Transferable antibiotic resistance plasmids from biogas plant digestates often belong to the IncP-1ε subgroup.

Birgit Wolters,Martina Kyselkovã¡,Kornelia Smalla,Robert Kreuzig,Ellen Krã¶Gerrecklenfort

doi:10.3389/fmicb.2014.00765

Abstract

Manure is known to contain residues of antibiotics administered to farm animals as well as bacteria carrying antibiotic resistance genes (ARGs). These genes are often located on mobile genetic elements. In biogas plants (BGPs), organic substrates such as manure and plant material are mixed and fermented in order to provide energy, and resulting digestates are used for soil fertilization. The fate of plasmid carrying bacteria from manure during the fermentation process is unknown. The present study focused on transferable antibiotic resistance plasmids from digestates of seven BGPs, using manure as a co-substrate, and their phenotypic and genotypic characterization. Plasmids conferring resistance to either tetracycline or sulfadiazine were captured by means of exogenous plasmid isolation from digestates into Pseudomonas putida KT2442 and Escherichia coli CV601 recipients, at transfer frequencies ranging from 10-5 to 10-7. Transconjugants (n = 101) were screened by PCR-Southern blot hybridization and real-time PCR for the presence of IncP-1, IncP-1ε, IncW, IncN, IncP-7, IncP-9, LowGC, and IncQ plasmids. While 61 plasmids remained unassigned, 40 plasmids belonged to the IncP-1ε subgroup. All these IncP-1ε plasmids were shown to harbor the genes tet(A), sul1, qacEΔ1, intI1, and integron gene cassette amplicons of different size. Further analysis of 16 representative IncP-1ε plasmids showed that they conferred six different multiple antibiotic resistance patterns and their diversity seemed to be driven by the gene cassette arrays. IncP-1ε plasmids displaying similar restriction and antibiotic resistance patterns were captured from different BGPs, suggesting that they may be typical of this environment. Our study showed that BGP digestates are a potential source of transferable antibiotic resistance plasmids, and in particular the broad host range IncP-1ε plasmids might contribute to the spread of ARGs when digestates are used as fertilizer.

Highlights

Due to the promotion of renewable energies the number of biogas plants (BGPs) in Germany is on the rise during the last years (Fachverband Biogas e.V., 20141)
Our study showed that BGP digestates are a potential source of transferable antibiotic resistance plasmids, and in particular the broad host range IncP-1ε plasmids might contribute to the spread of antibiotic resistance genes (ARGs) when digestates are used as fertilizer
TRANSFERABLE ANTIBIOTIC RESISTANCE PLASMIDS CAPTURED FROM BIOGAS DIGESTATES Exogenous plasmid isolations from the digestates were performed in order to capture transferable plasmids conferring resistance toward TET or SDZ into P. putida KT2442 gfp+ and E. coli CV601 gfp+ recipients, respectively

Summary

Introduction

Due to the promotion of renewable energies the number of biogas plants (BGPs) in Germany is on the rise during the last years (Fachverband Biogas e.V., 20141). In BGPs organic substrates of different origin, such as manures, sludge or plant material, are mixed and fermented anaerobically under usually mesophilic conditions (approximately 40◦C) in order to provide energy (Weiland, 2010). The digestates resulting from this process are often stored for several months before being applied to agricultural fields as fertilizer similar to manures. Depending on their chemical structure, antibiotics applied to livestock, as well as their partially still active metabolites, were detected in the excrements of treated animals in several studies (Boxall et al, 2004; Sarmah et al, 2006; Lamshöft et al, 2007).

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Conclusion