Comparative genomics of gut-derived Shewanella species reveals unexpected co-resistance to carbapenems and colistin in asymptomatic human carriers

The human gut is increasingly recognized as a major reservoir of antimicrobial resistance (AMR), yet the contribution of atypical environmental bacteria carrying last-line resistance determinants remains poorly understood. Shewanella species, ubiquitous in aquatic ecosystems, are emerging opportunistic pathogens and natural progenitors of chromosomal bla OXA carbapenemases. However, their detection, genomic diversity, and resistance ecology within the human intestinal tract have not been systematically characterized. We performed whole-genome sequencing and comprehensive comparative genomic analyses on 13 human gut-derived Shewanella isolates, including six strains newly recovered from healthy non-diarrheal individuals in Zhejiang, China, and seven publicly available isolates from diarrheal patients in Beijing, China (BioProject PRJNA1208244). Species delineation was achieved through average nucleotide identity (ANI), and phylogenetic relationships were reconstructed using core-genome single nucleotide polymorphism (cgSNP)-based maximum-likelihood inference. Antimicrobial susceptibility was determined via automated broth microdilution, and resistome-virulome mapping was conducted using ResFinder/CARD and VFDB databases. ANI-based taxonomic reassignment substantially corrected MALDI-TOF MS misidentifications, revealing that five of the six asymptomatic isolates belonged to Shewanella algae , whereas the remaining isolate could not be confidently assigned to any currently described Shewanella species. The diarrheal isolates comprised Shewanella indica , Shewanella chilikensis , and Shewanella algae . Notably, five of six asymptomatic isolates were resistant to imipenem, one additionally exhibited meropenem resistance, and three displayed resistance to colistin (MIC up to > 64 µg/mL), representing an unprecedented observation of concurrent resistance to both last-line antibiotics among Shewanella isolates detected in healthy hosts. Resistome analysis revealed lineage-specific partitioning of bla OXA variants and qnrA alleles, yet all qnrA -carrying isolates remained phenotypically susceptible to fluoroquinolones, highlighting a marked genotype-phenotype discordance. Importantly, colistin resistance in XW79, XW81 and XW84 could not be attributed to any known mcr genes, implicating the presence of alternative chromosomal mechanisms. Virulence profiling identified a conserved core of host interaction-associated genes shared across all isolates, while no distinct diarrheagenic signature differentiated symptomatic from asymptomatic strains, suggesting that host or ecological factors, rather than intrinsic virulence traits, may determine clinical outcomes. This study provides preliminary genomic evidence that the healthy human gut can occasionally harbor Shewanella strains co-resistant to carbapenems and colistin. While based on a limited number of isolates from a single regional center, these findings suggest that this environmental genus may act as a potential cryptic reservoir for last-line AMR determinants, yet longitudinal studies are needed to distinguish true colonization from transient intestinal passage. Our findings challenge the clinical dismissal of Shewanella species as transient contaminants and underscore the urgent need to expand One Health AMR monitoring to include atypical gut-derived bacteria. The unresolved mechanistic basis of colistin resistance and the functional silence of qnr genes in this genus demand further investigation and may reveal previously undescribed resistance pathways in non-fermenting Gram-negative bacilli.