Biology subjects

Grad, Y. H.

Publications and source records attributed to Grad, Y. H..

At least 19 recordsLinked to original sources

bioRxiv2026

In vitro evaluation of the activity of pairwise combinations of zoliflodacin, gepotidacin, and ciprofloxacin against Neisseria gonorrhoeae

Two new antibiotics, zoliflodacin and gepotidacin, were recently approved for the treatment of urogenital gonorrhea. While combination therapy could, in principle, delay the emergence and spread of resistance, doing so depends on the absence of antagonism between the co-administered drugs. Using in vitro checkerboard testing, we observed no evidence of antagonism for all pairwise combinations of zoliflodacin, gepotidacin, and ciprofloxacin, including in strains with elevated ciprofloxacin MICs.

microbiology↗

bioRxiv2026

Fluoroquinolone resistance-conferring gyrA variants alter the fitness cost and potentiate the resistance of the zoliflodacin resistance mutation gyrB D429N in Neisseria gonorrhoeae

Neisseria gonorrhoeae is a major public health concern due to its high global prevalence and rapid evolution of antibiotic resistance. A first-in-class topoisomerase inhibitor, zoliflodacin (a spiropyrimidinetrione) recently received FDA approval for treatment of gonorrhea, but its potential for cross-resistance with another topoisomerase inhibitor, the fluoroquinolone antibiotic ciprofloxacin, remains poorly understood. Here, we investigated how genetic diversity in the fluoroquinolone target gyrA influences the resistance and fitness effects of the zoliflodacin resistance mutation gyrBD429N. We constructed an isogenic panel of N. gonorrhoeae to determine how the resistance and fitness effects of the gyrBD429N mutation are modulated by the most common ciprofloxacin resistance-associated variants in gyrA. In the presence of gyrBD429N, the zoliflodacin minimum inhibitory concentration (MIC) was 2-4-fold higher in strains that also contained ciprofloxacin resistance-associated gyrA alleles, and the gyrBD429N mutation reciprocally increased ciprofloxacin MICs of these strains 3-6-fold. Fitness cost of the gyrBD429N mutation varied from modest to severe across gyrA backgrounds, with the largest cost in ciprofloxacin resistant gyrA91F/95G and gyrA91F/95N backgrounds and comparatively minimal cost in the ciprofloxacin resistant gyrA91F/95A background. These results demonstrate the capacity for epistatic interactions among resistance-associated gyrA and gyrB mutations, underscoring the need for genomic surveillance to monitor high-risk combinations of resistance determinants as new therapies are deployed.

microbiology↗

bioRxiv2026

Aerocavin is an antibiotic with potent and specific anti-Neisserial activity

Gonorrhea, caused by N. gonorrhoeae, is a widespread sexually transmitted disease that is becoming resistant to all currently used antibiotics. Therefore, new therapeutics against gonorrhea are desperately needed. Here, we show that a natural product - aerocavin, is highly potent and specific against Neisseria. Aerocavin accumulates in N. gonorrhoeae at high levels and inhibits bacterial RNA polymerase (RNAP) by binding the switch region. Aerocavin resistance mutations evolve in N. gonorrhoeae at a low rate and are absent in clinical isolates. Previously overlooked narrow-spectrum antimicrobials like aerocavin may enable microbiome-sparing treatments of gonorrhea.

microbiology↗

bioRxiv2026

Diversity and evolution of a phase-variable multi-locus antigen in Neisseria gonorrhoeae

Neisseria gonorrhoeae is a sexually transmitted bacterial pathogen that deploys multiple mechanisms to evade the immune system, including rapid variation in surface antigens. One of the most abundant and diverse antigens is the Opacity (Opa) protein, a surface protein that mediates gonococcal attachment to host receptors. Studies of Opa diversity and evolution have been limited by the inability of short-read sequencing to resolve the multiple copies of opa in each genome, preventing a comprehensive understanding of antigenic variation for vaccine design and immunology studies. We assembled a dataset of 219 complete genomes from diverse clinical isolates using long-read sequencing and developed bioinformatics and phylogenetics tools to assess opa variation quantitatively. Each genome had on average 7 distinct opa alleles at 9 to 12 opa loci, and almost all isolates had at least one pair of identical or near-identical opa genes. Fewer opa genes were in frame, and thus inferred to be expressed, than expected due to chance. While genomic distance between isolates correlated with overall opa allele sequence similarity, opa genes were on average 74 times more diverse than the rest of the genome. One opa locus evolved more rapidly than the other loci. There was little evidence that interspecies recombination contributed to N. gonorrhoeae opa diversity. Our findings reveal a continuously evolving opa repertoire that leads to diverse opa alleles even in closely related strains and indicate that there are likely unknown biological factors modulating opa expression. Author SummaryThe rising levels of antibiotic resistance in Neisseria gonorrhoeae make controlling the spread of this sexually transmitted pathogen a public health priority. N. gonorrhoeae rapidly varies surface proteins to evade recognition by the human adaptive immune system. Understanding how these proteins evolve may help us design better vaccines and control measures for curbing the spread of gonorrhea. One of the most abundant surface proteins is the Opacity protein (Opa), which helps N. gonorrhoeae bind to host cells upon colonization. Research efforts to understand the evolution of Opa have been limited because it is encoded by multiple genes in the genome that are not resolved by short-read sequencing technologies. Here, we resolved the genes that encode Opa using a dataset of 132 publicly available complete genomes and 87 genomes that we completed using long-read sequencing of diverse clinical isolates. We found that Opa evolves rapidly to generate different versions of the protein in the same isolate, but very few of these protein versions appear expressed. We also found evidence that there may be other, uncharacterized mechanisms that control how these proteins evolve over longer timescales.

evolutionary biology↗

bioRxiv2025

Zoliflodacin and gepotidacin cross-resistance in Neisseria gonorrhoeae

Resistance to zoliflodacin, a first-in-class antibiotic for gonorrhea treatment, can occur through gyrBD429N, but this mutations impact on fitness and resistance to other topoisomerase targeting drugs, including gepotidacin, have been unclear. Here, we show that gyrBD429N confers cross-resistance to gepotidacin in some clinical isolates and that its fitness effect varies with strain back-ground. These findings inform strategies for introducing the new topoisomerase inhibitors into clinical use and for surveillance of resistance.

microbiology↗

bioRxiv2025

The role of the L421P mutation in Penicillin-Binding Protein 1 (PBP1) in the evolution of chromosomally mediated penicillin resistance in Neisseria gonorrhoeae

ponAL421P encodes a mutated variant of penicillin-binding protein 1 (PBP1) and is a key resistance determinant that increases the penicillin MIC (MICPEN) above the clinical breakpoint in Neisseria gonorrhoeae. Despite the removal of penicillin from treatment guidelines for gonococcal infections in the 1980s, ponAL421P is present in nearly 50% of current N. gonorrhoeae isolates in the PubMLST database. Bioinformatic analysis indicates that ponAL421P is exclusive to N. gonorrhoeae isolates, whereas Leu-421 is 100% conserved in other Neisseria species. To understand the involvement of ponAL421P in antibiotic resistance, we introduced ponA variants encoding 16 different amino acids at position-421 into FA6140, a penicillin-resistant gonococcal isolate that naturally harbors ponAL421P. Proline-421 was the only mutation that increased the MICPEN to the same level as FA6140. We also assessed the fitness of strains with the 16 mutant ponA alleles over multiple serial passages, both with and without sub-MIC levels of penicillin. There was no fitness defect attributed to ponAL421P under these experimental conditions; instead, our analyses suggest that the widespread occurrence of ponAL421P is driven by its capacity to increase the MICpen above the clinical breakpoint. In FA6140 transformed with the mosaic penA allele from strain H041, a ceftriaxone-resistant isolate, ponAL421P increased the MIC of ceftriaxone, suggesting that ceftriaxone targets PBP1 in this strain. We conclude that the ponAL421P allele emerged in gonococcal isolates, increasing the MICPEN above the clinical breakpoint, and has remained in the population even after the removal of penicillin from treatment guidelines. ImportanceThe emergence of antibiotic-resistant Neisseria gonorrhoeae threatens effective treatment of gonorrhea, one of the most common sexually transmitted infections worldwide. Understanding the genetic changes that drive and maintain resistance is crucial for anticipating future resistance trends. Here, we investigated the impact of a key resistance mutation in PBP1 (encoded by ponAL421P). Although penicillin has not been used to treat gonorrhea for decades, this mutation remains widespread even in recent N. gonorrhoeae isolates. ponAL421P confers clinically relevant penicillin resistance without imposing an in vitro fitness cost. ponAL421P also increases resistance to ceftriaxone in strains with penA alleles that are associated with ceftriaxone resistance. This work highlights the role of the ponAL421P allele in shaping the current antibiotic resistance landscape and supports the need for ongoing surveillance and evolutionary studies of such mutations in the gonococcal population.

microbiology↗

bioRxiv2025

Quantifying the impact of genetic determinants of antibiotic resistance on bacterial lineage dynamics

The dynamics of antimicrobial resistance in bacterial populations are informed by the fitness impact of genetic determinants of resistance and antibiotic pressure. However, estimates of real-world fitness impact have been lacking. To address this gap, we developed a hierarchical Bayesian phylodynamic model to quantify contributions of resistance determinants to strain success in a 20-year collection of Neisseria gonorrhoeae isolates. Fitness contributions varied with antibiotic use, and genetic pathways to phenotypically identical resistance conferred distinct fitness effects. These findings were supported by in vitro and experimental infection competition. Quantifying these fitness contributions to lineage dynamics reveals opportunities for investigation into other genetic and environmental drivers of fitness. This work thus establishes a method for linking pathogen genomics and antibiotic use to define factors shaping ecological trends.

microbiology↗

bioRxiv2024

Axial asymmetry organizes division plane orthogonality in Neisseria gonorrhoeae

For rod-shaped bacterial model organisms, the division plane is defined by the geometry of the cell. However, for Neisseria gonorrhoeae, a coccoid organism that most commonly exists as a diplococcus and that possesses genes coding for rod-based cell division systems, the relationship between cell geometry and division is unclear. Here, we characterized the organization of N. gonorrhoeae division using a combination of fluorescent probes, genetics, and time-lapse microscopy. We found that the planes of successive cell divisions are orthogonal and temporally overlapping, thereby maintaining diplococcal morphology. Division takes place perpendicular to a subtle long-axis in each coccus. In keeping with the ParABS and the MinCDE systems reading the long-axis of rod-shaped bacteria, in the coccoid N. gonorrhoeae, ParB segregates along this subtle long-axis and cells lacking minCDE have severe morphological consequences, including an inability to perform orthogonal division and aberrant assembly of the division plane at the cell poles. Taken together, this stresses the central role of even slight dimensional asymmetry as a general organizational principle in bacterial cell division.

microbiology↗

bioRxiv2023

The inter-continental population dynamics of Neisseria gonorrhoeae

Neisseria gonorrhoeae is a globally distributed sexually transmitted bacterial pathogen. Recent studies have revealed that its evolution has been shaped by antibiotic use, while molecular surveillance efforts have demonstrated large changes in lineage composition over relatively short time-spans. However, the global population dynamics of N. gonorrhoeae remain unsatisfyingly characterized. To reconstruct recent large-scale population dynamics, we generated a dated phylogeny from 9,732 N. gonorrhoeae genomes and found the effective population size of the species to have expanded gradually over the past two centuries. While the effective population size of clades with reduced susceptibility to extended-spectrum cephalosporins started declining around 2010, a major clade containing a mosaic mtr operon associated with cephalosporin susceptibility and decreased azithromycin did not display any reduction in population size. Using ancestral trait reconstruction analyses, we delineated transmission lineages, defined as groups of sequences in which all the samples can be traced back to the same import event to a given location. Import, export and local transmission dynamics across two densely sampled locations (Norway and Victoria, Australia) were investigated in detail. Norway exhibited substantially higher rates of strain import and export compared to Victoria, where incidence was to a larger extent fuelled by locally transmitted lineages. Taken together, our work highlights the power of large-scale phylogenomic analyses to uncover the complex dynamics of lineage transmission in N. gonorrhoeae.

microbiology↗

bioRxiv2023

Bayesian modeling of the impact of antibiotic resistance on the efficiency MRSA decolonization

Methicillin-resistant Staphylococcus aureus (MRSA) is a major cause of morbidity and mortality and one of the most serious infectious disease threats. Colonization by MRSA increases the risk of infection and transmission, underscoring the importance of decolonization efforts. However, success of these decolonization protocols varies, raising the possibility that some MRSA strains may be more persistent than others. Here, we studied how the persistence of MRSA colonization correlates with genomic presence of antibiotic resistance genes. Our analysis using a Bayesian mixed effects survival model found that high-level resistance to mupirocin was strongly associated with failure of the decolonization protocol. However, we did not see a similar effect with resistance to chlorhexidine or other antibiotics. Including strain-specific random effects improved the predictive performance, indicating that some strain characteristics other than resistance also contributed to persistence. Study subject-specific random effects did not improve the model. Our results highlight the need to consider the properties of the colonizing MRSA strain when deciding which treatments to include in the decolonization protocol. Author summaryMethicillin-resistant Staphylococcus aureus (MRSA) is responsible for a high burden of morbidity and mortality. MRSA colonization incurs risk of MRSA infection and transmission, highlighting the need for highly effective decolonization protocols. However, decolonization protocols have had mixed success. The extent to which this mixed success is attributable to MRSA strain variations and their resistance to antibiotics, including those like mupirocin that are commonly used for decolonization, versus study subject factors, has been unclear. Here, we characterized the effect of antibiotic resistance genes on the efficiency of an MRSA decolonization protocol. We found that mupirocin resistance and strain-specific effects were associated with reduced effectiveness of an MRSA decolonization protocol, but that resistance to other antibiotics, including purported chlorhexidine resistance genes, and subject-specific effects had no discernible impact on protocol success. Our results highlight the need to consider the properties of the colonizing MRSA strain when deciding which treatments to include in the decolonization protocol.

microbiology↗

bioRxiv2022

Estimating the fitness cost and benefit of antimicrobial resistance from pathogen genomic data

Increasing levels of antibiotic resistance in many bacterial pathogen populations is a major threat to public health. Resistance to an antibiotic provides a fitness benefit when the bacteria is exposed to this antibiotic, but resistance also often comes at a cost to the resistant pathogen relative to susceptible counterparts. We lack a good understanding of these benefits and costs of resistance for many bacterial pathogens and antibiotics, but estimating them could lead to better use of antibiotics in a way that reduces or prevents the spread of resistance. Here, we propose a new model for the joint epidemiology of susceptible and resistant variants, which includes explicit parameters for the cost and benefit of resistance. We show how Bayesian inference can be performed under this model using phylogenetic data from susceptible and resistant lineages and that by combining data from both we are able to disentangle and estimate the resistance cost and benefit parameters separately. We applied our inferential methodology to several simulated datasets to demonstrate good scalability and accuracy. We analysed a dataset of Neisseria gonorrhoeae genomes collected between 2000 and 2013 in the USA. We found that two unrelated lineages resistant to fluoroquinolones shared similar epidemic dynamics and resistance parameters. Fluoroquinolones were abandoned for the treatment of gonorrhoea due to increasing levels of resistance, but our results suggest that they could be used to treat a minority of around 10% of cases without causing resistance to grow again.

evolutionary biology↗

bioRxiv2022

Recent O-antigen diversification masks highly pathogenic STEC O104:H4

Shiga toxin-producing E. coli (STEC) can give rise to a range of clinical outcomes from diarrhea to the life-threatening systemic condition, hemolytic uremic syndrome (HUS). Although STEC O157:H7 is the serotype most frequently associated with HUS, a major outbreak of HUS occurred in 2011 in Germany, and was caused by a rare serotype, STEC O104:H4. Prior to 2011 and since the outbreak, STEC O104:H4 strains have only rarely been associated with human infections. From 2012 to 2020 intensified STEC surveillance was performed in Germany where subtyping of [~]8,000 clinical isolates by molecular methods including whole genome sequencing was carried out. A rare STEC serotype O181:H4 associated with HUS was identified and, like the STEC O104:H4 outbreak strain, this strain belongs to sequence type (ST) 678. Genomic and virulence comparisons revealed that the two strains are phylogenetically related and differ principally in the gene cluster encoding their respective lipopolysaccharide O-antigens but exhibit similar virulence phenotypes. In addition, five other serotypes belonging to ST678 from human clinical infection, such as OX13:H4, O127:H4, OgN-RKI9:H4, O131:H4, and O69:H4, were identified from diverse locations worldwide. ImportanceOur data suggest the high virulence ensemble of the STEC O104:H4 outbreak strain remains a global threat because genomically similar strains cause disease worldwide, but that horizontal acquisition of O-antigen gene clusters has diversified the O-antigens of strains belonging to ST678. Thus, identification of these highly pathogenic strains is masked by diverse and rare O-antigens, thereby confounding the interpretation of their potential risk.

microbiology↗

bioRxiv2022

Neisseria gonorrhoeae diagnostic escape from a gyrA-based test for ciprofloxacin susceptibility can lead to increased zoliflodacin resistance

The etiologic agent of gonorrhea, Neisseria gonorrhoeae, has become resistant to each of the first line antibiotics used to treat it. However, most isolates in the US remain susceptible to ciprofloxacin, leading to the hypothesis that a diagnostic that accurately reports susceptibility to ciprofloxacin may allow its reintroduction into clinical use. One diagnostic approach is to identify codon 91 in gyrA, where coding for the wildtype serine is associated with ciprofloxacin susceptibility and phenylalanine with resistance. Here, we found that escape from this diagnostic method can occur through gyrA F91S reversion and expansion of pre-existing ciprofloxacin non-susceptible N. gonorrhoeae lineages with gyrA 91S. Furthermore, we found that mutations conferring ciprofloxacin resistance in gyrA 91S isolates led to cross-resistance with zoliflodacin, an antibiotic in phase 3 trial for treatment of gonorrhea. These findings define N. gonorrhoeae mutations that should be monitored via surveillance systems, particularly in populations and regions deploying gyrA 91-based diagnostics, and demonstrate how diagnostics that guide antibiotic therapy can have unintended consequences.

microbiology↗

bioRxiv2022

Variation in supplemental carbon dioxide requirements defines lineage-specific antibiotic resistance acquisition in Neisseria gonorrhoeae

The evolution of the obligate human pathogen Neisseria gonorrhoeae has been shaped by selective pressures from diverse host niche environments1,2 as well as antibiotics3,4. The varying prevalence of antibiotic resistance across N. gonorrhoeae lineages5 suggests that underlying metabolic differences may influence the likelihood of acquisition of specific resistance mutations6,7. We hypothesized that the requirement for supplemental CO2, present in approximately half of isolates8, reflects one such example of metabolic variation. Here, using a genome-wide association study and experimental investigations, we show that CO2-dependence is attributable to a single substitution in a {beta}-carbonic anhydrase, canB. CanB19E is necessary and sufficient for growth in the absence of CO2, and the hypomorphic CanB19G variant confers CO2-dependence. Furthermore, ciprofloxacin resistance is correlated with CanB19G in clinical isolates, and the presence of CanB19G increases the likelihood of acquisition of ciprofloxacin resistance. Together, our results suggest that metabolic variation has impacted the acquisition of fluoroquinolone resistance.

microbiology↗

bioRxiv2021

Genome wide association studies define minimal set of loci for prediction of penicillin and tetracycline susceptibility in Neisseria gonorrhoeae

BackgroundWhile Neisseria gonorrhoeae poses an urgent public health threat because of increasing antimicrobial resistance, 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 limited to ciprofloxacin, for which susceptibility can be predicted from a single locus. MethodsWe assembled a dataset of 12,045 N. gonorrhoeae genomes with phenotypic resistance data for tetracycline (n = 3,611) and penicillin (n = 6,935). Using conditional genome wide association studies (GWAS), we sought to define genetic variants associated with susceptibility to penicillin and tetracycline. We evaluated the sensitivity and specificity of these variants for predicting susceptibility and non-resistance in our collection of gonococcal genomes. FindingsIn our conditional penicillin GWAS, the presence of a genetic variant defined by a non-mosaic penA allele without an insertion at codon 345 was significantly associated with penicillin susceptibility and had the highest negative effect size of significant variants (p = 5.0 x 10-14, {beta} = -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 wild type allele at rpsJ codon 57, encoding valine, was significantly associated with tetracycline susceptibility (p = 5.6 x 10-16, {beta} = -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%). InterpretationAs 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 gonorrhea. FundingNational Institute of Allergy and Infectious Diseases, the National Science Foundation, and the Smith Family Foundation Research in ContextO_ST_ABSEvidence before this studyC_ST_ABSWe searched PubMed with the terms "Neisseria gonorrhoeae" and "diagnostic" or "assay" plus "penicillin" or "tetracycline" for reports in any language published up to July 1, 2021. We additionally searched for "Neisseria gonorrhoeae" and "genome wide association study". We found that previously proposed molecular diagnostics for penicillin and tetracycline susceptibility either exclusively focused on plasmid-mediated resistance (i.e., targeting blaTEM or tetM) or did not include variants in genes encoding antibiotic targets (e.g., did not include penA or rpsJ). Targets for molecular surveillance have focused on resistance-associated alleles rather than susceptibility-associated alleles. We did not find any previous penicillin or tetracycline conditional genome wide association studies (GWAS) in N. gonorrhoeae. Added value of this studyTo identify targets for molecular diagnostics that predict penicillin and tetracycline susceptibility, we conducted GWAS conditioning on the presence of plasmid-mediated resistance determinants to detect chromosomal loci with the highest association with susceptibility. We discovered a sequence (penA_01) that differentiates susceptible isolates from those with a resistance-associated insertion at codon 345 and from those with mosaic penA alleles, which is associated with penicillin susceptibility. We also found that rpsJ codon 57 was the chromosomal locus contributing the most to tetracycline susceptibility. The combination of these chromosomal loci and the absence of plasmid encoded determinants predicts penicillin and tetracycline susceptibility with high specificity in both a large global collection of N. gonorrhoeae and a validation dataset consisting of recently published genomes from CDCs Gonococcal Isolate Surveillance Program (GISP) surveillance collected in 2018. Implications of all the available evidenceThe chromosomal loci penA_01 and rpsJ codon 57 in combination with plasmid loci blaTEM and tetM are candidates for the development of point-of-care molecular diagnostics for penicillin and tetracycline susceptibility. The loci may be combined with the currently available ciprofloxacin susceptibility diagnostics to predict susceptibility to multiple antibiotics. Additionally, our study suggests that conditional GWAS focused on variants associated with susceptibility may be a promising approach to identify minimal sets of loci for molecular diagnostics and surveillance.

microbiology↗

bioRxiv2020

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

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 treatment with third-generation cephalosporins mainly due to the emergence of mosaic penA alleles. These two STs were first detected in Japan; however, when and how the mosaic penA alleles emerged and spread to other countries remains unknown. Here, we addressed the evolution of penA alleles by obtaining 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 1,075 strains sampled from 37 countries. We also sequenced the genomes of 103 Japanese ST-7363 N. gonorrhoeae isolates from 1996-2005 and reconstructed a phylogeny including 88 previously sequenced genomes. 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 a second recombination event 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, possibly due to independent mutations rather than horizontal gene transfer. Literature data suggest the emergence of these resistance determinants may reflect selection from the standard treatment regimens in Japan at that time. Our findings highlight how recombination and antibiotic use across and within Neisseria species intersect in driving the emergence and spread of drug-resistant gonorrhea. Author summaryAntimicrobial resistance is recognized as one of the greatest threats to human health, and Neisseria gonorrhoeae resistance is classified as one of the most urgent. The two major internationally spreading lineages resistant. to first line drugs likely originated in Japan, but when and how their genetic resistance determinants emerged remain unknown. In this study, we conducted an evolutionary analysis using clinical N. gonorrhoeae isolates from 37 countries, including a historical collection of Japanese isolates, to investigate the emergence of resistance in each of the two major lineages. We showed that the penA allele responsible for resistance to cephalosporins, the first-line treatment for gonorrhea, was possibly generated by two recombination events, one from another Neisseria species and one from another N. gonorrhoeae lineage. We also showed that mutations responsible for resistance to a previously widely used antibiotic treatment occurred twice independently in one of the two major lineages. The emergence of the genetic resistance determinants potentially reflects selection from the standard treatment regimen at that time. Our findings highlight how recombination (horizontal gene transfer) and antibiotic use across and within a bacterial species intersect in driving the emergence and spread of antimicrobial resistance genes and mutations.

microbiology↗

bioRxiv2020

A community-driven resource for genomic surveillance of Neisseria gonorrhoeae at Pathogenwatch

BackgroundAntimicrobial 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, 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. MethodsHere 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. ResultsAMR 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, emphasizing the power of Pathogenwatch to explore and evaluate genomic epidemiology questions of public health concern. ConclusionsThe N. gonorrhoeae scheme in Pathogenwatch provides customized 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.

genomics↗

bioRxiv2020

Efflux pump antibiotic binding site mutations are associated with azithromycin nonsusceptibility in clinical Neisseria gonorrhoeae isolates

Lyu and Moseng et al. used cryo-electron microscopy to characterize key residues involved in drug binding by mosaic-like MtrD efflux pump alleles in Neisseria gonorrhoeae (1). Isogenic experiments introducing key MtrD substitutions R714G and K823E increased macrolide MICs, leading the authors to predict that non-mosaic MtrD "gonococcal strains bearing both the mtrR promoter and amino acid changes at MtrD positions 714 or 823 could lead to clinically significant levels of Azi nonsusceptibility resistance". We tested this hypothesis by analyzing a global meta-analysis collection of 4852 N. gonorrhoeae genomes (2). In support of their prediction, we identified clinical isolates with novel non-mosaic MtrD drug binding site substitutions across multiple genetic backgrounds associated with elevated azithromycin MICs (Table).

microbiology↗