PCR quantification of antibiotic resistance genes in composted and liquid stored manures

Antimicrobial resistance is becoming increasingly widespread, seriously undermining the effectiveness of antibiotics used to treat human and animal infections.

A variety of antibiotics are used in swine, poultry and cattle feed at subtherapeutic levels to improve growth. This has been shown to increase concentrations of antibiotic resistance genes in manure. Tetracyclines, erythromycin and tylosin are among the most widely used antibiotics in food animal production in the USA. Most of the antibiotic resistance originating from food animals ends up in the manure, which constitutes the largest reservoir of antibiotic resistance on animal farms. Reduction of this antibiotic resistance reservoir could minimize the environmental dissemination of antibiotic resistance.
Most animal farms have an operational manure management system on farm designed for nutrient recovery and sanitation. However, the potential persistence or reduction of antibiotic resistance in these manure management systems remains largely unknown. We report here the use of real-time PCR assays that can quantify antibiotic resistance genes in animal manure and manure treatment systems. The antibiotic resistance genes quantified include six classes of erm genes (classes A through C, F, T, and X) that encode the major mechanism of resistance to macrolides-lincosamidesstreptogramin B (MLSB) and three groups of tetracycline resistance genes: tet(A) and tet(C); tet(G); and tet genes encoding ribosomal protection proteins, including tet(M), tet(O), tetB(P), tet(Q), tet(S), tet(T), and tet(W). These realtime PCR assays were used to quantify these gene classes in bovine manure, swine manure, compost of swine manure, and effluent and lagoons of a conventional liquid manure system. In addition, the CFU of culturable tetracycline and erythromycin resistant bacteria in swine manure and composted and liquid stored swine manure was determined. The bovine manures were found to contain fewer copies of all three groups of tet genes and erm genes than the swine manures. Results showed that the abundance of tet(A,C), tet(G), ribosomal protection protein (RPP) tet, erm(A), erm(B), erm(C), erm(F), erm(T), and erm(X) in the swine manure lagoon samples was comparable to that determined for untreated swine manures as well as that for hog house effluent. There were no significant differences within the respective tet or erm gene groups in terms of their abundance in these samples. In contrast, the composted swine manure samples had up to six orders of magnitude less tet and erm gene abundances as compared to the untreated swine manure. However, different classes of tet and erm genes had varying reductions in the same samples, suggesting that different resistance genes may have different persistences during composting. Results of experiments to determine the extent of antibiotic resistance among culturable bacteria in composted and liquid stored swine manure revealed that erythromycin resistant and tetracycline resistant bacteria which were initially present at 2x107 and 2x108 CFU/g, respectively, dropped by one order of magnitude during liquid storage for 50 days and by 4-5 orders of magnitude during composting.
Overall the results suggest that the method of manure storage and treatment may have a substantial impact on the persistence and decline of tet and MLSB resistance originating from food animals in agricultural environments. The results also indicate that land application of lagoon stored swine manures may lead to the wide dispersion of antibiotic resistance genes in the environment and that composting may greatly reduce this impact.



Copyright: © European Compost Network ECN e.V.
Quelle: Orbit 2008 (Oktober 2008)
Seiten: 13
Preis inkl. MwSt.: € 10,50
Autor: Jr. Frederick C. Michel
Jing Chen
Lingling Wang
Srinand Sreevatsan
Mark Morrison

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