Novel Quantitative PCR for Rhodococcus equi and Macrolide Resistance Detection in Equine Respiratory Samples

Abstract

R. equi is an important veterinary pathogen that takes the lives of many foals every year. With the emergence and spread of MDR R. equi to current antimicrobial treatment, new tools that can provide a fast and accurate diagnosis of the disease and antimicrobial resistance profile are needed. Here, we have developed and analytically validated a multiplex qPCR for the simultaneous detection of R. equi and related macrolide resistance genes in equine respiratory samples. The three sets of oligos designed in this study to identify R. equi housekeeping gene choE and macrolide resistance genes erm(46) and erm(51) showed high analytic sensitivity with a limit of detection (LOD) individually and in combination below 12 complete genome copies per PCR reaction, and an amplification efficiency between 90% and 147%. Additionally, our multiplex qPCR shows high specificity in in-silico analysis. Furthermore, it did not present any cross-reaction with normal flora from the equine respiratory tract, nor commonly encountered respiratory pathogens in horses or other genetically close organisms. Our new quantitative PCR is a trustable tool that will improve the speed of R. equi infection diagnosis, as well as helping in treatment selection.

Keywords: Rhodococcus equi; diagnosis; erm(46); erm(51); macrolide resistance; qPCR.

Figure 1

Conventional PCR with temperature gradient for oligo sets Rhodo_Dlab, Erm46_Dlab, and Erm51_Dlab. Expected band of ~200 bp size.

Figure 2

Testing the analytic sensitivity of Rhodo_Dlab, Erm46_Dlab, and Erm51_Dlab sets in singleplex. qPCR curves for the three oligo sets in singleplex assays. In the Y-exe ΔRn, or fluorescence signal. In the X-exe, PCR amplification cycle. Colors indicate different 10-fold dilution of R.equi DNA (ng/µL).

Figure 3

Testing the analytic specificity of Rhodo_Dlab, Erm46_Dlab, and Erm51_Dlab sets in the multiplex. qPCR curves for the three oligo sets in multiplex assays. In the Y-exe ΔRn, or fluorescence signal. In the X-exe, PCR amplification cycle. Colors indicate different bacteria species and PCR controls: pink- R.equi mix DNA (R.e); purple- nasal swab DNA (matrix); dark blue, M. avium subsp. paratuberculosis (M.a.p); light blue- N. asteorides (N.a); dark green-S. equi subsp. zooepidermicus (S.e.z); light green-S. equi subsp. equi (S.e.e); yellow- C. pseudotuberculosis (C.p); red negative control (no DNA, Mmix).