Development of a multiplex real-time PCR assay for predicting macrolide and tetracycline resistance associated with bacterial pathogens of bovine respiratory disease

Authors: DUTTA, ENAKSHY - University Of Nebraska; LOY, JOHN - University Of Nebraska; DEAL, CAITLYNA - University Of Nebraska; Wynn, Emily; Clawson, Michael - Mike; CLARKE, JENNIFER - University Of Nebraska; WANG, BING - University Of Nebraska

Submitted to: Pathogens
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/11/2021
Publication Date: 1/13/2021
Citation: Dutta, E., Loy, J.D., Deal, C.A., Wynn, E.L., Clawson, M.L., Clarke, J., Wang, B. 2021. Development of a multiplex real-time PCR assay for predicting macrolide and tetracycline resistance associated with bacterial pathogens of bovine respiratory disease. Pathogens. 10:64. https://doi.org/10.3390/pathogens10010064.
DOI: https://doi.org/10.3390/pathogens10010064

Interpretive Summary: Bovine respiratory disease (BRD) affects cattle throughout much of the United States and the world. In addition to an animal health and wellbeing issue, economic losses from BRD related prevention, treatment, and mortalities can cost over a billion dollars a year in the United States alone. There are a number of bacterial pathogens that cause or contribute to BRD. The main ones are Mannheimia haemolytica, Histophilus somni, and Pasteurella multocida. Antibiotics including macrolides and tetracyclines have been successfully used to treat and control BRD cases involving these bacteria. However, strains of M. haemolytica, H. somni, and P. multocida have all recently demonstrated levels of resistance to one or both of these antibiotic classes. Following a BRD diagnosis, timely treatments of cattle can reduce the risk of relapse and mortality. Thus, it is vital to know what antibiotic class will work most effectively for BRD cases. In this study, an assay was created that detects genes encoding macrolide or tetracycline resistance that are known to be carried in some strains of M. haemolytica, H. somni, or P. multocida. The assay was optimized for detection of the genes, and for prediction of either macrolide or tetracycline resistance by these bacteria. Importantly, this assay can be run very quickly on cattle samples. Consequently, it can be used to determine the potential effectiveness of tetracycline or macrolide treatments for BRD during outbreaks.

Technical Abstract: Antimicrobial resistance (AMR) in bovine respiratory disease (BRD) is an emerging concern that may threaten both animal and public health. Rapid and accurate detection of AMR is essential for prudent drug therapy selection during BRD outbreaks. This study aimed to develop a multiplex quantitative real-time polymerase chain reaction assay (qPCR) to provide culture-independent information regarding the phenotypic AMR status of BRD cases and an alternative to the gold-standard, culture-dependent test. Bovine clinical samples (297 lung and 111 nasal) collected in Nebraska were subjected to qPCR quantification of macrolide (MAC) and tetracycline (TET) resistance genes and gold-standard determinations of AMR of BRD pathogens. Receiver operating characteristic curve analysis was used to classify AMR based on the qPCR results. For lung tissues, the qPCR method showed good agreement with the gold-standard test for both MACs and TETs, with a sensitivity of 67–81% and a specificity higher than 80%. For nasal swabs, qPCR results passed validation criteria only for TET resistance detection, with a sensitivity of 88%, a specificity of 80% and moderate agreement. The culture-independent assay developed here provides the potential for more rapid AMR characterization of BRD cases directly from clinical samples at equivalent accuracy and higher time efficiency compared with the gold-standard, culture-based test.