Comparative genomic analysis of Streptococcus parasuis and Streptococcus suis reveals mobile element-associated enrichment of antimicrobial resistance and lack of detectable same-MGE colocalization with virulence-associated genes within stable species boundaries.

Streptococcus suis is a major porcine pathogen and a zoonotic agent that causes meningitis and septicemia in humans. Streptococcus parasuis, a recently recognized close relative, remains poorly characterized with regard to its clinical significance and genomic features. In this study, we generated a single-contig closed genome assembly with genome-wide DNA methylation profiles for S. parasuis strain A1, isolated from a diseased pig in Xinjiang, China, and complemented in silico genomic predictions with isolate-level experimental validation of antimicrobial resistance (AMR) genotypes, virulence genotypes, and phenotypic susceptibility for this reference strain. Using this high-quality genome as a reference anchor, we performed comparative genomic analyses across 195 streptococcal genomes, comprising 15 S. parasuis and 180 S. suis strains, to distinguish genome-level co-occurrence of resistance and virulence determinants from their physical colocalization on the same mobile genetic element (MGE).Species boundaries remained clearly delineated at the genomic level, with a median interspecies average nucleotide identity (ANI) of approximately 86.0%, compared with intraspecies ANI medians of 97.5% for S. parasuis and 96.2% for S. suis. Pangenome analysis identified 12,693 gene clusters, of which 1086 were core clusters, and functional annotation revealed significant differences in accessory gene repertoires between the two species. Within this stable genomic framework, S. parasuis genomes carried a higher AMR gene burden; strain A1 harbored 10 AMR genes, multiple virulence-associated genes, three genomic islands, and eight prophage regions. For strain A1, PCR validation confirmed six AMR genes and six virulence genes, and disk diffusion testing demonstrated a multidrug-resistant phenotype consistent with the genotypic profile.Among 235 predicted mobile elements, 19 harbored AMR genes and seven carried Virulence Factor Database (VFDB) homologs, but none carried both categories simultaneously. This finding reflects a lack of detectable same-MGE colocalization under the applied annotation and assembly framework; it should not be interpreted as evidence of biological physical decoupling. Under a random-placement model, the expected number of co-carrying regions was only 0.57, and the probability of observing zero co-carrying regions was P = 0.55. This negative result should be interpreted with caution, given the limited number of cargo-bearing regions and the predominantly draft status of most genomes. Furthermore, the A1 genome contained multiple restriction-modification systems, showed depletion of several methylation motif families in mobile regions, and had limited CRISPR spacer matching evidence, suggesting prior exposure to the relevant sequence space. None of the genomes met our predefined criteria for whole-genome convergence.Collectively, our results support a model in which S. parasuis accumulates AMR-related genes in a modular fashion via mobile elements within stable species boundaries, with no detectable same-MGE colocalization of AMR and virulence determinants under our analytical pipeline. These findings imply that AMR surveillance strategies for this species should prioritize tracking mobile genetic elements rather than inferring wholesale genomic convergence toward S. suis.