Composition of ARGs in Reclaimed Water-Soil-Plant System
The enumeration of ARGs and MGEs varied across samples, with a range of 28 to 38 detected in each (Figure 2A). Notably, the quantity of ARGs present in plant roots and leaves was less than that found in the corresponding rhizosphere soil. Furthermore, when comparing treatments, it was observed that seed dressing and soil application of CeO2 NPs notably reduced the abundance of ARGs within the rhizosphere soil, in contrast to treatments using reclaimed water containing CeO2 NPs.
The number and absolute abundances of ARGs genotypes. (A) Number of ARGs genotypes detected in different samples. (B) Absolute abundances of ARGs expressed as copies per g solid.
Abbreviation: ARGs=antibiotic resistance genes; MGE=mobile genetic element; MLSB= macrolide-lincosamide-streptogramin B.
The concentration of ARGs in the soil, which ranged from 5.21×109 to 7.74×109 copies/g soil, was approximately an order of magnitude higher than that observed in the phyllosphere, with values spanning from 5.04×108 to 7.31×108 copies/g plant tissue (Figure 2B). Moreover, the concentration of ARGs in root endophytes (ranging from 1.25×109 to 2.36×109 copies/g plant) was significantly lower than the concentration found in rhizosphere soil.
To ensure that variations in the native bacterial populations did not confound our results, we normalized the abundance of ARGs to the number of bacterial cells, quantified as 16S rDNA copy numbers (Supplementary Table S1). Given that the current average number of 16S rRNA genes per bacterial cell is approximately 4.1, the normalized ARG copy numbers varied from 0.10 to 0.35 copies per bacterial cell. The application of NPs had a discernible impact on the normalized abundance of ARGs, mirroring the effect seen with the absolute ARG copy numbers, with the exception of the rhizosphere soil subjected to the WT treatment (Supplementary Table S1).
Composition of MGEs in reclaimed water-soil-plant system
The abundance of mobile genetic elements (MGEs) exhibited considerable variability, spanning more than an order of magnitude, with counts ranging from 1.79×107 to 1.21×108 copies per gram of solid matter in the phyllosphere, and from 8.57×108 to 5.54×109 copies per gram of solid matter in the soil (Figure 2B). Similarly, reflecting the observed decline in the abundance of ARGs, the introduction of NPs was associated with a consequent reduction in the prevalence of MGEs within the water-soil-plant continuum.
Distribution of ARGs in Reclaimed Water-Soil-Plant System
If ARGs are identified solely in the roots or leaves of radishes when irrigated with reclaimed water, these ARGs are considered transmissible within the reclaimed water-soil-radish pathway. As depicted in Figure 3A, there are 30 ARGs capable of moving from reclaimed water to the leaves of radishes under the control condition. Relative to the control condition, we observed a reduction of 30.0%, 43.4%, and 40.0% in the number of transmissible ARGs from reclaimed water to radish leaves when NP applications in reclaimed water, seed dressing, and soil mixing interventions were implemented, respectively (Figure 3B–D).
Venn diagrams depicting the dissemination of ARGs from reclaimed water to radish seedlings. (A) control treatment, (B) water treatment, (C) seed dressing, and (D) soil treatment with NPs. (E) The fold change of the main 21 ARGs and MGEs which can spread from reclaimed water to radish.
Note: The outermost light blue circle represents the number of ARGs detected in reclaimed water, while the inner brown, green, and red circles represent the number of ARGs detected in soil, leaf endophytic, and root endophytic proportions, respectively. The dark blue circle in the middle represents the number of ARGs that can propagate in the reclaimed water-soil-radish system.
Abbreviation: NPs=nanoparticles; ARGs=antibiotic resistance genes; RW=reclaimed water; MGE=mobile genetic element; MLSB=macrolide-lincosamide-streptogramin B.
After the application of NPs, the majority of ARGs exhibited a decreasing trend in abundance. The aadA2 gene, associated with aminoglycoside resistance, exhibited the most substantial reduction, with a 48% decrease in the ST-RE group as shown in Figure 3E. Conversely, there was an upward trend observed in the abundance of vancomycin resistance genes, tetracycline resistance genes, and mexB, which is linked to multidrug resistance. Two ARGs, pbrT and qepA, showed a significant increase in the radish root when subjected to seed dressing and soil treatments. Following the NPs treatments, the abundance of three MGEs (Tn403, TrbC, and IS256) showed a significant decrease, particularly in the WT-RS, WT-LE, and SD-LE treatments.
Microbial Community Assembly
Upon merging overlapped read pairs and conducting quality filtering, we obtained a total of 12,412 high-quality sequences from all the samples. These sequences were subsequently denoised, yielding 1,219 OTUs. We organized the microbial communities into 17 phyla by using the 16S rRNA database (GreenGene, gg_13_5 version). Principal coordinate analysis (PCoA) at the genus level indicated distinct separation of soil, root, and leaf sample communities along the PC1, PC2, and PC3 axes, which accounted for 30.5%, 22.6%, and 5.3% of the variation, respectively (Figure 4A). The PCoA further revealed pronounced clustering of bacterial communities that correlated with the application of different NPs in the root and leaf samples. This finding suggests that NP application significantly influences the overall composition of bacterial communities in both roots and leaves.
Principal coordinate analysis (PCoA) based on genus level communities profile (using Bray-Curtis distance). (A) overall, (B) rhizosphere soil, and (C) root and (D) leaf endophytic proportions of radishes.
Understanding the specific members of a community that drive bacterial population shifts is crucial. As indicated in Supplementary Tables S2–S4, the genus Pseudomonas plays a pivotal role in the bacterial community dynamics of rhizosphere soil, as well as radish roots and leaves. Intriguingly, Pseudomonas was the taxonomic group that exhibited a significant decline across various NPs application methods in both soil and radish samples (Supplementary Figure S1). This reduction can likely be attributed to the pronounced antimicrobial properties of NPs and their ability to inhibit biofilm formation. It appears that NPs may reduce the competitive edge of certain bacteria, forcing them to relinquish their ecological niches and leading to an alteration in the structure of the bacterial community.
Bacterial diversity and MGEs accounting for antibiotic resistome
The absolute abundance of MGEs was found to be linearly and positively correlated with the absolute abundance of ARGs ( R2=0.64, P<0.001) (Figure 5A). This indicates that the variation within the antibiotic resistome of reclaimed water-soil-plant systems is closely associated with MGEs. Figure 5B–D demonstrates that Procrustes analysis, utilizing Bray-Curtis dissimilarity metrics, confirmed a significant correlation between the abundance of ARGs and bacterial diversity within reclaimed water-soil-plant systems (P<0.01). Considering the observed community variations attributed to NP application (Figure 4), we postulate that NPs could potentially reduce the risk of ARG transmission in these systems by fostering a more beneficial community structure. As illustrated in Figure 6, microbial communities primarily facilitate the transmission of ARGs targeting beta-lactam, aminoglycoside, and tetracycline antibiotics, with considerable statistical significance (P<0.01, R>0.8). MGEs are chiefly associated with the dissemination of ARGs against aminoglycoside, multidrug, and macrolide-lincosamide-streptogramin B (MLSB) antibiotics. The regulation of aminoglycoside ARGs appears to be governed not only by bacterial communities but also by MGEs. Specifically, the aadA2 gene, associated with aminoglycoside resistance, exhibited the most pronounced decrease following silver nanoparticle treatment (Figure 3E). This suggests that the reduction may result from the combined influence of microbial community structure and horizontal gene transfer mechanisms. Conversely, the tetracycline resistance genes tetG and tetR increased under WT and SD conditions, leading us to conjecture that microbial communities play a vital role in this dynamic. Overall, the findings indicate that modifications in the resistome of reclaimed water-soil-plant systems are determined by the interplay of bacterial communities and horizontal gene transfer, working in concert to shape antibiotic resistance patterns.
Correlation analysis of ARGs with MGEs and bacterial community. (A) Ordinary least squares (OLS) regression showing the relationship between total absolute abundance of ARGs and MGEs. (B–D) Procrustes analysis depicts the correlation between ARG content and bacterial community in different samples (B, Rhizosphere soil; C, Root endophytic; D, Leaf endophytic).
Abbreviation: ARGs=antibiotic resistance genes; MGEs=mobile genetic elements; 16s=16S ribosomal RNA.
Pairwise comparisons of ARGs.
Note: 16s (OTUs) and MGEs were related to each ARG by partial (geographic distance–corrected) Mantel tests. Edge width corresponds to Mantel’s R statistic for the corresponding distance correlations, and edge color denotes the statistical significance.
Abbreviation: Beta=beta-lactams; Amin=aminoglycoside; Multi=multidrug; Van=vancomycin; Tet=tetracycline; MLSB=macrolide-lincosamide-streptogramin B; Chl=chloramphenicol.