Comparative Genomic Analysis of Clonal Variants of a Spirabiliibacterium Lineage Recovered from a Chicken in the United States.

The family Pasteurellaceae includes pathogenic and opportunistic bacteria affecting poultry, yet taxonomic resolution remains challenging due to phenotypic heterogeneity, limited genomic representation, and the poor performance of routine diagnostic systems for uncommon taxa. At the Clemson Veterinary Diagnostic Center, a Gram-negative bacterium was isolated from a backyard hen. The isolate produced two distinct colony morphotypes with identical biochemical profiles. They were misidentified as Sphingomonas paucimobilis by VITEK® 2, while MALDI-TOF MS failed to identify them. Oxford Nanopore long-read sequencing with PacBio HiFi polishing generated near-complete chromosome-level assemblies. Comparative genomic analyses demonstrated that morphotypes CVDC-smooth and CVDC-rough represent clonal variants of a distinct Spirabiliibacterium lineage sharing 93% average nucleotide identity (ANI) and 52% digital DNA-DNA hybridization (dDDH) with the closest relative, Spirabiliibacterium mucosae. Copy-number variation within a tandemly duplicated ~20 kb genomic region may contribute to differences in colony morphology. Family-wide analysis identified lineage-associated gene patterns and a limited number of VFDB-matched virulence genes in the CVDC isolates. This study identifies a distinct lineage within the genus Spirabiliibacterium, reports the first isolation and chromosome-level genome assemblies of a Spirabiliibacterium lineage from a chicken in the United States, and highlights the limitations of routine diagnostic methods for identifying uncommon bacterial taxa.