Reclassification of the type B CAT family and identification of the novel chloramphenicol acetyltransferase CatB14 in Riemerella anatipestifer.

Chloramphenicol resistance in Riemerella anatipestifer is primarily mediated by chloramphenicol acetyltransferases (CATs). Inconsistent nomenclature of the CatB superfamily in public databases currently complicates the genomic surveillance of these resistance determinants. In this study, we tested the subtype scheme recorded in the NCBI Reference Gene Catalog against a complete pairwise amino acid identity matrix, applying a ≥ 80% identity criterion. The analysis supports the existing type B-1 to type B-5 subtypes, assigns two previously unassigned sequences to Type B-2, and recommends retirement of the polyphyletic catB12 designation. Under this framework, the catB gene previously reported in R. anatipestifer CH-2 is reclassified as Type B-6, for which we propose the designation catB13, and a novel variant forming an independent lineage (Type B-7) is identified, for which we propose the designation catB14. Functional analysis and site-directed mutagenesis of the predicted catalytic dyad (Y29/H78) confirmed that CatB14 is an active enzyme conferring chloramphenicol resistance; its activity decreased at 3 mM chloramphenicol concentrations relative to that at 2 mM, a pattern compatible with substrate inhibition. Comparative analysis of genetic contexts identified two arrangements of the catB14-floR module: one associated with mobA/mobB and ISCR2, the other chromosomal and associated with IS1182 within a multidrug-resistance region. We propose a two-configuration evolutionary model for the stabilization of the determinant, although this relationship is inferred from the comparison of genetic contexts rather than demonstrated experimentally. This work provides a standardized nomenclature framework for the CatB superfamily and describes the genetic contexts in which catB14 occurs.