Related Experiment Video
Updated: May 22, 2026
Multiplex PCR Assay for Typing of Staphylococcal Cassette Chromosome Mec Types I to V in Methicillin-resistant Staphylococcus aureus
Published on: September 5, 2013
Multiplex PCR to identify macrolide resistance determinants in Mannheimia haemolytica and Pasteurella multocida
Simon Rose1, Benoit Desmolaize, Puneet Jaju
1Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark.
Abstract:
The bacterial pathogens Mannheimia haemolytica and Pasteurella multocida are major etiological agents in respiratory tract infections of cattle. Although these infections can generally be successfully treated with veterinary macrolide antibiotics, a few recent isolates have shown resistance to these drugs. Macrolide resistance in members of the family Pasteurellaceae is conferred by combinations of at least three genes: erm(42), which encodes a monomethyltransferase and confers a type I MLS(B) (macrolide, lincosamide, and streptogramin B) phenotype; msr(E), which encodes a macrolide efflux pump; and mph(E), which encodes a macrolide-inactivating phosphotransferase. Here, we describe a multiplex PCR assay that detects the presence of erm(42), msr(E), and mph(E) and differentiates between these genes. In addition, the assay distinguishes P. multocida from M. haemolytica by amplifying distinctive fragments of the 23S rRNA (rrl) genes. One rrl fragment acts as a general indicator of gammaproteobacterial species and confirms whether the PCR assay has functioned as intended on strains that are negative for erm(42), msr(E), and mph(E). The multiplex system has been tested on more than 40 selected isolates of P. multocida and M. haemolytica and correlated with MICs for the veterinary macrolides tulathromycin and tilmicosin, and the newer compounds gamithromycin and tildipirosin. The multiplex PCR system gives a rapid and robustly accurate determination of macrolide resistance genotypes and bacterial genus, matching results from microbiological methods and whole-genome sequencing.
Insights
A new multiplex PCR assay rapidly detects macrolide resistance genes (erm(42), msr(E), mph(E)) in cattle pathogens Mannheimia haemolytica and Pasteurella multocida, aiding veterinary treatment decisions.
Area of Science:
- Veterinary Microbiology
- Molecular Diagnostics
- Antimicrobial Resistance
Background:
- Mannheimia haemolytica and Pasteurella multocida cause significant bovine respiratory disease.
- Veterinary macrolide antibiotics are effective, but emerging resistance threatens treatment efficacy.
- Key macrolide resistance genes include erm(42), msr(E), and mph(E) in Pasteurellaceae.
Purpose of the Study:
- To develop a multiplex PCR assay for simultaneous detection of macrolide resistance genes.
- To differentiate between P. multocida and M. haemolytica using 23S rRNA gene fragments.
- To provide a rapid and accurate diagnostic tool for antimicrobial resistance genotyping in cattle pathogens.
Main Methods:
- Design and implementation of a multiplex PCR assay targeting erm(42), msr(E), and mph(E).
- Inclusion of 23S rRNA gene targets for bacterial species identification.
- Validation of the assay on over 40 isolates of P. multocida and M. haemolytica.
- Correlation of PCR results with Minimum Inhibitory Concentrations (MICs) for veterinary macrolides.
Main Results:
- The multiplex PCR assay accurately detected erm(42), msr(E), and mph(E) genes.
- The assay successfully differentiated between P. multocida and M. haemolytica.
- Results correlated well with established microbiological methods and whole-genome sequencing.
- The assay demonstrated robustness and accuracy in identifying macrolide resistance genotypes.
Conclusions:
- The developed multiplex PCR assay is a reliable tool for detecting macrolide resistance in M. haemolytica and P. multocida.
- This diagnostic method aids in guiding veterinary therapeutic strategies against bovine respiratory infections.
- The assay facilitates rapid identification of bacterial pathogens and their resistance profiles.
Related Concept Videos
01:25Clinical Significance of Antibiotic Resistance
01:25Mechanism of Antibiotic Resistance in MRSA