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

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