Moving beyond the MIC: Evaluation of a novel multiplex real time PCR assay for detection of antimicrobial resistance genes in clinical bovine respiratory disease samples

Authors: LOY, JOHN - UNIVERSITY OF NEBRASKA; PAYNE, JOSHUA - UNIVERSITY OF NEBRASKA; DEAL, CAITLYN - UNIVERSITY OF NEBRASKA; DUTTA, ENAKSHY - UNIVERSITY OF NEBRASKA; BULUT, ECE - UNIVERSITY OF NEBRASKA; Clawson, Michael - Mike; WANG, BING - UNIVERSITY OF NEBRASKA

Submitted to: American Association of Veterinary Laboratory Diagnosticians
Publication Type: Abstract Only
Publication Acceptance Date: 9/1/2018
Publication Date: 10/26/2018
Citation: Loy, J.D., Payne, J., Deal, C., Dutta, E., Bulut, E., Clawson, M.L., Wang, B. 2018. Moving beyond the MIC: Evaluation of a novel multiplex real time PCR assay for detection of antimicrobial resistance genes in clinical bovine respiratory disease samples [abstract]. In: Proceedings of the American Association of Veterinary Laboratory Diagnosticians, October 18-22, 2018, Kansas City, Missouri. p. 7.

Interpretive Summary:

Technical Abstract: Bovine respiratory disease (BRD) is one of the most significant diseases of cattle production. BRD is multi-factorial, with viral and bacterial pathogen components. Mannheimia haemolytica is one of the predominant bacterial pathogens that causes BRD. Recently, multiple drug resistance has been reported among M. haemolytica and other BRD pathogens. Resistance in M. haemolytica can be conferred through the acquisition of mobile genetic elements (ICE) which can possess and/or acquire multiple antimicrobial resistance genes. A real time multiplex PCR assay (rtPCR) was designed that detects four genes conferring macrolide (erm(42), mph(E), msr(E)) or tetracycline (tetH) resistance. The assay was developed and validated using M. haemolytica isolates that had been subjected to whole genome sequencing and that were positive for an ICE containing one or more of the four resistance genes. A comparative analysis was undertaken with this assay and classical culture combined with MIC testing. Lung tissues from 136 animals submitted to the Nebraska Veterinary Diagnostic Center for bovine respiratory disease diagnosis were included in the analysis. The tissues were subjected to culture and susceptibility testing on isolates using routine methods. Additionally, DNA was extracted directly from lung tissue using a high-throughput DNA extraction method and then subjected to the rtPCR assay. Resistance or susceptibility of isolated M. haemolytica was determined by application of CLSI approved breakpoints for tilmicosin, tulathromycin and oxytetracycline. There were 25 cases in which isolated M. haemolytica were classified as resistant (R) to macrolides and 21 classified as susceptible (S), and 29 cases were classified as R for tetracyclines and 16 as S included in the analysis. Optimal Cq cutoff values that maximized sensitivity and specificity with isolation of a macrolide or tetracycline resistant M. haemolytica were calculated using Receiving Operating Characteristic (ROC) curve analysis. The optimal Cq cutoff value for testing macrolide resistant M. haemolytica ranged from 28.9-32.5, with Kappa statistic (K) showing the agreement between the rtPCR and culture-based methods ranging from 0.66-0.79 and with Sensitivity (Se) 80-100% and Specificity (Sp) 80-90%. For tetracycline resistance Cq cutoffs were calculated to be 31, with Se of 87.5 and Sp of 86.2 and K of 0.72. Overall detection of resistance genes by rtPCR had mostly moderate levels of agreement with isolation of resistant M. haemolytica from lung tissues. The presence of other organisms or strains with one or more of the four gene targets within the lung tissues may have increased the level of disagreement between the two assays. A rtPCR based assay that detects antimicrobial resistance genes in M. haemolytica and other BRD pathogens may provide more rapid results and allow for enhanced detection of potential antimicrobial resistance within clinical sample pathogens over culture-based approaches.