Bulk soil and maize rhizosphere resistance genes, mobile genetic elements and microbial communities are differently impacted by organic and inorganic fertilization.

Organic soil fertilizers, such as livestock manure and biogas digestate, frequently contain bacteria carrying resistance genes (RGs) to antimicrobial substances and mobile genetic elements (MGEs). The effects of different fertilizers (inorganic, manure, digestate) on RG and MGE abundance and microbial community composition were investigated in a field plot experiment. The relative abundances of RGs [sul1, sul2, tet(A), tet(M), tet(Q), tet(W), qacEΔ1/qacE] and MGEs [intI1, intI2, IncP-1, IncP-1ε and LowGC plasmids] in total community (TC)-DNA from organic fertilizers, bulk soil and maize rhizosphere were quantified by qPCR before/after fertilization and prior to maize harvest. Microbial communities were analyzed via Illumina sequencing of 16S rRNA gene fragments amplified from TC-DNA. Compared to inorganic fertilization, manure treatments increased relative abundances of all RGs analyzed, integrons and few genera affiliated to Bacteroidetes and Firmicutes in bulk soil, while digestate increased sul2, tet(W) and intI2. At harvest, treatment effects vanished in bulk soil. However, organic fertilizer effects were still detectable in the rhizosphere for RGs [manure: intI1, sul1; digestate: tet(W)] and Clostridium related sequences (digestates) with increased relative abundance. Our data indicated transient organic fertilizer effects on RGs, MGEs and microbial community composition in bulk soil with long-term history of digestate or manure application.

Contaminations of organic fertilizers with antibiotic residues, resistance genes, and mobile genetic elements mirroring antibiotic use in livestock?

Pig manures are frequently used as fertilizer or co-substrate in biogas plants (BGPs) and typically contain antibiotic residues (ARs), as well as bacteria carrying resistance genes (RGs) and mobile genetic elements (MGEs). A survey of manures from eight pig fattening and six pig breeding farms and digestates from eight BGPs in Lower Saxony, Germany was conducted to evaluate the link between antibiotic usage and ARs to RGs and MGEs present in organic fertilizers. In total, 11 different antibiotics belonging to six substance classes were applied in the farms investigated. Residue analysis revealed concentrations of tetracycline up to 300 mg kg-1 dry weight (DW) in manures and of doxycycline up to 10.1 mg kg-1 DW in digestates indicating incomplete removal during anaerobic digestion. RGs (sul1, sul2, tet(A), tet(M), tet(X), qacE∆1) were detected in total community DNA of all samples by PCR-Southern blot hybridization. Broad-host range plasmids (IncP-1, IncQ, IncN, and IncW) and integron integrase genes (intI1, intI2) were found in most manure samples with IncN and IncW plasmids being more abundant in manure from pig breeding compared to pig fattening farms. IntI1, IncQ, and IncW plasmids were also detected in all digestates, while IncP-1, IncN, and LowGC plasmids were detected only sporadically. Our findings strongly reinforce the need for further research to identify mitigation strategies to reduce the level of contamination of organic fertilizers with ARs and transferable RGs that are applied to soil and that might influence the mobile resistome of the plant microbiome.

From the application of antibiotics to antibiotic residues in liquid manures and digestates: A screening study in one European center of conventional pig husbandry.

In conventional pig husbandry, antibiotics are frequently applied. Together with excreta, antibiotic residues enter liquid manures finally used as organic soil fertilizers or input materials for biogas plants. Therefore, this first screening study was performed to survey the application patterns of antibiotics from fall 2011 until spring 2013. Manures and digestates were then analyzed for selected antibiotic residues from spring 2012 to 2013. The data analysis of veterinary drug application documents revealed the use of 34 different antibiotics belonging to 11 substance classes at 21 farms under study. Antibiotics, particularly tetracyclines, frequently administered to larger pig groups were detected in manure samples up to higher mg kg(-1) dry weight (DW) concentrations. Antibiotic residues in digestates, furthermore, show that a full removal capacity cannot be guaranteed through the anaerobic digestion process in biogas plants.

Laboratory testing on the removal of the veterinary antibiotic doxycycline during long-term liquid pig manure and digestate storage.

The veterinary antibiotic doxycycline (DOXY) is today frequently applied in conventional pig husbandry for the control of respiratory diseases. After the treatment, pigs excrete major amounts of DOXY as the unchanged active substance. Thus, DOXY residues were found in liquid manures and digestates of biogas plants at concentrations of mg kg(-1) dry weight. In order to assess the impact of field applications of contaminated manures and digestates on the entry of DOXY residues into arable and grassland soils, thorough information about the removal of DOXY during long-term storage of farm fertilizers is required. Since this aspect has been only less investigated for manures but not for digestates, first long-term storage simulation tests were performed at laboratory scale. Within the 170-d incubation periods under strictly anaerobic conditions, doxycycline was removed in liquid pig manure by 61% and in digestate by 76%. The calculated half-lives of 120 d and 91 d thus emphasized the persistence of doxycycline in both matrices. Due to the substance specific properties of DOXY, this removal was caused neither by mineralization, epimerization nor biotransformation. According to the high affinity of DOXY to manure and digestate solids, however, the formation of non-extractable residues has to be taken into account as the predominant concentration determining process. This was indicated by the sequential extraction procedure applied. Hence, these results confirmed that a full removal capacity for doxycycline cannot be reached through the long-term storage of farm fertilizers.