S353T as a Novel Synergistic Hub Driving Ceftriaxone Resistance in Mosaic penA Alleles of Neisseria gonorrhoeae

Neisseria Gonorrheae, which has been identified as a pathogen of concern in antibiotic resistance, has beenrecently displaying alarming levels of resistance to third generation cephalosporins, the only class of antibioticsthat have demonstrated effectiveness against it. The development of mosaic \textit{penA} alleles– recombinant allelesdisplaying resistance– plays a crucial role in the mechanism of said resistance. The \textit{penA} allele, which codes forPenicillin-binding-protein 2 (PBP2), is the main target of most classes of beta-lactam antibiotics, including thirdgeneration cephalosporins. As a preliminary investigation of the current development of antibiotic-resistant \textit{penA}alleles, we used various in silico tools to analyze seven strains of \textit{N}. \textit{Gonorrhoeae} experimentally identified todisplay resistance against the third-generation cephalosporin ceftriaxone. A total of 69 single nucleotidepolymorphisms in the \textit{penA} gene were identified, of which six combinations of 10 SNPs were identified to bemost synergistic in increasing the energy barrier to ceftriaxone binding. While literature values had identifiedhotspot regions in the $\Omega$-loop (residues 238--252) and the adjacent $\beta3$ -- $\beta4$ sheets, neural network analysis furtherrevealed another point mutation outside these regions, S353T, also served as a key synergistic hub for resistancemutations within the mutants analyzed, working with SNPs in the $\beta3$ -- $\beta4$ sheets to drastically increase the binding energy barrier. Aphenological analysis of these SNPs showed that they had been endogenously developed within the Gonorrhoeaegenus, and similarities between phenologically closer strains suggested an evolutionary gradient of resistanceacquisition. This is one of the first broad systematic computational assessments of the effect of mosaic \textit{penA} alleleson interactions with ceftriaxone, and can serve as a literature basis for strains that must not be treated with thirdgeneration cephalosporins, contributing to more effective treatment.