Development and application of real-time PCR assays for quantification of erm genes conferring resistance to macrolides-lincosamides-streptogramin B in livestock manure and manure management systems

Erythromycin and tylosin are commonly used in animal production, and such use is perceived to contribute to the overall antimicrobial resistance (AR) reservoirs. Quantitative measurements of this type of AR reservoir in microbial communities are required to understand AR ecology (e.g., emergence, persistence, and dissemination). We report here the development, validation, and use of six real-time PCR assays for quantifying six classes of erm genes (classes A through C, F, T, and X) that encode the major mechanism of resistance to macrolides-lincosamides-streptogramin B (MLS(B)). These real-time PCR assays were validated and used in quantifying the six erm classes in five types of samples, including those from bovine manure, swine manure, compost of swine manure, swine waste lagoons, and an Ekokan upflow biofilter system treating hog house effluents. The bovine manure samples were found to contain much smaller reservoirs of each of the six erm classes than the swine manure samples. Compared to the swine manure samples, the composted swine manure samples had substantially reduced erm gene abundances (by up to 7.3 logs), whereas the lagoon or the biofilter samples had similar erm gene abundances. These preliminary results suggest that the methods of manure storage and treatment probably have a substantial impact on the persistence and decline of MLS(B) resistance originating from food animals, thus likely affecting the dissemination of such resistance genes into the environment. The abundances of these erm genes appeared to be positively correlated with those of the tet genes determined previously among these samples. These real-time PCR assays provide a rapid, quantitative, and cultivation-independent measurement of six major classes of erm genes, which should be useful for ecological studies of AR.