Evaluation of Urethrotropic-Clade Meningococcal Infection by Urine Metagenomic Shotgun Sequencing.

Urethral infections caused by an emerging nongroupable (NG) urethrotropic clade of Neisseria meningitidis were first reported in the United States in 2015 (the "U.S. NmNG urethritis clade"). Here, we evaluate for the presence of other urethral pathogens in men with U.S. NmNG urethritis clade infection. We evaluated 129 urine specimens collected from men at a sexual health clinic, including 33 from patients with culture-confirmed or suspected urethral N. meningitidis infection and 96 specimens in which nucleic acid amplification test detected Neisseria gonorrhoeae, Chlamydia trachomatis, both pathogens, or neither pathogen. N. meningitidis was detected first by real-time PCR, followed by metagenomic shotgun sequencing of 91 specimens to identify coinfections. N. meningitidis genomes were sequenced following selective whole-genome amplification when possible. Metagenomic sequencing detected N. meningitidis in 16 of 17 specimens from culture-confirmed N. meningitidis cases, with no coinfection by other conventional urethral pathogens. Metagenomic sequencing also detected N. meningitidis in three C. trachomatis-positive specimens, one specimen positive for both N. gonorrhoeae and C. trachomatis, and nine specimens with negative N. gonorrhoeae and C. trachomatis results, eight of which had suspected Neisseria infections. N. meningitidis from culture-confirmed N. meningitidis cases belonged to the U.S. NmNG urethritis clade, while N. meningitidis identified in other specimens belonged to multiple clonal complexes. Additional urethral pathogens were predominant in non-N. meningitidis specimens, including N. gonorrhoeae, C. trachomatis, Mycoplasma genitalium, Ureaplasma urealyticum, and herpes simplex virus 2. Coinfection with other conventional urethral pathogens is rare in men with culture-confirmed U.S. NmNG urethritis clade infection and points to the strong association of this clade with disease.

Genomic Analysis of the Predominant Strains and Antimicrobial Resistance Determinants Within 1479 Neisseria gonorrhoeae Isolates From the US Gonococcal Isolate Surveillance Project in 2018.

BACKGROUND: The prevalence of Neisseria gonorrhoeae (GC) isolates with elevated minimum inhibitory concentrations to various antibiotics continues to rise in the United States and globally. Genomic analysis provides a powerful tool for surveillance of circulating strains, antimicrobial resistance determinants, and understanding of transmission through a population. METHODS: Neisseria gonorrhoeae isolates collected from the US Gonococcal Isolate Surveillance Project in 2018 (n = 1479) were sequenced and characterized. Whole-genome sequencing was used to identify sequence types, antimicrobial resistance profiles, and phylogenetic relationships across demographic and geographic populations. RESULTS: Genetic characterization identified that (1) 80% of the GC isolates were represented in 33 multilocus sequence types, (2) isolates clustered in 23 major phylogenetic clusters with select phenotypic and demographic prevalence, and (3) common antimicrobial resistance determinants associated with low-level or high-level decreased susceptibility or resistance to relevant antibiotics. CONCLUSIONS: Characterization of this 2018 Gonococcal Isolate Surveillance Project genomic data set, which is the largest US whole-genome sequence data set to date, sets the basis for future prospective studies, and establishes a genomic baseline of GC populations for local and national monitoring.

Full Molecular Typing of Neisseria meningitidis Directly from Clinical Specimens for Outbreak Investigation.

Neisseria meningitidis is a leading cause of bacterial meningitis and sepsis worldwide and an occasional cause of meningococcal urethritis. When isolates are unavailable for surveillance or outbreak investigations, molecular characterization of pathogens needs to be performed directly from clinical specimens, such as cerebrospinal fluid (CSF), blood, or urine. However, genome sequencing of specimens is challenging because of low bacterial and high human DNA abundances. We developed selective whole-genome amplification (SWGA), an isothermal multiple-displacement amplification-based method, to efficiently enrich, sequence, and de novo assemble N. meningitidis DNA from clinical specimens with low bacterial loads. SWGA was validated with 12 CSF specimens from invasive meningococcal disease cases and 12 urine specimens from meningococcal urethritis cases. SWGA increased the mean proportion of N. meningitidis reads by 2 to 3 orders of magnitude, enabling identification of at least 90% of the 1,605 N. meningitidis core genome loci for 50% of the specimens. The validated method was used to investigate two meningitis outbreaks recently reported in Togo and Burkina Faso. Twenty-seven specimens with low bacterial loads were processed by SWGA before sequencing, and 12 of 27 were successfully assembled to obtain the full molecular typing and vaccine antigen profile of the N. meningitidis pathogen, thus enabling thorough characterization of outbreaks. This method is particularly important for enhancing molecular surveillance in regions with low culture rates. SWGA produces enough reads for phylogenetic and allelic analysis at a low cost. More importantly, the procedure can be extended to enrich other important human bacterial pathogens.

Emergence of Localized Serogroup W Meningococcal Disease in the United States - Georgia, 2006-2016.

Several countries in Europe and Australia are reporting an increasing incidence of Neisseria meningitidis serogroup W (NmW) as a consequence of the rapid expansion of a single NmW clone belonging to clonal complex 11 (1-5). Because this clone is reported to be associated with more severe disease, unusual clinical presentations, and a high case fatality ratio (CFR), it is considered a hypervirulent strain (1,6). In the United States, NmW accounts for approximately 5% of meningococcal disease reported each year, and this proportion has remained stable for several years (7). However, localized increases in NmW have been reported, most notably in Florida during 2008-2009 (8). In Georgia, NmW accounted for only 3% of meningococcal disease cases reported during 2006-2013; however, between January 2014 and December 2016, 42% of all reported cases were NmW. Surveillance data from Georgia were analyzed to describe the epidemiology and clinical characteristics of NmW cases, and whole-genome sequencing of NmW isolates was performed for comparison with NmW strains circulating in the United States and worldwide. These data indicate that the U.S. NmW strains might have evolved from the same ancestor as the hypervirulent strain that is circulating globally. Genetic analysis demonstrates that these strains are closely related, which would suggest that genetic variation led to the rise of different strains from the same ancestor. Given the recent global expansion of this potentially hypervirulent NmW lineage, clinicians and public health officials need to remain vigilant in obtaining isolates to monitor changes in circulating strains.

Draft sequencing and assembly of the genome of the world's largest fish, the whale shark: Rhincodon typus Smith 1828.

BACKGROUND: The whale shark (Rhincodon typus) has by far the largest body size of any elasmobranch (shark or ray) species. Therefore, it is also the largest extant species of the paraphyletic assemblage commonly referred to as fishes. As both a phenotypic extreme and a member of the group Chondrichthyes - the sister group to the remaining gnathostomes, which includes all tetrapods and therefore also humans - its genome is of substantial comparative interest. Whale sharks are also listed as an endangered species on the International Union for Conservation of Nature's Red List of threatened species and are of growing popularity as both a target of ecotourism and as a charismatic conservation ambassador for the pelagic ecosystem. A genome map for this species would aid in defining effective conservation units and understanding global population structure. RESULTS: We characterised the nuclear genome of the whale shark using next generation sequencing (454, Illumina) and de novo assembly and annotation methods, based on material collected from the Georgia Aquarium. The data set consisted of 878,654,233 reads, which yielded a draft assembly of 1,213,200 contigs and 997,976 scaffolds. The estimated genome size was 3.44Gb. As expected, the proteome of the whale shark was most closely related to the only other complete genome of a cartilaginous fish, the holocephalan elephant shark. The whale shark contained a novel Toll-like-receptor (TLR) protein with sequence similarity to both the TLR4 and TLR13 proteins of mammals and TLR21 of teleosts. The data are publicly available on GenBank, FigShare, and from the NCBI Short Read Archive under accession number SRP044374. CONCLUSIONS: This represents the first shotgun elasmobranch genome and will aid studies of molecular systematics, biogeography, genetic differentiation, and conservation genetics in this and other shark species, as well as providing comparative data for studies of evolutionary biology and immunology across the jawed vertebrate lineages.

Overproduction of the MtrCDE efflux pump in Neisseria gonorrhoeae produces unexpected changes in cellular transcription patterns.

The global consequence of drug efflux gene overexpression in bacteria has not been specifically analyzed because strains showing high-level expression typically have mutations in genes encoding regulatory proteins that control other genes. Results from a transcriptional profiling study performed with a strain of Neisseria gonorrhoeae that is capable of high-level transcription of the mtrCDE efflux pump operon independently of control by cognate regulatory proteins revealed that its overexpression has ramifications for systems other than drug efflux.

Genomic characterization of the Bacillus cereus sensu lato species: backdrop to the evolution of Bacillus anthracis.

The key genes required for Bacillus anthracis to cause anthrax have been acquired recently by horizontal gene transfer. To understand the genetic background for the evolution of B. anthracis virulence, we obtained high-redundancy genome sequences of 45 strains of the Bacillus cereus sensu lato (s.l.) species that were chosen for their genetic diversity within the species based on the existing multilocus sequence typing scheme. From the resulting data, we called more than 324,000 new genes representing more than 12,333 new gene families for this group. The core genome size for the B. cereus s.l. group was ∼1750 genes, with another 2150 genes found in almost every genome constituting the extended core. There was a paucity of genes specific and conserved in any clade. We found no evidence of recent large-scale gene loss in B. anthracis or for unusual accumulation of nonsynonymous DNA substitutions in the chromosome; however, several B. cereus genomes isolated from soil and not previously associated with human disease were degraded to various degrees. Although B. anthracis has undergone an ecological shift within the species, its chromosome does not appear to be exceptional on a macroscopic scale compared with close relatives.

Phase-variable expression of lptA modulates the resistance of Neisseria gonorrhoeae to cationic antimicrobial peptides.

Phosphoethanolamine (PEA) decoration of lipid A produced by Neisseria gonorrhoeae has been linked to bacterial resistance to cationic antimicrobial peptides/proteins (CAMPs) and in vivo fitness during experimental infection. We now report that the lptA gene, which encodes the PEA transferase responsible for this decoration, is in an operon and that high-frequency mutation in a polynucleotide repeat within lptA can influence gonococcal resistance to CAMPs.

Functional genomic characterization of virulence factors from necrotizing fasciitis-causing strains of Aeromonas hydrophila.

The genomes of 10 Aeromonas isolates identified and designated Aeromonas hydrophila WI, Riv3, and NF1 to NF4; A. dhakensis SSU; A. jandaei Riv2; and A. caviae NM22 and NM33 were sequenced and annotated. Isolates NF1 to NF4 were from a patient with necrotizing fasciitis (NF). Two environmental isolates (Riv2 and -3) were from the river water from which the NF patient acquired the infection. While isolates NF2 to NF4 were clonal, NF1 was genetically distinct. Outside the conserved core genomes of these 10 isolates, several unique genomic features were identified. The most virulent strains possessed one of the following four virulence factors or a combination of them: cytotoxic enterotoxin, exotoxin A, and type 3 and 6 secretion system effectors AexU and Hcp. In a septicemic-mouse model, SSU, NF1, and Riv2 were the most virulent, while NF2 was moderately virulent. These data correlated with high motility and biofilm formation by the former three isolates. Conversely, in a mouse model of intramuscular infection, NF2 was much more virulent than NF1. Isolates NF2, SSU, and Riv2 disseminated in high numbers from the muscular tissue to the visceral organs of mice, while NF1 reached the liver and spleen in relatively lower numbers on the basis of colony counting and tracking of bioluminescent strains in real time by in vivo imaging. Histopathologically, degeneration of myofibers with significant infiltration of polymorphonuclear cells due to the highly virulent strains was noted. Functional genomic analysis provided data that allowed us to correlate the highly infectious nature of Aeromonas pathotypes belonging to several different species with virulence signatures and their potential ability to cause NF.

Corrigendum: Patterns of within-host spread of Chlamydia trachomatis between vagina, endocervix and rectum revealed by comparative genomic analysis.

[This corrects the article DOI: 10.3389/fmicb.2023.1154664.].

Genome-wide upstream motif analysis of Cryptosporidium parvum genes clustered by expression profile.

BACKGROUND: There are very few molecular genetic tools available to study the apicomplexan parasite Cryptosporidium parvum. The organism is not amenable to continuous in vitro cultivation or transfection, and purification of intracellular developmental stages in sufficient numbers for most downstream molecular applications is difficult and expensive since animal hosts are required. As such, very little is known about gene regulation in C. parvum. RESULTS: We have clustered whole-genome gene expression profiles generated from a previous study of seven post-infection time points of 3,281 genes to identify genes that show similar expression patterns throughout the first 72 hours of in vitro epithelial cell culture. We used the algorithms MEME, AlignACE and FIRE to identify conserved, overrepresented DNA motifs in the upstream promoter region of genes with similar expression profiles. The most overrepresented motifs were E2F (5'-TGGCGCCA-3'); G-box (5'-G.GGGG-3'); a well-documented ApiAP2 binding motif (5'-TGCAT-3'), and an unknown motif (5'-[A/C] AACTA-3'). We generated a recombinant C. parvum DNA-binding protein domain from a putative ApiAP2 transcription factor [CryptoDB: cgd8_810] and determined its binding specificity using protein-binding microarrays. We demonstrate that cgd8_810 can putatively bind the overrepresented G-box motif, implicating this ApiAP2 in the regulation of many gene clusters. CONCLUSION: Several DNA motifs were identified in the upstream sequences of gene clusters that might serve as potential cis-regulatory elements. These motifs, in concert with protein DNA binding site data, establish for the first time the beginnings of a global C. parvum gene regulatory map that will contribute to our understanding of the development of this zoonotic parasite.

Population genomics of Chlamydia trachomatis: insights on drift, selection, recombination, and population structure.

The large number of sexually transmitted diseases and ocular trachoma cases that are caused globally each year by Chlamydia trachomatis has made this organism a World Health Organization priority for vaccine development. However, there is no gene transfer system for Chlamydia to help identify potential vaccine targets. To accelerate discoveries toward this goal, here we analyzed the broadest diversity of C. trachomatis genomes to date, including 25 geographically dispersed clinical and seven reference strains representing 14 of the 19 known serotypes. Strikingly, all 32 genomes were found to have evidence of DNA acquisition by homologous recombination in their history. Four distinct clades were identified, which correspond to all C. trachomatis disease phenotypes: lymphogranuloma venereum (LGV; Clade 1); noninvasive urogenital infections (Clade 2); ocular trachoma (Clade 3); and protocolitis (Clade 4; also includes some noninvasive urogenital infections). Although the ancestral relationship between clades varied, most strains acted as donor and recipient of recombination with no evidence for barriers to genetic exchange. The niche-specific LGV and trachoma clades have undergone less recombination, although the opportunity for mixing with strains from other clades that infect the rectal and ocular mucosa, respectively, is evident. Furthermore, there are numerous occasions for gene conversion events through sequential infections at the same anatomic sites. The size of recombinant segments is relatively small (~357 bp) compared with in vitro experiments of various C. trachomatis strains but is consistent with in vitro estimates for other bacterial species including Escherichia coli and Helicobacter pylori. Selection has also played a crucial role during the diversification of the organism. Clade 2 had the lowest nonsynonymous to synonymous ratio (dN/dS) but the highest effect of recombination, which is consistent with the widespread occurrence of synonymous substitutions in recombined genomic segments. The trachoma Clade 3 had the highest dN/dS estimates, which may be caused by an increased effect of genetic drift from niche specialization and a reduced effective population size. The degree of drift, selection, and recombination in C. trachomatis suggests that the challenge will remain to identify genomic regions that are stable and cross protective for the development of an efficacious vaccine.

SNPxGE(2): a database for human SNP-coexpression associations.

MOTIVATION: Recently, gene-coexpression relationships have been found to be often conditional and dynamic. Many studies have suggested that single nucleotide polymorphisms (SNPs) have impacts on gene expression variations in human populations. RESULTS: The SNPxGE(2) database contains the computationally predicted human SNP-coexpression associations, i.e. the differential coexpression between two genes is associated with the genotypes of an SNP. These data were generated from a large-scale association study that was based on the HapMap phase I data, which covered 269 individuals from 4 human populations, 556 873 SNPs and 15 000 gene expression profiles. In order to reduce the computational cost, the SNP-coexpression associations were assessed using gap/substitution models, proven to have a comparable power to logistic regression models. The results, at a false discovery rate (FDR) cutoff of 0.1, consisted of 44 769 and 50 792 SNP-coexpression associations based on single and pooled populations, respectively, and can be queried in the SNPxGE(2) database via either gene symbol or reference SNP ID. For each reported association, a detailed information page is provided. AVAILABILITY: http://lambchop.ads.uga.edu/snpxge2/index.php CONTACT: wyp1125@uga.edu, rrekaya@uga.edu.

Patterns of within-host spread of Chlamydia trachomatis between vagina, endocervix and rectum revealed by comparative genomic analysis.

Chlamydia trachomatis , a gram-negative obligate intracellular bacterium, commonly causes sexually transmitted infections (STIs). Little is known about C. trachomatis transmission within the host, which is important for understanding disease epidemiology and progression. We used RNA-bait enrichment and whole-genome sequencing to compare rectal, vaginal and endocervical samples collected at the same time from 26 study participants who attended Fijian Ministry of Health and Medical Services clinics and tested positive for C. trachomatis at each anatomic site. The 78 C. trachomatis genomes from participants were from two major clades of the C. trachomatis phylogeny (the "prevalent urogenital and anorecta"l clade and "non-prevalent urogenital and anorectal" clade). For 21 participants, genome sequences were almost identical in each anatomic site. For the other five participants, two distinct C. trachomatis strains were present in different sites; in two cases, the vaginal sample was a mixture of strains. The absence of large numbers of fixed SNPs between C. trachomatis strains within many of the participants could indicate recent acquisition of infection prior to the clinic visit without sufficient time to accumulate significant variation in the different body sites. This model suggests that many C. trachomatis infections may be resolved relatively quickly in the Fijian population, possibly reflecting common prescription or over-the-counter antibiotics usage. Importance: Chlamydia trachomatis is a bacterial pathogen that causes millions of sexually transmitted infections (STIs) annually across the globe. Because C. trachomatis lives inside human cells, it has historically been hard to study. We know little about how the bacterium spreads between body sites. Here, samples from 26 study participants who had simultaneous infections in their vagina, rectum and endocervix were genetically analyzed using an improved method to extract C. trachomatis DNA directly from clinical samples for genome sequencing. By analyzing patterns of mutations in the genomes, we found that 21 participants shared very similar C. trachomatis strains in all three anatomic sites, suggesting recent infection and spread. For five participants two C. trachomatis strains were evident, indicating multiple infections. This study is significant in that improved enrichment methods for genome sequencing provides robust data to genetically trace patterns of C. trachomatis infection and transmission within an individual for epidemiologic and pathogenesis interrogations.

Patterns of within-host spread of Chlamydia trachomatis between vagina, endocervix and rectum revealed by comparative genomic analysis.

Introduction: Chlamydia trachomatis, a gram-negative obligate intracellular bacterium, commonly causes sexually transmitted infections (STIs). Little is known about C. trachomatis transmission within the host, which is important for understanding disease epidemiology and progression. Methods: We used RNA-bait enrichment and whole-genome sequencing to compare rectal, vaginal and endocervical samples collected at the same time from 26 study participants who attended Fijian Ministry of Health and Medical Services clinics and tested positive for C. trachomatis at each anatomic site. Results: The 78 C. trachomatis genomes from participants resolved into two major clades of the C. trachomatis phylogeny (the "prevalent urogenital and anorectal" clade and "non-prevalent urogenital and anorectal" clade). For 21 participants, genome sequences were almost identical in each anatomic site. For the other five participants, two distinct C. trachomatis strains were present in different sites; in two cases, the vaginal sample was a mixture of strains. Discussion: The absence of large numbers of fixed SNPs between C. trachomatis genomes within many of the participants could indicate recent acquisition of infection prior to the clinic visit without sufficient time to accumulate significant genetic variation in different body sites. This model suggests that many C. trachomatis infections may be resolved relatively quickly in the Fijian population, possibly reflecting common prescription or over-the-counter antibiotics usage.

Genomic analysis of 1710 surveillance-based Neisseria gonorrhoeae isolates from the USA in 2019 identifies predominant strain types and chromosomal antimicrobial-resistance determinants.

This study characterized high-quality whole-genome sequences of a sentinel, surveillance-based collection of 1710 Neisseria gonorrhoeae (GC) isolates from 2019 collected in the USA as part of the Gonococcal Isolate Surveillance Project (GISP). It aims to provide a detailed report of strain diversity, phylogenetic relationships and resistance determinant profiles associated with reduced susceptibilities to antibiotics of concern. The 1710 isolates represented 164 multilocus sequence types and 21 predominant phylogenetic clades. Common genomic determinants defined most strains' phenotypic, reduced susceptibility to current and historic antibiotics (e.g. bla TEM plasmid for penicillin, tetM plasmid for tetracycline, gyrA for ciprofloxacin, 23S rRNA and/or mosaic mtr operon for azithromycin, and mosaic penA for cefixime and ceftriaxone). The most predominant phylogenetic clade accounted for 21 % of the isolates, included a majority of the isolates with low-level elevated MICs to azithromycin (2.0 µg ml-1), carried a mosaic mtr operon and variants in PorB, and showed expansion with respect to data previously reported from 2018. The second largest clade predominantly carried the GyrA S91F variant, was largely ciprofloxacin resistant (MIC ≥1.0 µg ml-1), and showed significant expansion with respect to 2018. Overall, a low proportion of isolates had medium- to high-level elevated MIC to azithromycin ((≥4.0 µg ml-1), based on C2611T or A2059G 23S rRNA variants). One isolate carried the penA 60.001 allele resulting in elevated MICs to cefixime and ceftriaxone of 1.0 µg ml-1. This high-resolution snapshot of genetic profiles of 1710 GC sequences, through a comparison with 2018 data (1479 GC sequences) within the sentinel system, highlights change in proportions and expansion of select GC strains and the associated genetic mechanisms of resistance. The knowledge gained through molecular surveillance may support rapid identification of outbreaks of concern. Continued monitoring may inform public health responses to limit the development and spread of antibiotic-resistant gonorrhoea.

Phylogenomic Comparison of Neisseria gonorrhoeae Causing Disseminated Gonococcal Infections and Uncomplicated Gonorrhea in Georgia, United States.

Disseminated gonococcal infection (DGI) is a rare complication caused by the systemic dissemination of Neisseria gonorrhoeae to normally sterile anatomical sites. Little is known about the genetic diversity of DGI gonococcal strains and how they relate to other gonococcal strains causing uncomplicated mucosal infections. We used whole genome sequencing to characterize DGI isolates (n = 30) collected from a surveillance system in Georgia, United States, during 2017-2020 to understand phylogenetic clustering among DGI as well as uncomplicated uro- and extragenital gonococcal infection (UGI) isolates (n = 110) collected in Fulton County, Georgia, during 2017-2019. We also investigated the presence or absence of genetic markers related to antimicrobial resistance (AMR) as well as surveyed the genomes for putative virulence genetic factors associated with normal human-serum (NHS) resistance that might facilitate DGI. We found that DGI strains demonstrated significant genetic variability similar to the population structure of isolates causing UGI, with sporadic incidences of geographically clustered DGI strains. DGI isolates contained various AMR markers and genetic mechanisms associated with NHS resistance. DGI isolates had a higher frequency of the porB1A allele compared with UGI (67% vs 9%, P < .0001); however, no single NHS resistance marker was found in all DGI isolates. Continued DGI surveillance with genome-based characterization of DGI isolates is necessary to better understand specific factors that promote systemic dissemination.

Global Emergence and Dissemination of Neisseria gonorrhoeae ST-9363 Isolates with Reduced Susceptibility to Azithromycin.

Neisseria gonorrhoeae multilocus sequence type (ST) 9363 core-genogroup isolates have been associated with reduced azithromycin susceptibility (AZMrs) and show evidence of clonal expansion in the United States. Here, we analyze a global collection of ST-9363 core-genogroup genomes to shed light on the emergence and dissemination of this strain. The global population structure of ST-9363 core-genogroup falls into three lineages: Basal, European, and North American; with 32 clades within all lineages. Although, ST-9363 core-genogroup is inferred to have originated from Asia in the mid-19th century; we estimate the three modern lineages emerged from Europe in the late 1970s to early 1980s. The European lineage appears to have emerged and expanded from around 1986 to 1998, spreading into North America and Oceania in the mid-2000s with multiple introductions, along with multiple secondary reintroductions into Europe. Our results suggest two separate acquisition events of mosaic mtrR and mtrR promoter alleles: first during 2009-2011 and again during the 2012-2013 time, facilitating the clonal expansion of this core-genogroup with AZMrs in the United States. By tracking phylodynamic evolutionary trajectories of clades that share distinct demography as well as population-based genomic statistics, we demonstrate how recombination and selective pressures in the mtrCDE efflux operon granted a fitness advantage to establish ST-9363 as a successful gonococcal lineage in the United States and elsewhere. Although it is difficult to pinpoint the exact timing and emergence of this young core-genogroup, it remains critically important to continue monitoring it, as it could acquire additional resistance markers.

Naegleria genus pangenome reveals new structural and functional insights into the versatility of these free-living amoebae.

Introduction: Free-living amoebae of the Naegleria genus belong to the major protist clade Heterolobosea and are ubiquitously distributed in soil and freshwater habitats. Of the 47 Naegleria species described, N. fowleri is the only one being pathogenic to humans, causing a rare but fulminant primary amoebic meningoencephalitis. Some Naegleria genome sequences are publicly available, but the genetic basis for Naegleria diversity and ability to thrive in diverse environments (including human brain) remains unclear. Methods: Herein, we constructed a high-quality Naegleria genus pangenome to obtain a comprehensive catalog of genes encoded by these amoebae. For this, we first sequenced, assembled, and annotated six new Naegleria genomes. Results and Discussion: Genome architecture analyses revealed that Naegleria may use genome plasticity features such as ploidy/aneuploidy to modulate their behavior in different environments. When comparing 14 near-to-complete genome sequences, our results estimated the theoretical Naegleria pangenome as a closed genome, with 13,943 genes, including 3,563 core and 10,380 accessory genes. The functional annotations revealed that a large fraction of Naegleria genes show significant sequence similarity with those already described in other kingdoms, namely Animalia and Plantae. Comparative analyses highlighted a remarkable genomic heterogeneity, even for closely related strains and demonstrate that Naegleria harbors extensive genome variability, reflected in different metabolic repertoires. If Naegleria core genome was enriched in conserved genes essential for metabolic, regulatory and survival processes, the accessory genome revealed the presence of genes involved in stress response, macromolecule modifications, cell signaling and immune response. Commonly reported N. fowleri virulence-associated genes were present in both core and accessory genomes, suggesting that N. fowleri's ability to infect human brain could be related to its unique species-specific genes (mostly of unknown function) and/or to differential gene expression. The construction of Naegleria first pangenome allowed us to move away from a single reference genome (that does not necessarily represent each species as a whole) and to identify essential and dispensable genes in Naegleria evolution, diversity and biology, paving the way for further genomic and post-genomic studies.