpF, and pmpH where substitutions were concentrated and associated with a specific disease group. Thus, our data suggest a possible structural or functional role that may vary among pmp genes in promoting antigenic polymorphisms and/or diverse adhesions-receptors that may be involved in immune evasion and differential tissue tropism.

Insights into the genomic adaptive traits of Treponema pallidum, the causative bacterium of syphilis, have long been hampered due to the absence of in vitro culture models and the constraints associated with its propagation in rabbits. Here, we have bypassed the culture bottleneck by means of a targeted strategy never applied to uncultivable bacterial human pathogens to directly capture whole-genome T. pallidum data in the context of human infection. This strategy has unveiled a scenario of discreet T. pallidum interstrain single-nucleotide-polymorphism-based microevolution, contrasting with a rampant within-patient genetic heterogeneity mainly targeting multiple phase-variable loci and a major antigen-coding gene (tprK). TprK demonstrated remarkable variability and redundancy, intra- and interpatient, suggesting ongoing parallel adaptive diversification during human infection. Some bacterial functions (for example, flagella- and chemotaxis-associated) were systematically targeted by both inter- and intrastrain single nucleotide polymorphisms, as well as by ongoing within-patient phase variation events. Finally, patient-derived genomes possess mutations targeting a penicillin-binding protein coding gene (mrcA) that had never been reported, unveiling it as a candidate target to investigate the impact on the susceptibility to penicillin. Our findings decode the major genetic mechanisms by which T. pallidum promotes immune evasion and survival, and demonstrate the exceptional power of characterizing evolving pathogen subpopulations during human infection.

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Genome sequencing of Chlamydia trachomatis serovar D has identified polymorphic membrane proteins (Pmp) that are a newly recognized protein family unique to the Chlamydiaceae family. Cumulative data suggest that these diverse proteins are expressed on the cell surface and might be immunologically important. We performed phylogenetic analyses and statistical modeling with 18 reference serovars and 1 genovariant of C. trachomatis to examine the evolutionary characteristics and comparative genetics of PmpC and pmpC, the gene that encodes this protein. We also examined 12 recently isolated ocular and urogenital clinical samples, since reference serovars are laboratory adapted and may not represent strains that are presently responsible for human disease. Phylogenetic reconstructions revealed a clear distinction for disease groups, corresponding to levels of tissue specificity and virulence of the organism. Further, the most prevalent serovars, E, F, and Da, formed a distinct clade. According to the results of comparative genetic analyses, these three genital serovars contained two putative insertion sequence (IS)-like elements with 10-and 15-bp direct repeats, respectively, while all other genital serovars contained one IS-like element. Ocular trachoma serovars also contained both insertions. Previously, no IS-like elements have been identified for Chlamydiaceae. Surprisingly, 7 (58%) of 12 clinical isolates revealed pmpC sequences that were identical to the sequences of other serovars, providing clear evidence for a high rate of whole-gene recombination. Recombination and the differential presence of IS-like elements among distinct disease and prevalence groups may contribute to genome plasticity, which may lead to adaptive changes in tissue tropism and pathogenesis over the course of the organism's evolution.Chlamydia trachomatis is an obligate intracellular bacterium and a major cause of ocular and urogenital human infections worldwide (13). C. trachomatis has been divided into two human biological variants (biovars) on the basis of the nature of the disease that each group causes. To date, 18 serological variants (serovars) of these biovars have been identified by monoclonal antibody (MAb) typing of the major outer membrane protein (MOMP) of the organism. The oculogenital or trachoma biovar consists of serovars A to C and Ba that are responsible for trachoma, a chronic ocular inflammatory disease found in developing countries, and serovars Ba,

BackgroundChlamydia trachomatis is an obligate intracellular human pathogen causing ocular and urogenital infections that are a significant clinical and public health concern. This bacterium uses a type III secretion (T3S) system to manipulate host cells, through the delivery of effector proteins into their cytosol, membranes, and nucleus. In this work, we aimed to find previously unidentified C. trachomatis T3S substrates.ResultsWe first analyzed the genome of C. trachomatis L2/434 strain for genes encoding mostly uncharacterized proteins that did not appear to possess a signal of the general secretory pathway and which had not been previously experimentally shown to be T3S substrates. We selected several genes with these characteristics and analyzed T3S of the encoding proteins using Yersinia enterocolitica as a heterologous system. We identified 23 C. trachomatis proteins whose first 20 amino acids were sufficient to drive T3S of the mature form of β-lactamase TEM-1 by Y. enterocolitica. We found that 10 of these 23 proteins were also type III secreted in their full-length versions by Y. enterocolitica, providing additional support that they are T3S substrates. Seven of these 10 likely T3S substrates of C. trachomatis were delivered by Y. enterocolitica into host cells, further suggesting that they could be effectors. Finally, real-time quantitative PCR analysis of expression of genes encoding the 10 likely T3S substrates of C. trachomatis showed that 9 of them were clearly expressed during infection of host cells.ConclusionsUsing Y. enterocolitica as a heterologous system, we identified 10 likely T3S substrates of C. trachomatis (CT053, CT105, CT142, CT143, CT144, CT161, CT338, CT429, CT656, and CT849) and could detect translocation into host cells of CT053, CT105, CT142, CT143, CT161, CT338, and CT429. Therefore, we revealed several C. trachomatis proteins that could be effectors subverting host cell processes.

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