Plasmids are important vehicles for rapid adaptation of bacterial populations to changing environmental conditions. It is thought that to reduce the cost of plasmid carriage, only a fraction of a local population carries plasmids or is permissive to plasmid uptake. Plasmids provide various accessory traits which might be beneficial under particular conditions. The genetic variation generated by plasmid carriage within populations ensures the robustness toward environmental changes. Plasmid-mediated gene transfer plays an important role not only in the mobilization and dissemination of antibiotic resistance genes but also in the spread of degradative pathways and pathogenicity determinants of pathogens. Here we summarize the state-of-the-art methods to study the occurrence, abundance, and diversity of plasmids in environmental bacteria. Increasingly, cultivation-independent total-community DNA-based methods are being used to characterize and quantify the diversity and abundance of plasmids in relation to various biotic and abiotic factors. An improved understanding of the ecology of plasmids and their hosts is crucial in the development of intervention strategies for antibiotic-resistance-gene spread. We discuss the potentials and limitations of methods used to determine the host range of plasmids, as the ecology of plasmids is tightly linked to their hosts. The recent advances in sequencing technologies provide an enormous potential for plasmid classification, diversity, and evolution studies, but numerous challenges still exist.

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.

Quaternary ammonium compounds (QACs) are surface-active, antimicrobial, high production volume (HPV) chemicals with a broad application in agriculture. This review provides a comprehensive overview of (1) predicted and measured concentrations of QACs in soils including their analysis, (2) sequestration mechanisms in soils based on their physicochemical properties and chemical structure, and (3) implications of concentrations and fate of QACs in soils for the proliferation of antibiotic resistance in the environment. Predicted environmental concentrations (PEC) for QACs that are applied to soils with manure are in the order of 3.5 mg kg⁻¹. Based on literature data, the median PEC of QAC in sewage sludge amended soils is 25 µg kg⁻¹. The positively charged QACs are mainly sorbed to clay minerals. We propose that QACs might be sequestered in the interlayer regions of layered silicates in clay-rich soils, reducing their acute toxicity, while increasing their persistence. The release of sequestered QACs from soil can still potentially maintain concentration levels that are sufficient to develop antibiotic resistance in the environment.

The role of broad-host range IncP-1ε plasmids in the dissemination of antibiotic resistance in agricultural systems has not yet been investigated. These plasmids were detected in total DNA from all of 16 manure samples and in arable soil based on a novel 5′-nuclease assay for real-time PCR. A correlation between IncP-1ε plasmid abundance and antibiotic usage was revealed. In a soil microcosm experiment the abundance of IncP-1ε plasmids was significantly increased even 127 days after application of manure containing the antibiotic compound sulfadiazine, compared to soil receiving only manure, only sulfadiazine, or water. Fifty IncP-1ε plasmids that were captured in E. coli CV601gfp from bacterial communities of manure and arable soil were characterized by PCR and hybridization. All plasmids carried class 1 integrons with highly varying sizes of the gene cassette region and the sul1 gene. Three IncP-1ε plasmids captured from soil bacteria and one from manure were completely sequenced. The backbones were nearly identical to that of the previously described IncP-1ε plasmid pKJK5. The plasmids differed mainly in the composition of a Tn402-like transposon carrying a class 1 integron with varying gene cassettes, IS1326, and in three of the plasmids the tetracycline resistance transposon Tn1721 with various truncations. Diverse Beta- and Gammaproteobacteria were revealed as hosts of one of the IncP-1ε plasmids in soil microcosms. Our data suggest that IncP-1ε plasmids are important vectors for horizontal transfer of antibiotic resistance in agricultural systems.

Large amounts of manure have been applied to arable soils as fertilizer worldwide. Manure is often contaminated with veterinary antibiotics which enter the soil together with antibiotic resistant bacteria. However, little information is available regarding the main responders of bacterial communities in soil affected by repeated inputs of antibiotics via manure. In this study, a microcosm experiment was performed with two concentrations of the antibiotic sulfadiazine (SDZ) which were applied together with manure at three different time points over a period of 133 days. Samples were taken 3 and 60 days after each manure application. The effects of SDZ on soil bacterial communities were explored by barcoded pyrosequencing of 16S rRNA gene fragments amplified from total community DNA. Samples with high concentration of SDZ were analyzed on day 193 only. Repeated inputs of SDZ, especially at a high concentration, caused pronounced changes in bacterial community compositions. By comparison with the initial soil, we could observe an increase of the disturbance and a decrease of the stability of soil bacterial communities as a result of SDZ manure application compared to the manure treatment without SDZ. The number of taxa significantly affected by the presence of SDZ increased with the times of manure application and was highest during the treatment with high SDZ-concentration. Numerous taxa, known to harbor also human pathogens, such as Devosia, Shinella, Stenotrophomonas, Clostridium, Peptostreptococcus, Leifsonia, Gemmatimonas, were enriched in the soil when SDZ was present while the abundance of bacteria which typically contribute to high soil quality belonging to the genera Pseudomonas and Lysobacter, Hydrogenophaga, and Adhaeribacter decreased in response to the repeated application of manure and SDZ.