Occurrence and abundance of antibiotic resistance genes in agricultural soil receiving dairy manure

Animal manures are commonly used to enhance soil fertility, but there are growing concerns over the impact of this practice on the development and dissemination of antibiotic resistance. The aim of this study was to determine the effect of annual dairy manure applications on the occurrence and abundance of antibiotic resistance genes (ARGs) in an agricultural soil under crop production. The experimental design was a randomized complete block with four replications, which included the following treatments: i) control (no fertilizer); ii) inorganic fertilizer; and iii) stockpiled dairy manure. Inorganic fertilizer was applied in the spring, while dairy manure was applied in the fall of 2012–2015 at 17.2, 34.5, and 52.0 Mg (dry wt.)/ha. Soil samples were collected in the spring (pre-plant) and fall (post-harvest) to a depth of 120 cm. DNA extracted from the soils was used in a quantitative real-time PCR reaction to determine absolute abundances (per g dry soil) and relative abundances (per 16S rRNA gene copies) of ARGs. The ARGs targeted were blaCTX-M-1, erm(B), sul1, tet(A), tet(W), and tet(X) and a class 1 integron-integrase gene (intI1), but only sul1, tet(W), tet(X), and intI1 were detected in enough samples to run statistics and draw conclusions. This study found that: i) manure application increases ARG abundances above background soil levels; ii) the higher the manure application rate, the higher the ARG abundance in soil; iii) the amount of manure applied is more important than reoccurring annual applications of the same amount of manure; iv) absolute abundance and occurrence of ARGs decreases with increasing soil depth, but relative abundances remained constant; and v) ARGs correlated with each other and a number of soil chemical parameters, but not with soil moisture or 16S rRNA gene. Overall, this study demonstrated that dairy manure applications significantly increase the abundance of clinically relevant ARGs when compared to soil that received inorganic fertilizer or no fertilizer/manure