Accumulation of sulfonamide resistance genes in arable soils due to repeated application of manure containing sulfadiazine

Abstract

Two soils were amended three times with pig manure. The abundance of sulfonamide resistance genes was determined by quantitative PCR 2 months after each application. In both soils treated with sulfadiazine-containing manure, the numbers of copies of sul1 and sul2 significantly increased compared to numbers after treatments with antibiotic-free manure or a control and accumulated with repeated applications.

FIG. 1.

Effects of antibiotic-free manure and manure containing sulfadiazine (SDZ¹⁰ or SDZ¹⁰⁰, 10 or 100 mg kg⁻¹ soil) on the abundances of the sulfonamide resistance genes sul1 and sul2 relative to ribosomal rrn gene abundances in two soils, as quantified by 5′-nuclease assays using quantitative real-time PCR. Manure was applied at days 0, 63, and 133. Samples were analyzed 2 months after each application (days 60, 123, and 193). Error bars indicate the standard deviations (n = 5). Values over arrows show the percentages of alterations due to the repeated applications of the respective manures within a treatment (±estimated errors of slopes), as revealed by linear-regression analysis.

FIG. 2.

Sulfadiazine concentrations in soil microcosms after treatment S10 (upper graph) and S100 (lower graph) during the 60-day periods after each of three manure applications. Black boxes represent the temporal averages for each period, which were calculated from interpolated concentrations with a time step of 1 day. Box whisker plots show the medians, quartiles, and ranges of measured concentrations.