Isolation, identification, and whole-genome sequencing analysis of a serotype D Pasteurella multocida strain from sheep in Xinjiang, China

Abstract Purpose Pneumonia resulting from the co-infection of Pasteurella multocida (Pm) with Mannheimia haemolytica and Mycoplasma pneumoniae is a primary cause of mortality in sheep. Specifically, serotypes A and D of Pm induce enzootic pneumonia, causing substantial economic losses. This study aims to investigate the pathogenic mechanisms and molecular basis of the Pm-DY strain. Methods Whole-genome sequencing (WGS) combined with systematic bioinformatics analyses using NR, COG, GO, KEGG, TCDB, OMR, CAZy, and VFDB databases was employed to evaluate Pm-DY. A phylogenetic tree was constructed based on core-genome single-nucleotide polymorphisms (SNPs) derived from whole-genome sequencing data to determine its evolutionary relationships. A Kunming (KM) mouse model was employed to verify its virulence. Results The draft genome of Pm-DY was determined to be 2,301,255 bp. A total of 2,324 protein-coding genes were predicted in the Pasteurella multocida genome. Functional annotation of these 947 DEGs against public databases showed that 943 (99.58%) were assigned to COG functional categories, 508 (53.64%) had significant matches in the NR database, and 23 (2.43%) were mapped to KEGG pathways. In the COG functional classification, genes associated with amino acid and carbohydrate transport and metabolism exhibited the highest abundance. GO and KEGG analyses indicated that the gene functions were primarily enriched in metabolic activities, membrane transport, and catalytic activities. Annotation against the TCDB database revealed that Pm-DY harbors multiple classes of efflux transporters, predominantly of the ABC superfamily, including macB and oleC associated with macrolide efflux, and msbA involved in transport and multidrug tolerance, suggesting potential multidrug resistance capabilities. Notably, the antimicrobial resistance gene repertoire identified in the Pm-DY genome was partially consistent with its phenotypic resistance profile determined by disk diffusion assay. Within the OMR family, the identification of iron uptake-related genes provides molecular evidence for the strain’s blood-dependent growth and its ability to overcome the host’s iron-restricted environment. Furthermore, eight glycoside hydrolase genes were identified via the CAZy database, which may participate in the degradation of host cellular structures. Virulence analysis using the VFDB identified 128 potential virulence genes, covering functional categories such as adhesion, invasion, iron uptake, and anti-phagocytosis. The high abundance of oligosaccharide- and capsule-related genes suggests that Pm-DY may utilize these surface structures for immune evasion and host colonization. Phylogenetic analysis demonstrated that Pm-DY is most closely related to the HN01 strain. Pathogenicity experiments showed that Pm-DY infection induced typical lesions in mice, including multi-organ hemorrhage, pulmonary consolidation, and splenic necrosis, confirming its strong virulence. The enrichment of metabolic and transport systems, virulence factors, and resistance-associated genes in the Pm-DY genome provides a genetic foundation for its rapid proliferation, host adaptation, and pathogenicity. This study offers crucial insights to elucidate the pathogenic mechanisms of Pasteurella multocida and to develop effective prevention and control strategies.