Extension of penA diversity within the genus Neisseria: characterization of a <i>penA/ftsI</i> homolog in <i>Neisseria bacilliformis</i> associated with elevated ceftriaxone MICs

Reduced susceptibility to extended-spectrum cephalosporins in Neisseria gonorrhoeae is primarily mediated by structural alterations in penicillin-binding protein 2 (PBP2), encoded by penA, frequently arising through interspecies recombination with commensal Neisseria. While mosaic penA diversity has been extensively characterized in coccoid Neisseria species, rod-shaped commensals such as Neisseria bacilliformis remain poorly explored. Here, we investigated the beta-lactam target repertoire of N. bacilliformis using whole-genome sequencing, comparative genomics, mutation profiling, and structural modeling. Fifteen N. bacilliformis genomes were analyzed, including eight isolates with elevated ceftriaxone minimum inhibitory concentrations (MIC) (range 0.094-1 & micro;g/mL; median 0.5 & micro;g/mL). Interrogation of the PubMLST locus NEIS1753 did not identify matches under default parameters. However, de novo annotation revealed a syntenic high-molecular-mass transpeptidase annotated as ftsI, occupying the conserved rsmH-ftsL-ftsI-murE chromosomal context observed in N. gonorrhoeae. BLASTP analysis demonstrated similar to 59%-61% amino-acid identity to gonococcal PBP2. Structural modeling using AlphaFold and template-based approaches confirmed preservation of the canonical class B penicillin-binding protein fold, including conserved catalytic motifs. Across 24 positions previously associated with altered cephalosporin susceptibility in gonococci, multiple canonical substitutions were conserved in N. bacilliformis, alongside lineage-specific variants not reported in N. gonorrhoeae. Interestingly, polymorphism at positions A328 and A341 was observed in isolates with higher MICs (1 & micro;g/mL). Definitive genotype-phenotype associations could not be established. Recombination analysis did not identify discrete parental donors but suggested a complex evolutionary history. These findings demonstrate that N. bacilliformis encodes a structurally conserved PBP2 homolog annotated as FtsI and expand the landscape of beta-lactam target diversity within the genus Neisseria.