Retrospective characterization of Rhodococcus equi clinical isolates: Integrating phenotypic antimicrobial resistance with vapA, erm(46), and erm(51) genotyping

Abstract

Rhodococcus equi pneumonia remains a leading cause of mortality in foals, with treatment outcomes critically dependent on early detection of virulent strains and antimicrobial resistance. The emergence of macrolide-resistant strains carrying erm(46) has altered therapeutic success rates, calling for rapid molecular diagnostic approaches. The objective of this study was to develop and validate a multiplex PCR assay for simultaneous detection of R. equi (choE), and determining virulence (vapA), and macrolide resistance (erm(46), erm(51)), and to characterize resistance prevalence and temporal trends in clinical isolates. A total of 229 clinical R. equi isolates collected from February 2021 to December 2023 were analyzed using quantitative PCRs targeting the genes: choE, vapA, erm(46) and erm(51). Antimicrobial susceptibility testing was performed using broth microdilution for 12 agents. Sequence validation was conducted using MEGA (MUSCLE) alignment analysis. Geographic distribution, temporal trends, and phenotypic-genotypic concordance were evaluated. The PCRs demonstrated high specificity with 100% sequence identity to reference strains. Among 229 isolates, vapA was detected in 82.1% (188/229), confirming the majority were virulent strains. The erm(46) gene showed high prevalence (55.9%, 128/229) with significant temporal variation: 48.3% in 2021, peaking at 81.5% in 2022, then declining to 69.6% in 2023 (p<0.05). In contrast, erm(51) was rare (0.4%, 1/229), consistent with its environmental origin. Phenotypic-genotypic concordance was high for macrolides (>55%) but low for non- MLSB antibiotics (35-38%), confirming mechanistic specificity. Gentamicin, ceftiofur, minocycline, and doxycycline maintained universal or near-universal susceptibility (97-100%) regardless of erm(46) status. This study demonstrates high erm(46) prevalence with significant temporal fluctuation, while erm(51) remains primarily an environmental concern. The PCR assays enabled rapid identification of virulent, resistant strains within 12 hours compared to 72 hours for conventional methods. Concordance between erm(46) detection and macrolide resistance validates molecular screening for guiding therapeutic decisions. The sustained efficacy of alternative antimicrobials provides critical treatment options for resistant infections. These findings support integration of molecular diagnostics into routine R. equi pneumonia management and highlight the need for enhanced antimicrobial stewardship on endemic farms.