Novel Recombinase Polymerase Amplification Assay Is Sensitive for Detection of Macrolide Resistance Genes Relevant to Bovine Respiratory Disease Management in Feedlot Calves

Macrolides are crucial for the management and treatment of bovine respiratory disease (BRD). However, antimicrobial resistance (AMR) threatens the efficacy of these and other antimicrobials. We developed real-time recombinase polymerase amplification (RPA) assays targeting three clinically relevant macrolide antimicrobial resistance genes (ARGs)-<i>msrE</i>-<i>mphE</i> and <i>erm42</i>-in ≤30 min using extracted DNA. A set of 199 deep nasopharyngeal swabs (DNPS) collected from feedlot calves near the time of arrival were selected based on bacterial culture (BC) results for <i>Mannheimia haemolytica</i>, <i>Pasteurella multocida</i>, and <i>Histophilus somni</i> and antimicrobial susceptibility testing (AST) for tulathromycin, tilmicosin, tildipirosin, or gamithromycin. Samples were also tested for the same targets using RPA and polymerase chain reaction (PCR). In samples that were culture-positive for one or more macrolide-resistant BRD-associated bacteria (n = 101), <i>msrE-mphE</i> and/or <i>erm42</i> were detected in 95% of cases using RPA. The remaining 98 samples were either culture-negative, or the recovered bacteria were macrolide-susceptible: 43% of these were RPA-positive for at least one macrolide ARG. Together with BC-AST and PCR, Bayesian latent class modelling estimated the clinical sensitivity of RPA for macrolide ARGs to be 95% and specificity to be 58%, with moderate agreement between RPA and BC-AST (κ = 0.52) or PCR (κ = 0.55). The estimated sensitivity of the RPA multiplex assay for the targeted macrolide ARGs was very good, although estimated specificity was limited. However, Sanger sequencing confirmed RPA detection of <i>msrE-mphE</i> in BC-AST/PCR-negative samples (n = 23), reflecting the presence of this locus in non-target bacteria, as well as potential ARG variants among BRD bacteria. These findings support the potential of RPA for rapid ARG detection from extracted DNA. Continued assay optimization and evaluation for detection of respiratory bacteria and ARGs will further enhance its diagnostic utility.