Impact of Soil Fertilization with Pig Slurry on Antibiotic Residues and Resistance Genes: A Longitudinal Study

Abstract

The impact of soil fertilization with animal manure on the spread and persistence of antibiotic resistance in the environment is far from being fully understood. To add knowledge about persistence and correlations between antibiotic residues and antibiotic resistance genes (ARGs) in fertilized soil, a longitudinal soil mesocosm study was conducted. Soil samples were collected from the mesocosms immediately before spreading and then afterward at fifteen time points during a 320-day observation period. Eight ARGs (ermB, sul1, tetA, tetG, tetM, cfr, fexA, and optrA) and the class 1 integron-integrase gene, intI1, were determined in both pig slurry and soil, as well as residues of 36 antibiotics. Soil chemical and biochemical parameters were also measured. Twelve antibiotics were detected in the slurry in the range of 3 µg kg^-1-3605 µg kg^-1, with doxycycline, lincomycin, and tiamulin being the most abundant, whereas ermB, sul1, and tetM were the predominant ARGs. Before spreading, neither antibiotic residues nor ARGs were detectable in the soil; afterwards, their concentrations mirrored those in the slurry, with a gradual decline over the duration of the experiment. After about three months, the effect of the amendment was almost over, and no further evolution was observed.

Keywords: antibiotic residues; antibiotic resistance genes; manure treatment; slurry; swine.

Figure 1

Extracted ion chromatograms: (a) soil after two days of spreading (t2); (b) soil before slurry spreading (t0). The peaks of the four most abundant antibiotics (lincomycin, oxytetracycline, doxycycline, and tiamulin) are shown.

Figure 2

Soil concentrations (µg kg⁻¹ dw) of doxycycline (DOX), tiamulin (TIA), and lincomycin (LIN) during the incubation experiment.

Figure 3

Relative abundances of ermB, sul1 (a), cfr, fexA, and optrA (b). In both figures the copy gene number/copy number of 16S rRNA-log scale in soil during the incubation experiment are reported.

Figure 4

Principal Component Analysis Factor Map: Cluster 1 (●) grouped the observations relative to t9–t15 time points, Cluster 2 (▲) the observations relative to t5–t8 time points, and Cluster 3 (■) the observations relative to t1–t4 time points. Before the underscore is reported, the time point number (t1, t2, t3 … t15) and, after, the number of replicates (1, 2, 3).