Genetic Manipulation of Neisseria gonorrhoeae and Commensal Neisseria Species.

The sexually transmitted pathogen, Neisseria gonorrhoeae, undergoes natural transformation at high frequency. This property has led to the rapid dissemination of antibiotic resistance markers and the panmictic structure of the gonococcal population. However, high-frequency transformation also makes N. gonorrhoeae one of the easiest bacterial species to manipulate genetically in the laboratory. Techniques have been developed that result in transformation frequencies >50%, allowing the identification of mutants by screening and without selection. Constructs have been created to take advantage of this high-frequency transformation, facilitating genetic mutation, complementation, and heterologous gene expression. Similar methods have been developed for N. meningitidis and nonpathogenic Neisseria including N. mucosa and N. musculi. Techniques are described for genetic manipulation of N. gonorrhoeae and commensal Neisseria species, as well as for growth of these fastidious organisms. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Spot transformation of Neisseria gonorrhoeae on agar plates Basic Protocol 2: Spot transformation of commensal Neisseria on agar plates Basic Protocol 3: Transformation of Neisseria gonorrhoeae in liquid culture Basic Protocol 4: Electroporation of Neisseria gonorrhoeae Basic Protocol 5: Creation of unmarked mutations using a positive and negative selection cassette Basic Protocol 6: In vitro mutagenesis of Neisseria gonorrhoeae chromosomal DNA using EZ-Tn5 Basic Protocol 7: Chemical mutagenesis Basic Protocol 8: Complementation on the Neisseria gonorrhoeae chromosome Alternate Protocol 1: Complementation with replicating plasmids Alternate Protocol 2: Complementation on the Neisseria musculi or Neisseria mucosa chromosome Basic Protocol 9: Preparation of chromosomal DNA from Neisseria gonorrhoeae grown on solid medium Alternate Protocol 3: Preparation of chromosomal DNA from Neisseria gonorrhoeae grown in broth Support Protocol: Preparing PCR templates from Neisseria gonorrhoeae colonies.

Structural basis of Cas3 activation in type I-C CRISPR-Cas system.

CRISPR-Cas systems function as adaptive immune mechanisms in bacteria and archaea and offer protection against phages and other mobile genetic elements. Among many types of CRISPR-Cas systems, Type I CRISPR-Cas systems are most abundant, with target interference depending on a multi-subunit, RNA-guided complex known as Cascade that recruits a transacting helicase nuclease, Cas3, to degrade the target. While structural studies on several other types of Cas3 have been conducted long ago, it was only recently that the structural study of Type I-C Cas3 in complex with Cascade was revealed, shedding light on how Cas3 achieve its activity in the Cascade complex. In the present study, we elucidated the first structure of standalone Type I-C Cas3 from Neisseria lactamica (NlaCas3). Structural analysis revealed that the histidine-aspartate (HD) nuclease active site of NlaCas3 was bound to two Fe2+ ions that inhibited its activity. Moreover, NlaCas3 could cleave both single-stranded and double-stranded DNA in the presence of Ni2+ or Co2+, showing the highest activity in the presence of both Ni2+ and Mg2+ ions. By comparing the structural studies of various Cas3 proteins, we determined that our NlaCas3 stays in an inactive conformation, allowing us to understand the structural changes associated with its activation and their implication.

Antimicrobial susceptibility of commensal Neisseria spp. in parents and their children in Belgium: a cross-sectional survey.

BACKGROUND: commensal Neisseria species are part of the oropharyngeal microbiome and play an important role in nitrate reduction and protecting against colonization by pathogenic bacteria. They do, however, also serve as a reservoir of antimicrobial resistance. Little is known about the prevalence of these species in the general population, how this varies by age and how antimicrobial susceptibility varies between species. METHODS: we assessed the prevalence and antimicrobial susceptibility of commensal Neisseria species in the parents (n = 38) and children (n = 50) of 35 families in Belgium. RESULTS: various commensal Neisseria (n = 5) could be isolated from the participants. Most abundant were N. subflava and N. mucosa. Neisseria subflava was detected in 77 of 88 (87.5%) individuals and N. mucosa in 64 of 88 (72.7%). Neisseria mucosa was more prevalent in children [41/50 (82%)] than parents [23/38 (60.5%); P < .05], while N. bacilliformis was more prevalent in parents [7/36 (19.4%)] than children [2/50 (4%); P < .05]. Neisseria bacilliformis had high ceftriaxone minimum inhibitory concentrations (MICs; median MIC 0.5 mg/l; IQR 0.38-0.75). The ceftriaxone MICs of all Neisseria isolates were higher in the parents than in the children. This could be explained by a higher prevalence of N. bacilliformis in the parents. INTERPRETATION: the N. bacilliformis isolates had uniformly high ceftriaxone MICs which warrant further investigation.

Genomic oropharyngeal Neisseria surveillance detects MALDI-TOF MS species misidentifications and reveals a novel Neisseria cinerea clade.

Introduction. Commensal Neisseria spp. are highly prevalent in the oropharynx as part of the healthy microbiome. N. meningitidis can colonise the oropharynx too from where it can cause invasive meningococcal disease. To identify N. meningitidis, clinical microbiology laboratories often rely on Matrix Assisted Laser Desorption/Ionisation Time of Flight Mass Spectrometry (MALDI-TOF MS).Hypothesis/Gap statement. N. meningitidis may be misidentified by MALDI-TOF MS.Aim. To conduct genomic surveillance of oropharyngeal Neisseria spp. in order to: (i) verify MALDI-TOF MS species identification, and (ii) characterize commensal Neisseria spp. genomes.Methodology. We analysed whole genome sequence (WGS) data from 119 Neisseria spp. isolates from a surveillance programme for oropharyngeal Neisseria spp. in Belgium. Different species identification methods were compared: (i) MALDI-TOF MS, (ii) Ribosomal Multilocus Sequence Typing (rMLST) and (iii) rplF gene species identification. WGS data were used to further characterize Neisseria species found with supplementary analyses of Neisseria cinerea genomes.Results. Based on genomic species identification, isolates from the oropharyngeal Neisseria surveilence study were composed of the following species: N. meningitidis (n=23), N. subflava (n=61), N. mucosa (n=15), N. oralis (n=8), N. cinerea (n=5), N. elongata (n=3), N. lactamica (n=2), N. bacilliformis (n=1) and N. polysaccharea (n=1). Of these 119 isolates, four isolates identified as N. meningitidis (n=3) and N. subflava (n=1) by MALDI-TOF MS, were determined to be N. polysaccharea (n=1), N. cinerea (n=2) and N. mucosa (n=1) by rMLST. Phylogenetic analyses revealed that N. cinerea isolates from the general population (n=3, cluster one) were distinct from those obtained from men who have sex with men (MSM, n=2, cluster two). The latter contained genomes misidentified as N. meningitidis using MALDI-TOF MS. These two N. cinerea clusters persisted after the inclusion of published N. cinerea WGS (n=42). Both N. cinerea clusters were further defined through pangenome and Average Nucleotide Identity (ANI) analyses.Conclusion. This study provides insights into the importance of genomic genus-wide Neisseria surveillance studies to improve the characterization and identification of the Neisseria genus.

Neisseria leonii sp. nov., isolated from the nose, lung, and liver of rabbits.

A taxogenomic study of three strains (3986T, 51.81, and JF 2415) isolated from rabbits between 1972 and 2000 led to the description of a new Neisseria species. The highest sequence similarity of the 16S rRNA gene was found to Neisseria animalis NCTC 10212T (96.7 %). The 16S rRNA gene similarity above 99 % and average nucleotide identity (ANI) values above 96 % among the strains, indicated that they belong to the same species. At the same time, the strains shared ANI values below 81 % and dDDH values below 24 % with all described Neisseria species. In the bac120 gene phylogenetic tree, the three strains clustered near Neisseria elongata and Neisseria bacilliformis in the Neisseria clade. However, the Neisseria clade is not monophyletic, and includes the type strains of Morococcus cerebrosus, Bergeriella denitrificans, Kingella potus, Uruburuella suis, and Uruburuella testudinis. Neisseria shayeganii clustered outside the clade with members of the genus Eikenella. Amino acid identity (AAI) values were calculated, and a threshold of 71 % was used to circumscribe the genus Neisseria. According to this proposed AAI threshold, strains 3986T, 51.81, and JF 2415 were placed within the genus Neisseria. The cells of the three strains were Gram-stain-negative diplococcobacilli and non-motile. Optimal growth on trypticase soy agar occurred at 37 °C and pH 8.5 in aerobic conditions. Notably, all strains exhibited indole production in the API-NH test, which is atypical for Neisseria and the family Neisseriaceae. The strains exhibited a common set of 68 peaks in their MALDI-TOF MS profiles, facilitating the swift and accurate identification of this species. Based on genotypic and phenotypic data, it is proposed that strains 3986T, 51.81, and JF 2415 represent a novel species within the genus Neisseria, for which the name Neisseria leonii sp. nov. is proposed (type strain 3986T=R726T=CIP 109994T=LMG 32907T).

Oral microbiome sequencing revealed the enrichment of Fusobacterium sp., Porphyromonas sp., Campylobacter sp., and Neisseria sp. on the oral malignant fibroma surface of giant panda.

Introduction: Microbial community composition is closely associated with host disease onset and progression, underscoring the importance of understanding host-microbiota dynamics in various health contexts. Methods: In this study, we utilized full-length 16S rRNA gene sequencing to conduct species-level identification of the microorganisms in the oral cavity of a giant panda (Ailuropoda melanoleuca) with oral malignant fibroma. Results: We observed a significant difference between the microbial community of the tumor side and non-tumor side of the oral cavity of the giant panda, with the latter exhibiting higher microbial diversity. The tumor side was dominated by specific microorganisms, such as Fusobacterium simiae, Porphyromonas sp. feline oral taxon 110, Campylobacter sp. feline oral taxon 100, and Neisseria sp. feline oral taxon 078, that have been reported to be associated with tumorigenic processes and periodontal diseases in other organisms. According to the linear discriminant analysis effect size analysis, more than 9 distinct biomarkers were obtained between the tumor side and non-tumor side samples. Furthermore, the Kyoto Encyclopedia of Genes and Genomes analysis revealed that the oral microbiota of the giant panda was significantly associated with genetic information processing and metabolism, particularly cofactor and vitamin, amino acid, and carbohydrate metabolism. Furthermore, a significant bacterial invasion of epithelial cells was predicted in the tumor side. Discussion: This study provides crucial insights into the association between oral microbiota and oral tumors in giant pandas and offers potential biomarkers that may guide future health assessments and preventive strategies for captive and aging giant pandas.

Peptidoglycan fragment release and NOD activation by commensal Neisseria species from humans and other animals.

Neisseria gonorrhoeae, a human restricted pathogen, releases inflammatory peptidoglycan (PG) fragments that contribute to the pathophysiology of pelvic inflammatory disease. The genus Neisseria is also home to multiple species of human- or animal-associated Neisseria that form part of the normal microbiota. Here we characterized PG release from the human-associated nonpathogenic species Neisseria lactamica and Neisseria mucosa and animal-associated Neisseria from macaques and wild mice. An N. mucosa strain and an N. lactamica strain were found to release limited amounts of the proinflammatory monomeric PG fragments. However, a single amino acid difference in the PG fragment permease AmpG resulted in increased PG fragment release in a second N. lactamica strain examined. Neisseria isolated from macaques also showed substantial release of PG monomers. The mouse colonizer Neisseria musculi exhibited PG fragment release similar to that seen in N. gonorrhoeae with PG monomers being the predominant fragments released. All the human-associated species were able to stimulate NOD1 and NOD2 responses. N. musculi was a poor inducer of mouse NOD1, but ldcA mutation increased this response. The ability to genetically manipulate N. musculi and examine effects of different PG fragments or differing amounts of PG fragments during mouse colonization will lead to a better understanding of the roles of PG in Neisseria infections. Overall, we found that only some nonpathogenic Neisseria have diminished release of proinflammatory PG fragments, and there are differences even within a species as to types and amounts of PG fragments released.

Characterizing the diversity and commensal origins of penA mosaicism in the genus Neisseria.

Mosaic penA alleles formed through horizontal gene transfer (HGT) have been instrumental to the rising incidence of ceftriaxone-resistant gonococcal infections. Although interspecies HGT of regions of the penA gene between Neisseria gonorrhoeae and commensal Neisseria species has been described, knowledge concerning which species are the most common contributors to mosaic penA alleles is limited, with most studies examining only a small number of alleles. Here, we investigated the origins of recombinant penA alleles through in silico analyses that incorporated 1700 penA alleles from 35 513 Neisseria isolates, comprising 15 different Neisseria species. We identified Neisseria subflava and Neisseria cinerea as the most common source of recombinant sequences in N. gonorrhoeae penA. This contrasted with Neisseria meningitidis penA, for which the primary source of recombinant DNA was other meningococci, followed by Neisseria lactamica. Additionally, we described the distribution of polymorphisms implicated in antimicrobial resistance in penA, and found that these are present across the genus. These results provide insight into resistance-related changes in the penA gene across human-associated Neisseria species, illustrating the importance of genomic surveillance of not only the pathogenic Neisseria, but also of the oral niche-associated commensals from which these pathogens are sourcing key genetic variation.

Gonococcal resistance to zoliflodacin could emerge via transformation from commensal Neisseria species. An in-vitro transformation study.

One of the most promising new treatments for gonorrhoea currently in phase 3 clinical trials is zoliflodacin. Studies have found very little resistance to zoliflodacin in currently circulating N. gonorrhoeae strains, and in-vitro experiments demonstrated that it is difficult to induce resistance. However, zoliflodacin resistance may emerge in commensal Neisseria spp., which could then be transferred to N. gonorrhoeae via transformation. In this study, we investigated this commensal-resistance-pathway hypothesis for zoliflodacin. To induce zoliflodacin resistance, ten wild-type susceptible isolates belonging to 5 Neisseria species were serially passaged for up to 48 h on gonococcal agar plates containing increasing zoliflodacin concentrations. Within 7 to 10 days, all strains except N. lactamica, exhibited MICs of ≥ 4 µg/mL, resulting in MIC increase ranging from 8- to 64-fold. The last passaged strains and their baseline were sequenced. We detected mutations previously reported to cause zoliflodacin resistance in GyrB (D429N and S467N), novel mutations in the quinolone resistance determining region (QRDR) (M464R and T472P) and mutations outside the QRDR at amino acid positions 28 and 29 associated with low level resistance (MIC 2 µg/mL). Genomic DNA from the laboratory evolved zoliflodacin-resistant strains was transformed into the respective baseline wild-type strain, resulting in MICs of ≥ 8 µg/mL in most cases. WGS of transformants with decreased zoliflodacin susceptibility revealed presence of the same zoliflodacin resistance determinants as observed in the donor strains. Two inter-species transformation experiments were conducted to investigate whether zoliflodacin resistance determinants of commensal Neisseria spp. could be acquired by N. gonorrhoeae. N. gonorrhoeae strain WHO P was exposed to (i) pooled genomic DNA from the two resistant N. mucosa strains and (ii) a gyrB amplicon of the resistant N. subflava strain 45/1_8. Transformants of both experiments exhibited an MIC of 2 µg/mL and whole genome analysis revealed uptake of the mutations detected in the donor strains. This is the first in-vitro study to report that zoliflodacin resistance can be induced in commensal Neisseria spp. and subsequently transformed into N. gonorrhoeae.

Type IV pilus retraction is required for Neisseria musculi colonization and persistence in a natural mouse model of infection.

IMPORTANCE: We describe the importance of Type IV pilus retraction to colonization and persistence by a mouse commensal Neisseria, N. musculi, in its native host. Our findings have implications for the role of Tfp retraction in mediating interactions of human-adapted pathogenic and commensal Neisseria with their human host due to the relatedness of these species.

Distributed genotyping and clustering of Neisseria strains reveal continual emergence of epidemic meningococcus over a century.

Core genome multilocus sequence typing (cgMLST) is commonly used to classify bacterial strains into different types, for taxonomical and epidemiological applications. However, cgMLST schemes require central databases for the nomenclature of new alleles and sequence types, which must be synchronized worldwide and involve increasingly intensive calculation and storage demands. Here, we describe a distributed cgMLST (dcgMLST) scheme that does not require a central database of allelic sequences and apply it to study evolutionary patterns of epidemic and endemic strains of the genus Neisseria. We classify 69,994 worldwide Neisseria strains into multi-level clusters that assign species, lineages, and local disease outbreaks. We divide Neisseria meningitidis into 168 endemic lineages and three epidemic lineages responsible for at least 9 epidemics in the past century. According to our analyses, the epidemic and endemic lineages experienced very different population dynamics in the past 100 years. Epidemic lineages repetitively emerged from endemic lineages, disseminated worldwide, and apparently disappeared rapidly afterward. We propose a stepwise model for the evolutionary trajectory of epidemic lineages in Neisseria, and expect that the development of similar dcgMLST schemes will facilitate epidemiological studies of other bacterial pathogens.

Evolution and exchange of plasmids in pathogenic Neisseria.

IMPORTANCE: Horizontal gene transfer (HGT) is a major influence in driving the spread of antimicrobial resistance (AMR) in many bacteria. A conjugative plasmid which is widespread in Neisseria gonorrhoeae, pConj, prevented the use of tetracycline/doxycycline for treating gonococcal infection. Here, we show that pConj evolved in the related pathogen, Neisseria meningitidis, and has been acquired by the gonococcus from the meningococcus on multiple occasions. Following its initial acquisition, pConj spread to different gonococcal lineages; changes in the plasmid's conjugation machinery associated with another transfer event limit spread in the gonococcal populations. Our findings have important implications for the use of doxycycline to prevent bacterial sexually transmitted disease which is likely to exacerbate the spread of AMR through HGT in pathogenic bacteria.

Neisseria lisongii sp. nov. and Neisseria yangbaofengii sp. nov., isolated from the respiratory tracts of marmots.

Four Gram-stain-negative, oxidase-positive, non-motile, cocci-shaped bacteria strains (ZJ106T, ZJ104, ZJ785T and ZJ930) were isolated from marmot respiratory tracts. Phylogenetic analyses based on 16S rRNA genes, 53 ribosomal protein sequences and 441 core genes supported that all four strains belonged to the genus Neisseria with close relatives Neisseria weixii 10022T and Neisseria iguanae ATCC 51483T. Average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values were below the species-level thresholds (95-96 % for ANI, and 70 % for dDDH). The major fatty acids of all four strains were C16 : 1 ω7c /C16 : 1 ω6c, C16 : 0 and C18 : 1 ω9c. Major polar lipids were composed of diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylglycerol. MK-8 was the major menaquinone. Based on Virulence Factor Database analysis, the four strains were found to contain NspA and PorB H-factor binding proteins that promote evasion of host immunity. Strains ZJ106T and ZJ104 contained structures similar to the capsule synthesis manipulator of Neisseria meningitidis. Based on phenotypic and phylogenetic evidence, we propose that strains ZJ106T and ZJ785T represent two novel species of the genus Neisseria, respectively, with the names Neisseria lisongii sp. nov. and Neisseria yangbaofengii sp. nov. The type strains are ZJ106T (=GDMCC 1.3111T=JCM 35323T) and ZJ785T (=GDMCC 1.1998T=KCTC 82336T).

Neisseria montereyensis sp. nov., Isolated from Oropharynx of California Sea Lion (Zalophus californianus): Genomic, Phylogenetic, and Phenotypic Study.

A novel Neisseria strain, designated CSL10203-ORH2T, was isolated from the oropharynx of a wild California sea lion (Zalophus californianus) that was admitted to The Marine Mammal Center in California, USA. The strain was originally cultured from an oropharyngeal swab on BD Phenylethyl Alcohol (PEA) agar with 5% sheep blood under aerobic conditions. Phylogenetic analyses based on 16S rRNA, rplF, and rpoB gene sequences and the core genome sequences indicated that the strain was most closely related to only N. zalophi CSL 7565T. The average nucleotide identity and digital DNA-DNA hybridization values between strain CSL10203-ORH2T and the closely related species N. zalophi CSL 7565T were 89.84 and 39.70%, respectively, which were significantly lower than the accepted species-defined thresholds for describing novel prokaryotic species at the genomic level. Both type strains were phenotypically similar but can be easily and unambiguously distinguished between each other by the analysis of their housekeeping genes, e.g., rpoB, gyrB, or argF. The major fatty acids in both type strains were C12:0, C16:0, C16:1-c9, and C18:1-c11. Based on the genomic, phenotypic, and phylogenetic properties, the novel strain represents a novel species of the genus Neisseria, for which the name Neisseria montereyensis sp. nov. with the type strain CSL10203-ORH2T (= DSM 114706T = CCUG 76428T = NCTC 14721T) is proposed. The genome G + C content is 45.84% and the complete draft genome size is 2,310,535 bp.

Anti-CRISPR Protein AcrIIC5 Inhibits CRISPR-Cas9 by Occupying the Target DNA Binding Pocket.

Anti-CRISPR proteins inhibit CRISPR-Cas immune systems through diverse mechanisms. Previously, the anti-CRISPR protein AcrIIC5Smu was shown to potently inhibit a type II-C Cas9 from Neisseria meningitidis (Nme1Cas9). In this work, we explore the mechanism of activity of the AcrIIC5 homologue from Neisseria chenwenguii (AcrIIC5Nch) and show that it prevents Cas9 binding to target DNA. We show that AcrIIC5Nch targets the PAM-interacting domain (PID) of Nme1Cas9 for inhibition, agreeing with previous findings for AcrIIC5Smu, and newly establish that strong binding of the anti-CRISPR requires guide RNA be pre-loaded on Cas9. We determined the crystal structure of AcrIIC5Nch using X-ray crystallography and identified amino acid residues that are critical for its function. Using a protein docking algorithm we show that AcrIIC5Nch likely occupies the Cas9 DNA binding pocket, thereby inhibiting target DNA binding through a mechanism similar to that previously described for AcrIIA2 and AcrIIA4.

Penicillin and Cefotaxime Resistance of Quinolone-Resistant Neisseria meningitidis Clonal Complex 4821, Shanghai, China, 1965-2020.

Clonal complex 4821 (CC4821) Neisseria meningitidis, usually resistant to quinolones but susceptible to penicillin and third-generation cephalosporins, is increasing worldwide. To characterize the penicillin-nonsusceptible (PenNS) meningococci, we analyzed 491 meningococci and 724 commensal Neisseria isolates in Shanghai, China, during 1965-2020. The PenNS proportion increased from 0.3% in 1965-1985 to 7.0% in 2005-2014 and to 33.3% in 2015-2020. Of the 26 PenNS meningococci, 11 (42.3%) belonged to the CC4821 cluster; all possessed mutations in penicillin-binding protein 2, mostly from commensal Neisseria. Genetic analyses and transformation identified potential donors of 6 penA alleles. Three PenNS meningococci were resistant to cefotaxime, 2 within the CC4821 cluster. With 96% of the PenNS meningococci beyond the coverage of scheduled vaccination and the cefotaxime-resistant isolates all from toddlers, quinolone-resistant CC4821 has acquired penicillin and cefotaxime resistance closely related to the internationally disseminated ceftriaxone-resistant gonococcal FC428 clone, posing a greater threat especially to young children.

Whole Genome Analyses Accurately Identify Neisseria spp. and Limit Taxonomic Ambiguity.

Genome sequencing facilitates the study of bacterial taxonomy and allows the re-evaluation of the taxonomic relationships between species. Here, we aimed to analyze the draft genomes of four commensal Neisseria clinical isolates from the semen of infertile Lebanese men. To determine the phylogenetic relationships among these strains and other Neisseria spp. and to confirm their identity at the genomic level, we compared the genomes of these four isolates with the complete genome sequences of Neisseria gonorrhoeae and Neisseria meningitidis and the draft genomes of Neisseria flavescens, Neisseria perflava, Neisseria mucosa, and Neisseria macacae that are available in the NCBI Genbank database. Our findings revealed that the WGS analysis accurately identified and corroborated the matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) species identities of the Neisseria isolates. The combination of three well-established genome-based taxonomic tools (in silico DNA-DNA Hybridization, Ortho Average Nucleotide identity, and pangenomic studies) proved to be relatively the best identification approach. Notably, we also discovered that some Neisseria strains that are deposited in databases contain many taxonomical errors. The latter is very important and must be addressed to prevent misdiagnosis and missing emerging etiologies. We also highlight the need for robust cut-offs to delineate the species using genomic tools.

Genomic sequence of the non-pathogen Neisseria sp. strain MA1-1 with antibiotic resistance and virulence factors isolated from a head and neck cancer patient.

Recent research has claimed virulence factors or antimicrobial resistance in commensal or non-pathogenic Neisseria spp. This study aimed to isolate and analyze commensal microorganisms related to the genus Neisseria from the oral cavity of a patient with head and neck cancer. We successfully isolated strain MA1-1 and identified its functional gene contents. Although strain MA1-1 was related to Neisseria flava based on 16S rRNA gene sequence similarity, genomic relatedness analysis revealed that strain MA1-1 was closely related to Neisseria mucosa, reported as a commensal Neisseria species. The strain MA1-1 genome harbored genes for microaerobic respiration and the complete core metabolic pathway with few transporters for nutrients. A number of genes have been associated with virulence factors and resistance to various antibiotics. In addition, the comparative genomic analysis showed that most genes identified in the strain MA1-1 were shared with other Neisseria spp. including two well-known pathogens, Neisseria gonorrhoeae and Neisseria meningitidis. This indicates that the gene content of intra-members of the genus Neisseria has been evolutionarily conserved and is stable, with no gene recombination with other microbes in the host. Finally, this study provides more fundamental interpretations for the complete gene sequence of commensal Neisseria spp. and will contribute to advancing public health knowledge.

Canary in the Coal Mine: How Resistance Surveillance in Commensals Could Help Curb the Spread of AMR in Pathogenic Neisseria.

Antimicrobial resistance (AMR) is widespread within Neisseria gonorrhoeae populations. Recent work has highlighted the importance of commensal Neisseria (cN) as a source of AMR for their pathogenic relatives through horizontal gene transfer (HGT) of AMR alleles, such as mosaic penicillin binding protein 2 (penA), multiple transferable efflux pump (mtr), and DNA gyrase subunit A (gyrA) which impact beta-lactam, azithromycin, and ciprofloxacin susceptibility, respectively. However, nonpathogenic commensal species are rarely characterized. Here, we propose that surveillance of the universally carried commensal Neisseria may play the role of the "canary in the coal mine," and reveal circulating known and novel antimicrobial resistance determinants transferable to pathogenic Neisseria. We summarize the current understanding of commensal Neisseria as an AMR reservoir, and call to increase research on commensal Neisseria species, through expanding established gonococcal surveillance programs to include the collection, isolation, antimicrobial resistance phenotyping, and whole-genome sequencing (WGS) of commensal isolates. This will help combat AMR in the pathogenic Neisseria by: (i) determining the contemporary AMR profile of commensal Neisseria, (ii) correlating AMR phenotypes with known and novel genetic determinants, (iii) qualifying and quantifying horizontal gene transfer (HGT) for AMR determinants, and (iv) expanding commensal Neisseria genomic databases, perhaps leading to the identification of new drug and vaccine targets. The proposed modification to established Neisseria collection protocols could transform our ability to address AMR N. gonorrhoeae, while requiring minor modifications to current surveillance practices. IMPORTANCE Contemporary increases in the prevalence of antimicrobial resistance (AMR) in Neisseria gonorrhoeae populations is a direct threat to global public health and the effective treatment of gonorrhea. Substantial effort and financial support are being spent on identifying resistance mechanisms circulating within the gonococcal population. However, these surveys often overlook a known source of resistance for gonococci-the commensal Neisseria. Commensal Neisseria and pathogenic Neisseria frequently share DNA through horizontal gene transfer, which has played a large role in rendering antibiotic therapies ineffective in pathogenic Neisseria populations. Here, we propose the expansion of established gonococcal surveillance programs to integrate a collection, AMR profiling, and genomic sequencing pipeline for commensal species. This proposed expansion will enhance the field's ability to identify resistance in and from nonpathogenic reservoirs and anticipate AMR trends in pathogenic Neisseria.

Neisseria species as pathobionts in bronchiectasis.

Neisseria species are frequently identified in the bronchiectasis microbiome, but they are regarded as respiratory commensals. Using a combination of human cohorts, next-generation sequencing, systems biology, and animal models, we show that bronchiectasis bacteriomes defined by the presence of Neisseria spp. associate with poor clinical outcomes, including exacerbations. Neisseria subflava cultivated from bronchiectasis patients promotes the loss of epithelial integrity and inflammation in primary epithelial cells. In vivo animal models of Neisseria subflava infection and metabolipidome analysis highlight immunoinflammatory functional gene clusters and provide evidence for pulmonary inflammation. The murine metabolipidomic data were validated with human Neisseria-dominant bronchiectasis samples and compared with disease in which Pseudomonas-, an established bronchiectasis pathogen, is dominant. Metagenomic surveillance of Neisseria across various respiratory disorders reveals broader importance, and the assessment of the home environment in bronchiectasis implies potential environmental sources of exposure. Thus, we identify Neisseria species as pathobionts in bronchiectasis, allowing for improved risk stratification in this high-risk group.