Increased abundance and transferability of resistance genes after field application of manure from sulfadiazine-treated pigs

Abstract

Spreading manure containing antibiotics in agriculture is assumed to stimulate the dissemination of antibiotic resistance in soil bacterial populations. Plant roots influencing the soil environment and its microflora by exudation of growth substrates might considerably increase this effect. In this study, the effects of manure from pigs treated with sulfadiazine (SDZ), here called SDZ manure, on the abundance and transferability of sulfonamide resistance genes sul1 and sul2 in the rhizosphere of maize and grass were compared to the effects in bulk soil in a field experiment. In plots that repeatedly received SDZ manure, a significantly higher abundance of both sul genes was detected compared to that in plots where manure from untreated pigs was applied. Significantly lower abundances of sul genes relative to bacterial ribosomal genes were encountered in the rhizosphere than in bulk soil. However, in contrast to results for bulk soil, the sul gene abundance in the SDZ manure-treated rhizosphere constantly deviated from control treatments over a period of 6 weeks after manuring, suggesting ongoing antibiotic selection over this period. Transferability of sulfonamide resistance was analyzed by capturing resistance plasmids from soil communities into Escherichia coli. Increased rates of plasmid capture were observed in samples from SDZ manure-treated bulk soil and the rhizosphere of maize and grass. More than 97% of the captured plasmids belonged to the LowGC type (having low G+C content), giving further evidence for their important contribution to the environmental spread of antibiotic resistance. In conclusion, differences between bulk soil and rhizosphere need to be considered when assessing the risks associated with the spreading of antibiotic resistance.

Fig 1

Time course of the relative abundance of sulfadiazine (SDZ) resistance genes sul1 (a) and sul2 (b) in bulk soil and rhizosphere of maize and grass. Soils treated with manure from pigs treated with SDZ or not are indicated by boxes connected by solid lines and triangles connected by dashed lines, respectively. Data points are connected by mere convenience, while no connection was drawn between data points taken before and after application of manure on days 0, 49, and 133. Error bars indicate standard deviations of results for 3 or 4 experimental replicates.

Fig 2

Effect of SDZ on relative sul1 and sul2 abundance in bulk soil (a) and rhizosphere (b) expressed by plotting the days after the previous application of manure against the differences in log units between results for control and SDZ treatment. Solid lines correspond to a linear regression model, shaded areas to their associated 95% confidence interval, and dotted lines to their 95% prediction limits.

Fig 3

Effect of manure containing sulfadiazine (SDZ) on frequencies of sulfonamide resistance transfer from bulk soil (BS) and rhizosphere (RH) bacteria to E. coli K-12 CV601gfp. Transfer frequencies per recipient cell were determined by filter matings on days 48 and 132 for field plots sown with grass and maize. Differing letters indicate significant differences of means in pairwise comparisons (Tukey test; P < 0.05) for each day. Treatments of soil with control manure and manure containing SDZ are indicated by light and dark gray, respectively. Error bars indicate standard deviations of results for 4 replicates (except maize rhizosphere treatment without SDZ, day 48, where n = 3).