Intracellular and Extracellular Antimicrobial Resistance Genes in the Sludge of Livestock Waste Management Structures

Abstract

The sludge compartment in livestock waste management structures is a potential hotbed for the emergence and proliferation of antimicrobial resistance among bacteria. Little is known about the distribution of antimicrobial resistance genes (ARGs) between the intracellular and extracellular DNA pools in the sludge. The overall objective of this study was to assess the significance of extracellular ARGs to the total ARGs in the sludge of livestock waste management structures. In this study, sludge samples were collected from four cattle manure storage ponds and three swine waste treatment lagoons and analyzed for genetic indicators of resistance. Intracellular DNA (iDNA) and extracellular DNA (eDNA) in the sludge were separately extracted using an optimized protocol. ARGs [sul(I), sul(II), tet(O), tet(Q), and tet(X)] in both the iDNA and eDNA extracts were quantified using quantitative polymerase chain reaction (qPCR), and antimicrobials, including sulfonamides and tetracyclines, were measured using liquid chromatography tandem mass spectrometry. Results showed that eDNA constituted less than 1.5% of the total DNA in sludge. All ARGs tested were detected in nearly all eDNA and iDNA samples. Furthermore, every gram of dry sludge contained from 1.7 × 10(3) to 4.2 × 10(8) copies of extracellular ARG and from 3.2 × 10(7) to 3.2 × 10(10) copies of intracellular ARG. Chlortetracycline concentrations ranged between 187 and 2674 μg/g of sludge wet weight (ww), while sulfonamide concentrations were lower than 6.3 μg/g of sludge ww. The detection of ARGs in eDNA extracts suggests that transformation is a potential mechanism in ARG proliferation in the sludge of livestock waste management structures.