The quantification and identification of new plasmid-acquiring bacteria in representative mating conditions is critical to characterize the risk of horizontal gene transfer in the environment. This study aimed to quantify conjugation events resulting from manure application to soils and identify the transconjugants resulting from these events. Conjugation was quantified at multiple time points by plating and flow cytometry, and the transconjugants were recovered by fluorescence-activated cell sorting and identified by 16S rRNA sequencing. Overall, transconjugants were only observed within the first 4 days after manure application and at values close to the detection limits of this experimental system (1.00-2.49 log CFU/g of manured soil, ranging between 10-5 and 10-4 transconjugants-to-donor ratios). In the pool of recovered transconjugants, we found amplicon sequence variants (ASVs) of genera whose origin was traced to soils (Bacillus and Nocardioides) and manure (Comamonas and Rahnella). This work showed that gene transfer from fecal to soil bacteria occurred despite the less-than-optimal conditions faced by manure bacteria when transferred to soils, but these events were rare, mainly happened shortly after manure application, and the plasmid did not colonize the soil community. This study provides important information to determine the risks of AMR spread via manure application.

Application of animal manure to soils results in the introduction of manure-derived bacteria and their antimicrobial resistance genes (ARGs) into soils. ResCap is a novel targeted-metagenomic approach that allows the detection of minority components of the resistome gene pool without the cost-prohibitive coverage depths and can provide a valuable tool to study the spread of antimicrobial resistance (AMR) in the environment. We used high-throughput sequencing and qPCR for 16S rRNA gene fragments as well as ResCap to explore the dynamics of bacteria, and ARGs introduced to soils and adjacent water ditches, both at community and individual scale, over a period of three weeks. The soil bacteriome and resistome showed strong resilience to the input of manure, as manuring did not impact the overall structure of the bacteriome, and its effects on the resistome were transient. Initially, manure application resulted in a substantial increase of ARGs in soils and adjacent waters, while not affecting the overall bacterial community composition. Still, specific families increased after manure application, either through the input of manure (e.g., Dysgonomonadaceae) or through enrichment after manuring (e.g., Pseudomonadaceae). Depending on the type of ARG, manure application resulted mostly in an increase (e.g., aph(6)-Id), but occasionally also in a decrease (e.g., dfrB3) of the absolute abundance of ARG clusters (FPKM/kg or L). This study shows that the structures of the bacteriome and resistome are shaped by different factors, where the bacterial community composition could not explain the changes in ARG diversity or abundances. Also, it highlights the potential of applying targeted metagenomic techniques, such as ResCap, to study the fate of AMR in the environment.

The occurrence and removal patterns of 24 antimicrobial agents and antimicrobial resistant determinants namely 6 antibiotic resistance genes (ARGs) and 2 mobile genetic elements (MGEs), and the fecal indicator E. coli were investigated in three full-scale wastewater treatment plants. Their waterlines and biosolids lines (including secondary treatment based on both granular and activated sludge) were sampled monthly throughout one year. Samples were analyzed by means of LC-MS/MS, qPCR and cell enumeration, respectively. The influence of rainfall, temperature, and turbidity on the occurrence and removal of the aforementioned agents was assessed through statistical linear mixed models. Ten of the antimicrobial agents (macrolides, fluoroquinolones, tetracyclines, and sulfonamides) were commonly found in influent in concentrations of 0.1-2 µg L⁻¹, and the predominant ARGs were ermB and sul1 (6.4 and 5.9 log₁₀ mL⁻¹ respectively). Warmer temperatures slightly reduced gene concentrations in influent whilst increasing that of E. coli and produced an uneven effect on the antimicrobial concentrations across plants. Rainfall diluted both E. coli (-0.25 logs, p < 0.001) and antimicrobials but not genes. The wastewater treatment reduced the absolute abundance of both genes (1.86 logs on average) and E. coli (2.31 logs on average). The antimicrobials agents were also partly removed, but 8 of them were still detectable after treatment, and 6 accumulated in the biosolids. ARGs were also found in biosolids with patterns resembling those of influent. No significant differences in the removal of antimicrobials, genes and E. coli were observed when comparing conventional activated sludge with aerobic granular sludge. Irrespective of the type of sludge treatment, the removal of genes was significantly reduced with increasing hydraulic loads caused by rainfall (-0.35 logs per ∆ average daily flow p < 0.01), and slightly decreased with increasing turbidity (-0.02 logs per ∆1 nephelometric turbidy unit p < 0.05).

Plasmid-mediated dissemination of antibiotic resistance among fecal Enterobacteriaceae in natural ecosystems may contribute to the persistence of antibiotic resistance genes in anthropogenically impacted environments. Plasmid transfer frequencies measured under laboratory conditions might lead to overestimation of plasmid transfer potential in natural ecosystems. This study assessed differences in the conjugative transfer of an IncP-1 (pKJK5) plasmid to three natural Escherichia coli strains carrying extended-spectrum beta-lactamases, by filter mating. Matings were performed under optimal laboratory conditions (rich LB medium and 37°C) and environmentally relevant temperatures (25, 15 and 9°C) or nutrient regimes mimicking environmental conditions and limitations (synthetic wastewater and soil extract). Under optimal nutrient conditions and temperature, two recipients yielded high transfer frequencies (5 × 10^–1) while the conjugation frequency of the third strain was 1000-fold lower. Decreasing mating temperatures to psychrophilic ranges led to lower transfer frequencies, albeit all three strains conjugated under all the tested temperatures. Low nutritive media caused significant decreases in transconjugants (−3 logs for synthetic wastewater; −6 logs for soil extract), where only one of the strains was able to produce detectable transconjugants. Collectively, this study highlights that despite less-than-optimal conditions, fecal organisms may transfer plasmids in the environment, but the transfer of pKJK5 between microorganisms is limited mainly by low nutrient conditions.

Manure application can spread antimicrobial resistance (AMR) from manure to soil and surface water. This study evaluated the role of the soil texture on the dynamics of antimicrobial resistance genes (ARGs) in soils and surrounding surface waters. Six dairy farms with distinct soil textures (clay, sand, and peat) were sampled at different time points after the application of manure, and three representative ARGs sul1, erm(B), and tet(W) were quantified with qPCR. Manuring initially increased levels of erm(B) by 1.5 ± 0.5 log copies/kg of soil and tet(W) by 0.8 ± 0.4 log copies/kg across soil textures, after which levels gradually declined. In surface waters from clay environments, regardless of the ARG, the gene levels initially increased by 2.6 ± 1.6 log copies/L, after which levels gradually declined. The gene decay in soils was strongly dependent on the type of ARG (erm(B) < tet(W) < sul1; half-lives of 7, 11, and 75 days, respectively), while in water, the decay was primarily dependent on the soil texture adjacent to the sampled surface water (clay < peat < sand; half-lives of 2, 6, and 10 days, respectively). Finally, recovery of ARG levels was predicted after 29–42 days. The results thus showed that there was not a complete restoration of ARGs in soils between rounds of manure application. In conclusion, this study demonstrates that rather than showing similar dynamics of decay, factors such as the type of ARG and soil texture drive the ARG persistence in the environment.

Wastewater treatment plants (WWTPs), linking human fecal residues and the environment, are considered as hotspots for the spread of antimicrobial resistance (AMR). In order to evaluate the role of WWTPs and underlying operational parameters for the removal of AMR, the presence and removal efficiency of a selected set of 6 antimicrobial resistance genes (ARGs) and 2 mobile genetic elements (MGEs) was evaluated by means of qPCR in influent and effluent samples from 62 Dutch WWTPs. The role of possible factors impacting the concentrations of ARGs and MGEs in the influent and their removal was identified through statistical analysis. ARGs and the class I integron-integrase gene (intI1) were, on average, removed to a similar extent (1.76 log reduction) or better (+0.30–1.90 logs) than the total bacteria (measured as 16S rRNA gene). In contrast, broad-host-range plasmids (IncP-1) had a significantly increased (p < 0.001) relative abundance after treatment. The presence of healthcare institutions in the area served did only slightly increase the concentrations of ARGs or MGEs in influent. From the extended panel of operational parameters, rainfall, increasing the hydraulic load of the plant, most significantly (p < 0.05) affected the treatment efficiency by decreasing it on average −0.38 logs per time the flow exceeded the average daily flow. Our results suggest that overall, WWTP treatments do not favor the proliferation of the assessed resistance genes but might increase the relative abundance of broad-host-range plasmids of the IncP-1 type.