Exploring the immediate and long-term impact on bacterial communities in soil amended with animal and urban organic waste fertilizers using pyrosequencing and screening for horizontal transfer of antibiotic resistance

Abstract

We investigated immediate and long-term effects on bacterial populations of soil amended with cattle manure, sewage sludge or municipal solid waste compost in an ongoing agricultural field trial. Soils were sampled in weeks 0, 3, 9 and 29 after fertilizer application. Pseudomonas isolates were enumerated, and the impact on soil bacterial community structure was investigated using 16S rRNA amplicon pyrosequencing. Bacterial community structure at phylum level remained mostly unaffected. Actinobacteria, Proteobacteria and Chloroflexi were the most prevalent phyla significantly responding to sampling time. Seasonal changes seemed to prevail with decreasing bacterial richness in week 9 followed by a significant increase in week 29 (springtime). The Pseudomonas population richness seemed temporarily affected by fertilizer treatments, especially in sludge- and compost-amended soils. To explain these changes, prevalence of antibiotic- and mercury-resistant pseudomonads was investigated. Fertilizer amendment had a transient impact on the resistance profile of the soil community; abundance of resistant isolates decreased with time after fertilizer application, but persistent strains appeared multiresistant, also in unfertilized soil. Finally, the ability of a P. putida strain to take up resistance genes from indigenous soil bacteria by horizontal gene transfer was present only in week 0, indicating a temporary increase in prevalence of transferable antibiotic resistance genes.

Long-term amendment of urban and animal wastes equivalent to more than 100 years of application had minimal effect on plant uptake of potentially toxic elements

“…Thus, Poulsen et al (2013a) showed that the effects on microbial diversity of cumulated waste application equivalent of approximately 65 years are very limited. These findings were confirmed by Riber et al (2014), in accelerated treatments having received waste application corresponding to more than 100 years of legal application. Riber et al (2014) also found that fertilizer amendment had only a transient impact on the antibiotic resistance profile of the soil community and that abundance of resistant isolates decreased rapidly with time after fertilizer application, while bacterial community structure at phylum level remained mostly unaffected.…”

“…These findings were confirmed by Riber et al (2014), in accelerated treatments having received waste application corresponding to more than 100 years of legal application. Riber et al (2014) also found that fertilizer amendment had only a transient impact on the antibiotic resistance profile of the soil community and that abundance of resistant isolates decreased rapidly with time after fertilizer application, while bacterial community structure at phylum level remained mostly unaffected. While changes in bacterial structure below phylum levels cannot be excluded, and risks from accumulation of persistent organic pollutants cannot be ruled out by the studies cited above, they indicate a strong resilience of these soils considering the very high doses applied.…”

“…As a result of the yearly accumulated input of the amendments, the elemental concentrations in soil slightly increased from those reported by Riber et al (2014) of the soils sampled in 2009. The long-term soil amendment with urban and animal wastes studied in this work showed an accumulation of nutrients and selected metals in soil based on the strong correlation found between the cumulated inputs of P, N, Zn and Cu by the fertilizers and their concentrations in the soil.…”

Impact of Sample Type and DNA Isolation Procedure on Genomic Inference of Microbiome Composition

“…we performed DESeq2 analyses. The read count tables from the 16S rRNA gene 461 22 profiling and metagenomics sequence analysis, respectively, were aggregated to the 462 family level in R (v. 3 The collection of human and pig fecal specimens as well as sewage was non-invasive, 481 and were performed in accordance with the Declaration of Helsinki, and complied with 482 Table S1A). (C) Ecological richness (Chao 1) and diversity (Shannon index) were determined based on contingency tables from 16S rRNA gene profiling and metagenomic sequencing data at OTU and species levels, respectively (See also Supplemental Table S1B).…”

“…Samples were quantified using Quant-iT™ PicoGreen® dsDNA Assay Kit (Invitrogen, Carlsbad, CA) on a Lightcycler 96 (Roche, Mannheim, Germany) and adjusted to equal concentrations. In the second PCR the same conditions were 6 used as in the first round, except the PCR was reduced to 15 cycles and the primers had a unique adaptor/linker/index sequence per sample (3). The PCR products were purified using Agencourt AmPure XP beads (Beckman Coulter Inc, A63881), and concentrations were measured using Quant-iT™ PicoGreen® dsDNA Assay Kit on a Lightcycler 96.…”