Emergence of tigecycline-resistant Raoultella ornithinolytica with tet(X)-carrying plasmid from swine wastewater in China

Background The increasing prevalence of antibiotic resistance genes (ARGs), such as the plasmid-mediated tigecycline-modifying enzyme tet (X), significantly hinders the treatment of infectious diseases in humans and animals. Livestock wastewater contributes to the transmission of these ARGs. Methods Between June 2023 and December 2024, 140 wastewater samples from 15 swine farms in Shandong, China, were screened for tet (X)-positive strains using PCR and 16S rRNA sequencing. Raoultella ornithinolytica SD8 was assessed for antimicrobial susceptibility, plasmid stability, conjugation, fitness cost, and pathogenicity in a BALB/c mouse model. Furthermore, this strain was subjected to whole-genome sequencing. Results tet (X4) was found to be located on a 78,159 bp IncFII(pCRY)-like plasmid (pSD8-1-2) in R. ornithinolytica SD8-1, exhibiting high stability (92% retention after 20 days) and conjugative transfer to Escherichia coli C600 and bla NDM -producing E218 at frequencies of 1.6 × 10 –5 and 4.3 × 10 –6 , respectively, with minimal fitness cost. Studies in mice showed that R. ornithinolytica SD8-1 caused severe organ damage. pSD8-1-2 led to tigecycline treatment failure, unlike the plasmid-cured strain. Database analysis identified pSD8-1-2-like plasmids or fragments were identified predominantly in Klebsiella pneumoniae , indicating a potential risk of dissemination. Conclusion The tet (X4)-carrying plasmid pSD8-1-2 in R. ornithinolytica SD8-1 exhibits high stability and cross-species transferability, exacerbating tigecycline resistance and treatment failure. Based on the “One Health” concept, the spread of this plasmid into humans in clinical settings should be closely monitored.