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.