erm(46) copy number determines macrolide resistance in Rhodococcus equi

To develop a multiplex real-time PCR assay for simultaneous detection of Rhodococcus equi (choE), virulence (vapA), and macrolide-resistance determinants (erm[46], erm[51]), and evaluate their clinical and temporal trends.

Clinical R equi isolates (n = 229) obtained from the Hagyard Equine Medical Institute laboratory were collected sequentially over a 3-year surveillance period (2021 to 2023) from 14 states and underwent broth microdilution susceptibility testing. A cohort of 214 isolates with high-quality DNA was characterized using quantitative PCR. Geographic distribution, seasonal trends, and genotypic-phenotypic concordance were evaluated.

The multiplex PCR assay demonstrated 100% analytical specificity. The virulence gene vapA was present in 94.4% (202 of 214) of isolates. The macrolide resistance determinant erm(46) was highly prevalent at 62.6% (134 of 214), showing a significant seasonal increase during July to December periods at 76.6% (vs 58.2% in January to June). Crucially, erm(51) was detected in a single isolate (0.5%), representing its first recorded clinical identification in R equi. Unstratified genotypic-phenotypic agreement for macrolides was fair (κ = 0.22 to 0.25). However, stratification by genomic copy number revealed near-perfect concordance (κ ≥ 0.97) exclusively among isolates harboring > 50,000 target copies. Alternative agents (gentamicin, ceftiofur, minocycline, doxycycline) maintained 97% to 100% susceptibility, regardless of erm(46) status.

While erm(46) is widely distributed across R equi-endemic farms, actual phenotypic resistance expression is strongly copy-number dependent. The clinical emergence of erm(51) expands the known resistome of this pathogen.

Incorporating quantitative molecular screening into routine R equi diagnostics provides crucial real-time therapeutic guidance, supporting precision medicine and targeted antimicrobial stewardship on endemic equine farms.