Whole-Genome Sequencing to Predict Antimicrobial Susceptibility Profiles in Neisseria gonorrhoeae.

BACKGROUND: Neisseria gonorrhoeae is a major public health problem due to increasing incidence and antimicrobial resistance. Genetic markers of reduced susceptibility have been identified; the extent to which those are representative of global antimicrobial resistance is unknown. We evaluated the performance of whole-genome sequencing (WGS) used to predict susceptibility to ciprofloxacin and other antimicrobials using a global collection of N. gonorrhoeae isolates. METHODS: Susceptibility testing of common antimicrobials and the recently developed zolifodacin was performed using agar dilution to determine minimum inhibitory concentrations (MICs). We identified resistance alleles at loci known to contribute to antimicrobial resistance in N. gonorrhoeae from WGS data. We tested the ability of each locus to predict antimicrobial susceptibility. RESULTS: A total of 481 N. gonorrhoeae isolates, collected between 2004 and 2019 and making up 457 unique genomes, were sourced from 5 countries. All isolates with demonstrated susceptibility to ciprofloxacin (MIC ≤0.06 µg/mL) had a wild-type gyrA codon 91. Multilocus approaches were needed to predict susceptibility to other antimicrobials. All isolates were susceptible to zoliflodacin, defined by an MIC ≤0.25 µg/mL. CONCLUSIONS: Single marker prediction can be used to inform ciprofloxacin treatment of N. gonorrhoeae infection. A combination of molecular markers may be needed to determine susceptibility for other antimicrobials.

A Genomic Perspective on the Near-term Impact of Doxycycline Post-exposure Prophylaxis on Neisseria gonorrhoeae Antimicrobial Resistance.

Pre-existing tetracycline resistance in Neisseria gonorrhoeae limits the effectiveness of post-exposure prophylaxis (PEP) with doxycycline against gonorrhea, and selection for tetracycline resistance may influence prevalence of multi-drug resistant strains. Using genomic and antimicrobial susceptibility data from N. gonorrhoeae, we assessed the near-term impact of doxycycline PEP on N. gonorrhoeae resistance.

The Distribution and Spread of Susceptible and Resistant Neisseria gonorrhoeae Across Demographic Groups in a Major Metropolitan Center.

BACKGROUND: Genomic epidemiology studies of gonorrhea in the United States have primarily focused on national surveillance for antibiotic resistance, and patterns of local transmission between demographic groups of resistant and susceptible strains are unknown. METHODS: We analyzed a convenience sample of genome sequences, antibiotic susceptibility, and patient data from 897 gonococcal isolates cultured at the New York City (NYC) Public Health Laboratory from NYC Department of Health and Mental Hygiene (DOHMH) Sexual Health Clinic (SHC) patients, primarily in 2012-2013. We reconstructed the gonococcal phylogeny, defined transmission clusters using a 10 nonrecombinant single nucleotide polymorphism threshold, tested for clustering of demographic groups, and placed NYC isolates in a global phylogenetic context. RESULTS: The NYC gonococcal phylogeny reflected global diversity with isolates from 22/23 of the prevalent global lineages (96%). Isolates clustered on the phylogeny by patient sexual behavior (P < .001) and race/ethnicity (P < .001). Minimum inhibitory concentrations were higher across antibiotics in isolates from men who have sex with men compared to heterosexuals (P < .001) and white heterosexuals compared to black heterosexuals (P < .01). In our dataset, all large transmission clusters (≥10 samples) of N. gonorrhoeae were susceptible to ciprofloxacin, ceftriaxone, and azithromycin, and comprised isolates from patients across demographic groups. CONCLUSIONS: All large transmission clusters were susceptible to gonorrhea therapies, suggesting that resistance to empiric therapy was not a main driver of spread, even as risk for resistance varied across demographic groups. Further study of local transmission networks is needed to identify drivers of transmission.

Using rapid point-of-care tests to inform antibiotic choice to mitigate drug resistance in gonorrhoea.

BackgroundThe first cases of extensively drug resistant gonorrhoea were recorded in the United Kingdom in 2018. There is a public health need for strategies on how to deploy existing and novel antibiotics to minimise the risk of resistance development. As rapid point-of-care tests (POCTs) to predict susceptibility are coming to clinical use, coupling the introduction of an antibiotic with diagnostics that can slow resistance emergence may offer a novel paradigm for maximising antibiotic benefits. Gepotidacin is a novel antibiotic with known resistance and resistance-predisposing mutations. In particular, a mutation that confers resistance to ciprofloxacin acts as the 'stepping-stone' mutation to gepotidacin resistance.AimTo investigate how POCTs detecting Neisseria gonorrhoeae resistance mutations for ciprofloxacin and gepotidacin can be used to minimise the risk of resistance development to gepotidacin.MethodsWe use individual-based stochastic simulations to formally investigate the aim.ResultsThe level of testing needed to reduce the risk of resistance development depends on the mutation rate under treatment and the prevalence of stepping-stone mutations. A POCT is most effective if the mutation rate under antibiotic treatment is no more than two orders of magnitude above the mutation rate without treatment and the prevalence of stepping-stone mutations is 1-13%.ConclusionMutation frequencies and rates should be considered when estimating the POCT usage required to reduce the risk of resistance development in a given population. Molecular POCTs for resistance mutations and stepping-stone mutations to resistance are likely to become important tools in antibiotic stewardship.

Epidemiological and genomic determinants of tuberculosis outbreaks in First Nations communities in Canada.

BACKGROUND: In Canada, tuberculosis disproportionately affects foreign-born and First Nations populations. Within First Nations' peoples, a high proportion of cases occur in association with outbreaks. Tuberculosis transmission in the context of outbreaks is thought to result from the convergence of several factors including characteristics of the cases, contacts, the environment, and the pathogen. METHODS: We examined the epidemiological and genomic determinants of two well-characterized tuberculosis outbreaks attributed to two super-spreaders among First Nations in the province of Alberta. These outbreaks were associated with two distinct DNA fingerprints (restriction fragment-length polymorphisms or RFLPs 0.0142 and 0.0728). We compared outbreak isolates with endemic isolates not spatio-temporarily linked to outbreak cases. We extracted epidemiological variables pertaining to tuberculosis cases and contacts from individual public health records and the provincial tuberculosis registry. We conducted group analyses using parametric and non-parametric statistical tests. We carried out whole-genome sequencing and bioinformatic analysis using validated protocols. RESULTS: We observed differences between outbreak and endemic groups in the mean number of total and child-aged contacts and the number of contacts with new positive and converted tuberculin skin tests in all group comparisons (p < 0.05). Differences were also detected in the proportion of cases with cavitation on a chest radiograph and the mean number of close contacts in selected group comparisons (p < 0.02). A phylogenetic network analysis of whole-genome sequencing data indicated that most outbreak and endemic strains were closely related to the source case for the 0.0142 fingerprint. For the 0.0728 fingerprint, the source case haplotype was circulating among endemic cases prior to the outbreak. Genetic and temporal distances were not correlated for either RFLP 0.0142 (r2 = - 0.05) or RFLP 0.0728 (r2 = 0.09) when all isolates were analyzed. CONCLUSIONS: We found no evidence that endemic strains acquired mutations resulting in their emergence in outbreak form. We conclude that the propagation of these outbreaks was likely driven by the combination of characteristics of the source cases, contacts, and the environment. The role of whole-genome sequencing in understanding mycobacterial evolution and in assisting public health authorities in conducting contact investigations and managing outbreaks is important and expected to grow in the future.

Diversity of Mycobacterium tuberculosis across Evolutionary Scales.

Tuberculosis (TB) is a global public health emergency. Increasingly drug resistant strains of Mycobacterium tuberculosis (M.tb) continue to emerge and spread, highlighting adaptability of this pathogen. Most studies of M.tb evolution have relied on 'between-host' samples, in which each person with TB is represented by a single M.tb isolate. However, individuals with TB commonly harbor populations of M.tb numbering in the billions. Here, we use analyses of M.tb genomic data from within and between hosts to gain insight into influences shaping genetic diversity of this pathogen. We find that the amount of M.tb genetic diversity harbored by individuals with TB can vary dramatically, likely as a function of disease severity. Surprisingly, we did not find an appreciable impact of TB treatment on M.tb diversity. In examining genomic data from M.tb samples within and between hosts with TB, we find that genes involved in the regulation, synthesis, and transportation of immunomodulatory cell envelope lipids appear repeatedly in the extremes of various statistical measures of diversity. Many of these genes have been identified as possible targets of selection in other studies employing different methods and data sets. Taken together, these observations suggest that M.tb cell envelope lipids are targets of selection within hosts. Many of these lipids are specific to pathogenic mycobacteria and, in some cases, human-pathogenic mycobacteria. We speculate that rapid adaptation of cell envelope lipids is facilitated by functional redundancy, flexibility in their metabolism, and their roles mediating interactions with the host.

A genomic perspective on the near-term impact of doxycycline post-exposure prophylaxis on Neisseria gonorrhoeae antimicrobial resistance.

Post-exposure prophylaxis with doxycycline (doxyPEP) is being introduced to prevent bacterial sexually transmitted infections (STIs). Pre-existing tetracycline resistance in Neisseria gonorrhoeae limits doxyPEP effectiveness against gonorrhea, and selection for tetracycline resistant lineages may influence prevalence of resistance to other antimicrobials via selection for multi-drug resistant strains. Using genomic and antimicrobial susceptibility data from 5,644 clinical isolates of N. gonorrhoeae , we assessed the near-term impact of doxyPEP on N. gonorrhoeae antimicrobial resistance. We found that the impact on antimicrobial resistance is likely to be influenced by the strength of selection for plasmid-encoded and chromosomally-encoded tetracycline resistance, as isolates with high-level, plasmid-encoded resistance had lower minimum inhibitory concentrations to other antimicrobials compared to isolates with low-level tetracycline resistance. The impact of doxyPEP may differ across demographic groups and geographic regions within the United States due to variation in pre-existing tetracycline resistance.

Neisseria gonorrhoeae diagnostic escape from a gyrA-based test for ciprofloxacin susceptibility and the effect on zoliflodacin resistance: a bacterial genetics and experimental evolution study.

BACKGROUND: The aetiological bacterial agent of gonorrhoea, Neisseria gonorrhoeae, has become resistant to each of the first-line antibiotics used to treat it, including ciprofloxacin. One diagnostic approach to identify ciprofloxacin-susceptible isolates is to determine codon 91 in the gene encoding the A subunit of DNA gyrase, gyrA, where coding for the wild-type serine (gyrA91S) is associated with ciprofloxacin susceptibility and phenylalanine (gyrA91F) with resistance. The aim of this study was to investigate the possibility of diagnostic escape from gyrA susceptibility testing. METHODS: We used bacterial genetics to introduce pairwise substitutions in GyrA positions 91 (S or F) and 95 (D, G, or N), which is a second site in GyrA associated with ciprofloxacin resistance, into five clinical isolates of N gonorrhoeae. All five isolates encoded GyrA S91F, an additional substitution in GyrA at position 95, substitutions in ParC that are known to cause an increased minimum inhibitory concentration (MIC) to ciprofloxacin, and GyrB 429D, which is associated with susceptibility to zoliflodacin (a spiropyrimidinetrione-class antibiotic in phase 3 trials for treatment of gonorrhoea). We evolved these isolates to assess for the existence of pathways to ciprofloxacin resistance (MIC ≥1 µg/mL) and measured MICs for ciprofloxacin and zoliflodacin. In parallel, we searched metagenomic data for 11 355 N gonorrhoeae clinical isolates with reported ciprofloxacin MICs that were publicly available from the European Nucleotide Archive for strains that would be identified as susceptible by gyrA codon 91-based assays. FINDINGS: Three clinical isolates of N gonorrhoeae with substitutions in GyrA position 95 associated with resistance (G or N) maintained intermediate ciprofloxacin MICs (0·125-0·5 µg/mL), which has been associated with treatment failure, despite reversion of GyrA position 91 from phenylalanine to serine. From an in-silico analysis of the 11 355 genomes from N gonorrhoeae clinical isolates, we identified 30 isolates with gyrA codon 91 encoding a serine and a ciprofloxacin resistance-associated mutation at codon 95. The reported MICs for these isolates varied from 0·023 µg/mL to 0·25 µg/mL, including four with intermediate ciprofloxacin MICs (associated with substantially increased risk of treatment failure). Finally, through experimental evolution, one clinical isolate of N gonorrhoeae bearing GyrA 91S acquired ciprofloxacin resistance through mutations in the gene encoding for the B subunit of DNA gyrase (gyrB) that also conferred reduced susceptibility to zoliflodacin (ie, MIC ≥2 µg/mL). INTERPRETATION: Diagnostic escape from gyrA codon 91 diagnostics could occur through either reversion of the gyrA allele or expansion of circulating lineages. N gonorrhoeae genomic surveillance efforts might benefit from including gyrB, given its potential for contributing to ciprofloxacin and zoliflodacin resistance, and diagnostic strategies that reduce the likelihood of escape, such as the incorporation of multiple target sites, should be investigated. Diagnostics that guide antibiotic therapy can have unintended consequences, including novel resistance determinants and antibiotic cross-resistance. FUNDING: US National Institutes of Health National Institute of Allergy and Infectious Diseases, National Institute of General Medical Sciences, and the Smith Family Foundation.

Machine learning models for Neisseria gonorrhoeae antimicrobial susceptibility tests.

Neisseria gonorrhoeae is an urgent public health threat due to the emergence of antibiotic resistance. As most isolates in the United States are susceptible to at least one antibiotic, rapid molecular antimicrobial susceptibility tests (ASTs) would offer the opportunity to tailor antibiotic therapy, thereby expanding treatment options. With genome sequence and antibiotic resistance phenotype data for nearly 20,000 clinical N. gonorrhoeae isolates now available, there is an opportunity to use statistical methods to develop sequence-based diagnostics that predict antibiotic susceptibility from genotype. N. gonorrhoeae, therefore, provides a useful example illustrating how to apply machine learning models to aid in the design of sequence-based ASTs. We present an overview of this framework, which begins with establishing the assay technology, the performance criteria, the population in which the diagnostic will be used, and the clinical goals, and extends to the choices that must be made to arrive at a set of features with the desired properties for predicting susceptibility phenotype from genotype. While we focus on the example of N. gonorrhoeae, the framework generalizes to other organisms for which large-scale genotype and antibiotic resistance data can be combined to aid in diagnostics development.

Interactions between Loci Contributing to Antimicrobial Resistance and Virulence in Neisseria gonorrhoeae.

In a recent mBio article, Ayala et al. (mBio 13:e00276-22, 2022, https://doi.org/10.1128/mbio.00276-22) identified a single nucleotide variant in the repressor gdhR in Neisseria gonorrhoeae that reduces binding to the promoter of the virulence factor lctP and thereby increases its expression. The allele (gdhR6) frequently co-occurs with mutations in the mtr operon promoter that reduce expression of another repressor, mtrR, resulting in overexpression of the efflux pump-encoding mtrCDE and increased antimicrobial resistance. Because mtrR also represses gdhR, a decline in mtrR would decrease expression of lctP. Hypothesizing that gdhR6 arose to circumvent the impact of mtrR promoter mutations on lctP expression, the authors analyzed these loci in genomes of N. gonorrhoeae isolates from the preantibiotic era. Surprisingly, they found isolates with gdhR6 prior to selection for mtrR resistance-associated alleles. These results suggest that independent and perhaps interacting pressures have influenced the co-occurrence of these alleles.

Loci for prediction of penicillin and tetracycline susceptibility in Neisseria gonorrhoeae: a genome-wide association study.

BACKGROUND: Neisseria gonorrhoeae poses an urgent public health threat because of increasing antimicrobial resistance; however, much of the circulating population remains susceptible to historical treatment regimens. Point-of-care diagnostics that report susceptibility could allow for reintroduction of these regimens, but development of such diagnostics has been restricted to ciprofloxacin, for which susceptibility can be predicted from a single locus. We aimed to define genetic variants associated with susceptibility to penicillin and tetracycline. METHODS: We collected publicly available global whole-genome sequencing data (n=12 045) from clinical N gonorrhoeae isolates, with phenotypic resistance data for penicillin (n=6935), and tetracycline (n=5727). Using conditional genome-wide association studies, we defined genetic variants associated with susceptibility to penicillin and tetracycline. We excluded isolates that could not be classified as either susceptible or resistant. To validate our results, we assembled 1479 genomes from the US Centers for Disease Control and Prevention (CDC)'s Gonococcal Isolate Surveillance Project, for which urethral specimens are collected at sentinel surveillance sites across the USA. We evaluated the sensitivity and specificity of susceptibility-associated alleles using Clinical & Laboratory Standards Institute breakpoints for susceptibility and non-resistance in both the global and validation datasets. FINDINGS: In our conditional penicillin genome-wide association study, the presence of a genetic variant defined by a non-mosaic penA allele without an insertion at codon 345 was associated with penicillin susceptibility and had the highest negative effect size (ß) of significant variants (p=5·0x10-14, ß -2·5). In combination with the absence of blaTEM, this variant predicted penicillin susceptibility with high specificity (99·8%) and modest sensitivity (36·7%). For tetracycline, the wildtype allele at rpsJ codon 57, encoding valine, was associated with tetracycline susceptibility (p=5·6x10-16, ß -1·6) after conditioning on the presence of tetM. The combination of rpsJ codon 57 allele and tetM absence predicted tetracycline susceptibility with high specificity (97·2%) and sensitivity (88·7%). INTERPRETATION: As few as two genetic loci can predict susceptibility to penicillin and tetracycline in N gonorrhoeae with high specificity. Molecular point-of-care diagnostics targeting these loci have the potential to increase available treatments for gonorrhoea. FUNDING: National Institute of Allergy and Infectious Diseases, the National Science Foundation, and the Smith Family Foundation.

Sculpting the Bacterial O-Glycoproteome: Functional Analyses of Orthologous Oligosaccharyltransferases with Diverse Targeting Specificities.

Protein glycosylation systems are widely recognized in bacteria, including members of the genus Neisseria. In most bacterial species, the molecular mechanisms and evolutionary contexts underpinning target protein selection and the glycan repertoire remain poorly understood. Broad-spectrum O-linked protein glycosylation occurs in all human-associated species groups within the genus Neisseria, but knowledge of their individual glycoprotein repertoires is limited. Interestingly, PilE, the pilin subunit of the type IV pilus (Tfp) colonization factor, is glycosylated in Neisseria gonorrhoeae and Neisseria meningitidis but not in the deeply branching species N. elongata subsp. glycolytica. To examine this in more detail, we assessed PilE glycosylation status across the genus and found that PilEs of commensal clade species are not modified by the gonococcal PglO oligosaccharyltransferase. Experiments using PglO oligosaccharyltransferases from across the genus expressed in N. gonorrhoeae showed that although all were capable of broad-spectrum protein glycosylation, those from a deep-branching group of commensals were unable to support resident PilE glycosylation. Further glycoproteomic analyses of these strains using immunoblotting and mass spectrometry revealed other proteins differentially targeted by otherwise remarkably similar oligosaccharyltransferases. Finally, we generated pglO allelic chimeras that begin to localize PglO protein domains associated with unique substrate targeting activities. These findings reveal previously unappreciated differences within the protein glycosylation systems of highly related bacterial species. We propose that the natural diversity manifest in the neisserial protein substrates and oligosaccharyltransferases has significant potential to inform the structure-function relationships operating in these and related bacterial protein glycosylation systems. IMPORTANCE Although general protein glycosylation systems have been well recognized in prokaryotes, the processes governing their distribution, function, and evolution remain poorly understood. Here, we have begun to address these gaps in knowledge by comparative analyses of broad-spectrum O-linked protein glycosylation manifest in species within the genus Neisseria that strictly colonize humans. Using N. gonorrhoeae as a well-defined model organism in conjunction with comparative genomics, intraspecies gene complementation, and glycoprotein phenotyping, we discovered clear differences in both glycosylation susceptibilities and enzymatic targeting activities of otherwise largely conserved proteins. These findings reveal previously unappreciated differences within the protein glycosylation systems of highly related bacterial species. We propose that the natural diversity manifest within Neisseria species has significant potential to elucidate the structure-function relationships operating in these and related systems and to inform novel approaches to applied glycoengineering strategies.

Rapid adaptation of a complex trait during experimental evolution of Mycobacterium tuberculosis.

Tuberculosis (TB), caused by Mycobacterium tuberculosis (M. tb), is a leading cause of death due to infectious disease. TB is not traditionally associated with biofilms, but M. tb biofilms are linked with drug and immune tolerance and there is increasing recognition of their contribution to the recalcitrance of TB infections. Here, we used M. tb experimental evolution to investigate this complex phenotype and identify candidate loci controlling biofilm formation. We identified novel candidate loci, adding to our understanding of the genetic architecture underlying M. tb biofilm development. Under selective pressure to grow as a biofilm, regulatory mutations rapidly swept to fixation and were associated with changes in multiple traits, including extracellular matrix production, cell size, and growth rate. Genetic and phenotypic paths to enhanced biofilm growth varied according to the genetic background of the parent strain, suggesting that epistatic interactions are important in M. tb adaptation to changing environments.

In many environments, bacteria live together in structures called biofilms. Cells in biofilms coordinate with each other to protect the group and allow it to survive difficult conditions. Mycobacterium tuberculosis, the bacterium that causes tuberculosis, forms biofilms when it infects the human body. Biofilms make the infection a lot more difficult to treat, which may be one of the reasons why tuberculosis is the deadliest bacterial infection in the world. Bacteria evolve rapidly over the course of a single infection, but bacteria forming biofilms evolve differently to bacteria living alone. This evolution happens through mutations to the bacterial DNA, which can be small (a single base in a DNA sequence changes to a different base) or larger changes (such as the deletion or insertion of several bases). Smith, Youngblom et al. studied the evolution of tuberculosis growing in biofilms in the lab. As the bacteria evolved, they tended to form thicker biofilms, an effect linked to 14 mutations involving single base DNA changes and four larger ones. Most of the changes were in regulatory regions of DNA, which control whether genes are 'read' by cells to produce proteins. These regions often change more though evolution than regions coding for proteins, because they have a coordinated effect on a group of related genes rather than randomly altering individual genes. Smith, Youngblom et al. also showed that biofilms made from different strains of tuberculosis evolved in different ways. Smith Youngblom et al.'s findings provide more information regarding how bacteria adapt to living in biofilms, which may reveal new ways to control them. This could have applications in water treatment, food production and healthcare. Learning how to treat bacteria growing in biofilms could also improve the outcomes for patients infected with tuberculosis.

Disseminated Gonococcal Infection Complicated by Prosthetic Joint Infection: Case Report and Genomic and Phylogenetic Analysis.

Neisseria gonorrhoeae infections have been increasing globally, with prevalence rising across age groups. In this study, we report a case of disseminated gonococcal infection (DGI) involving a prosthetic joint, and we use whole-genome sequencing to characterize resistance genes, putative virulence factors, and the phylogenetic lineage of the infecting isolate. We review the literature on sequence-based prediction of antibiotic resistance and factors that contribute to risk for DGI. We argue for routine sequencing and reporting of invasive gonococcal infections to aid in determining whether an invasive gonococcal infection is sporadic or part of an outbreak and to accelerate understanding of the genetic features of N gonorrhoeae that contribute to pathogenesis.

Draft Genome Sequences of Three Penicillin-Resistant Neisseria gonorrhoeae Strains Isolated in Cincinnati, Ohio, in 1994.

Here, we report the draft genome sequences of three penicillin-resistant Neisseria gonorrhoeae isolates. We include associated data on MICs and genetic relationships to other N. gonorrhoeae strains collected from across the United States. Resistance mutations known to contribute to reduced penicillin susceptibility are annotated in each genome.

Emergence and evolution of antimicrobial resistance genes and mutations in Neisseria gonorrhoeae.

BACKGROUND: Antimicrobial resistance in Neisseria gonorrhoeae is a global health concern. Strains from two internationally circulating sequence types, ST-7363 and ST-1901, have acquired resistance to third-generation cephalosporins, mainly due to mosaic penA alleles. These two STs were first detected in Japan; however, the timeline, mechanism, and process of emergence and spread of these mosaic penA alleles to other countries remain unknown. METHODS: We studied the evolution of penA alleles by obtaining the complete genomes from three Japanese ST-1901 clinical isolates harboring mosaic penA allele 34 (penA-34) dating from 2005 and generating a phylogenetic representation of 1075 strains sampled from 35 countries. We also sequenced the genomes of 103 Japanese ST-7363 N. gonorrhoeae isolates from 1996 to 2005 and reconstructed a phylogeny including 88 previously sequenced genomes. RESULTS: Based on an estimate of the time-of-emergence of ST-1901 (harboring mosaic penA-34) and ST-7363 (harboring mosaic penA-10), and > 300 additional genome sequences of Japanese strains representing multiple STs isolated in 1996-2015, we suggest that penA-34 in ST-1901 was generated from penA-10 via recombination with another Neisseria species, followed by recombination with a gonococcal strain harboring wildtype penA-1. Following the acquisition of penA-10 in ST-7363, a dominant sub-lineage rapidly acquired fluoroquinolone resistance mutations at GyrA 95 and ParC 87-88, by independent mutations rather than horizontal gene transfer. Data in the literature suggest that the emergence of these resistance determinants may reflect selection from the standard treatment regimens in Japan at that time. CONCLUSIONS: Our findings highlight how antibiotic use and recombination across and within Neisseria species intersect in driving the emergence and spread of drug-resistant gonorrhea.

A community-driven resource for genomic epidemiology and antimicrobial resistance prediction of Neisseria gonorrhoeae at Pathogenwatch.

BACKGROUND: Antimicrobial-resistant (AMR) Neisseria gonorrhoeae is an urgent threat to public health, as strains resistant to at least one of the two last-line antibiotics used in empiric therapy of gonorrhoea, ceftriaxone and azithromycin, have spread internationally. Whole genome sequencing (WGS) data can be used to identify new AMR clones and transmission networks and inform the development of point-of-care tests for antimicrobial susceptibility, novel antimicrobials and vaccines. Community-driven tools that provide an easy access to and analysis of genomic and epidemiological data is the way forward for public health surveillance. METHODS: Here we present a public health-focussed scheme for genomic epidemiology of N. gonorrhoeae at Pathogenwatch ( https://pathogen.watch/ngonorrhoeae ). An international advisory group of experts in epidemiology, public health, genetics and genomics of N. gonorrhoeae was convened to inform on the utility of current and future analytics in the platform. We implement backwards compatibility with MLST, NG-MAST and NG-STAR typing schemes as well as an exhaustive library of genetic AMR determinants linked to a genotypic prediction of resistance to eight antibiotics. A collection of over 12,000 N. gonorrhoeae genome sequences from public archives has been quality-checked, assembled and made public together with available metadata for contextualization. RESULTS: AMR prediction from genome data revealed specificity values over 99% for azithromycin, ciprofloxacin and ceftriaxone and sensitivity values around 99% for benzylpenicillin and tetracycline. A case study using the Pathogenwatch collection of N. gonorrhoeae public genomes showed the global expansion of an azithromycin-resistant lineage carrying a mosaic mtr over at least the last 10 years, emphasising the power of Pathogenwatch to explore and evaluate genomic epidemiology questions of public health concern. CONCLUSIONS: The N. gonorrhoeae scheme in Pathogenwatch provides customised bioinformatic pipelines guided by expert opinion that can be adapted to public health agencies and departments with little expertise in bioinformatics and lower-resourced settings with internet connection but limited computational infrastructure. The advisory group will assess and identify ongoing public health needs in the field of gonorrhoea, particularly regarding gonococcal AMR, in order to further enhance utility with modified or new analytic methods.

Lateral Gene Transfer Shapes Diversity of Gardnerella spp.

Gardnerella spp. are pathognomonic for bacterial vaginosis, which increases the risk of preterm birth and the transmission of sexually transmitted infections. Gardnerella spp. are genetically diverse, comprising what have recently been defined as distinct species with differing functional capacities. Disease associations with Gardnerella spp. are not straightforward: patients with BV are usually infected with multiple species, and Gardnerella spp. are also found in the vaginal microbiome of healthy women. Genome comparisons of Gardnerella spp. show evidence of lateral gene transfer (LGT), but patterns of LGT have not been characterized in detail. Here we sought to define the role of LGT in shaping the genetic structure of Gardnerella spp. We analyzed whole genome sequencing data for 106 Gardnerella strains and used these data for pan genome analysis and to characterize LGT in the core and accessory genomes, over recent and remote timescales. In our diverse sample of Gardnerella strains, we found that both the core and accessory genomes are clearly differentiated in accordance with newly defined species designations. We identified putative competence and pilus assembly genes across most species; we also found them to be differentiated between species. Competence machinery has diverged in parallel with the core genome, with selection against deleterious mutations as a predominant influence on their evolution. By contrast, the virulence factor vaginolysin, which encodes a toxin, appears to be readily exchanged among species. We identified five distinct prophage clusters in Gardnerella genomes, two of which appear to be exchanged between Gardnerella species. Differences among species are apparent in their patterns of LGT, including their exchange with diverse gene pools. Despite frequent LGT and co-localization in the same niche, our results show that Gardnerella spp. are clearly genetically differentiated and yet capable of exchanging specific genetic material. This likely reflects complex interactions within bacterial communities associated with the vaginal microbiome. Our results provide insight into how such interactions evolve and are maintained, allowing these multi-species communities to colonize and invade human tissues and adapt to antibiotics and other stressors.

Increased power from conditional bacterial genome-wide association identifies macrolide resistance mutations in Neisseria gonorrhoeae.

The emergence of resistance to azithromycin complicates treatment of Neisseria gonorrhoeae, the etiologic agent of gonorrhea. Substantial azithromycin resistance remains unexplained after accounting for known resistance mutations. Bacterial genome-wide association studies (GWAS) can identify novel resistance genes but must control for genetic confounders while maintaining power. Here, we show that compared to single-locus GWAS, conducting GWAS conditioned on known resistance mutations reduces the number of false positives and identifies a G70D mutation in the RplD 50S ribosomal protein L4 as significantly associated with increased azithromycin resistance (p-value = 1.08 × 10-11). We experimentally confirm our GWAS results and demonstrate that RplD G70D and other macrolide binding site mutations are prevalent (present in 5.42% of 4850 isolates) and widespread (identified in 21/65 countries across two decades). Overall, our findings demonstrate the utility of conditional associations for improving the performance of microbial GWAS and advance our understanding of the genetic basis of macrolide resistance.

Targeted surveillance strategies for efficient detection of novel antibiotic resistance variants.

Genotype-based diagnostics for antibiotic resistance represent a promising alternative to empiric therapy, reducing inappropriate antibiotic use. However, because such assays infer resistance based on known genetic markers, their utility will wane with the emergence of novel resistance. Maintenance of these diagnostics will therefore require surveillance to ensure early detection of novel resistance variants, but efficient strategies to do so remain undefined. We evaluate the efficiency of targeted sampling approaches informed by patient and pathogen characteristics in detecting antibiotic resistance and diagnostic escape variants in Neisseria gonorrhoeae, a pathogen associated with a high burden of disease and antibiotic resistance and the development of genotype-based diagnostics. We show that patient characteristic-informed sampling is not a reliable strategy for efficient variant detection. In contrast, sampling informed by pathogen characteristics, such as genomic diversity and genomic background, is significantly more efficient than random sampling in identifying genetic variants associated with resistance and diagnostic escape.