Neisseria meningitidis is the causative agent of invasive meningococcal disease. Although third-generation cephalosporins remain the recommended empirical therapy, there has been a growing number of globally reported isolates with resistance. This study aims to elucidate the genetic and evolutionary mechanisms underlying third-generation cephalosporin resistance in N. meningitidis . Whole-genome sequencing and phenotypic antibiotic susceptibility testing were performed, and the potential resistance determinants, genomic features, and evolutionary trajectories of resistant strains were comprehensively characterized. Seven PBP2 mutation sites potentially associated with resistance were identified, including A311V, R468K, E469K, D/N472E, L/P480A, T483S, and G545S. Genomic analysis revealed that strains possessing these seven potential resistance determinant mutations emerged within the past two decades and exhibited an increasing trend. The majority of these strains were isolated from northern regions of China. The predominant mutation strains were located in mainly epidemic strains, like cc4821, cc23, serogroup B and serogroup Y, respectively. Systematic phylogenetic analysis revealed that there has been a phenomenon of cross-regional transmission. Three potential cross-regional transmission third-generation cephalosporin-resistant strain types were identified, namely cc4821-ST-8920-NEIS1753_3923- penA795 , ST-14760-NEIS1753_3652- penA1357 , and cc23-ST-18108-NEIS1753_5058- penA1258 . This study systematically analyzed the phenotypic and genetic characteristics of third-generation cephalosporin-resistant N. meningitidis strains . It not only deepened the understanding of the resistance mechanism but also provided a scientific basis and practical guidance for rapid clinical detection, laboratory monitoring, as well as the prevention and control of cross-regional transmission risks.
