Influence of decades-long irrigation with secondary treated wastewater on soil microbial diversity, resistome dynamics, and antibiotrophy development

Abstract

In arid and semi-arid regions, the use of treated wastewater (TWW) for irrigation is gaining ground to alleviate pressure on natural water sources. Despite said treatment, the existing methods fail to eliminate potentially dangerous contaminants. As such, this study assessed the impact of long-term TWW irrigation (5 and 25 years) on soil physicochemical properties and bacterial resistance to heavy metals (Pb, Cu, Cd) and antibiotics (tetracycline and amoxicillin). The results revealed heightened salinity and conductivity and reduced pH in irrigated soils. TWW induces harmful effects by reducing microbial density and size, leading to the disappearance of sensitive populations. Conversely, resilient populations, which mainly utilize antibiotics as a carbon source, have adapted. Metagenomic 16S amplicon sequencing analysis demonstrated a shift, notably reducing Actinobacteria, Bacteroidetes, and Firmicutes while increasing Acidobacteriota and Patescibacteria in treated soils. Operational Taxonomic Units affiliated with either Halomonadacea, or Saccharimonadacea and Vicinamibacteracea, were defined as indicators of the absence or presence of TWW contamination, respectively. We conclude that TWW irrigation significantly increases bacterial resistance to heavy metals, whereas the impact of antibiotics is nuanced, with antibiotrophy leveraging lower concentrations in treated soils.

Keywords: Antibiotics; Antibiotrophy; Heavy metals; Resistance gene co-selection; Soil resistome; Wastewater irrigation.

Fig. 1

Enumeration of total mesophilic aerobic flora in TWW-irrigated soil samples in the presence of Cu, Pb, Cd, TET, and AMX (S1: control soil never irrigated with TWW; S2: soil irrigated for 5 years; S3: soil irrigated for 25 years with TWW). The results are presented as mean ± standard error of four independent repetitions. a–c: For each parameter, different letters indicate significant differences at p < 0.05, according to Tukey's test.

Fig. 2

Growth of strains isolated from S1, S2, and S3 in the presence of 1 g/L TET, and 1 g/L AMX in mineral medium for 2 days. The results are presented as mean ± standard error of four independent repetitions.

Fig. 3

Bar plots of relative abundance at the phylum level found in soils never irrigated with wastewater (S1), soils irrigated for 5 years (S2), and soils irrigated for 25 years (S3), based on relative frequency %.

Fig. 4

Heatmap of the relative abundance of bacterial genera found in soils never irrigated with wastewater (S1), soils irrigated for 5 years (S2), and soils irrigated for 25 years (S3).

Fig. 5

Venn diagram showing the overlap of bacterial communities found in soils never irrigated with wastewater (S1), soils irrigated for 5 years (S2), and soils irrigated for 25 years (S3) based on OTUs.