Carbendazim shapes microbiome and enhances resistome in the earthworm gut

Abstract

Background: It is worrisome that several pollutants can enhance the abundance of antibiotic resistance genes (ARGs) in the environment, including agricultural fungicides. As an important bioindicator for environmental risk assessment, earthworm is still a neglected focus that the effects of the fungicide carbendazim (CBD) residues on the gut microbiome and resistome are largely unknown. In this study, Eisenia fetida was selected to investigate the effects of CBD in the soil-earthworm systems using shotgun metagenomics and qPCR methods.

Results: CBD could significantly perturb bacterial community and enrich specific bacteria mainly belonging to the phylum Actinobacteria. More importantly, CBD could serve as a co-selective agent to elevate the abundance and diversity of ARGs, particularly for some specific types (e.g., multidrug, glycopeptide, tetracycline, and rifamycin resistance genes) in the earthworm gut. Additionally, host tracking analysis suggested that ARGs were mainly carried in some genera of the phyla Actinobacteria and Proteobacteria. Meanwhile, the level of ARGs was positively relevant to the abundance of mobile genetic elements (MGEs) and some representative co-occurrence patterns of ARGs and MGEs (e.g., cmx-transposase and sul1-integrase) were further found on the metagenome-assembled contigs in the CBD treatments.

Conclusions: It can be concluded that the enhancement effect of CBD on the resistome in the earthworm gut may be attributed to its stress on the gut microbiome and facilitation on the ARGs dissemination mediated by MGEs, which may provide a novel insight into the neglected ecotoxicological risk of the widely used agrochemicals on the gut resistome of earthworm dwelling in soil. Video abstract.

Keywords: Antibiotic resistance genes; Fungicide; Gut microbiota; Manure; Mobile genetic elements; Soil animal.

Fig. 1

Bioaccumulation concentration (a) and bioaccumulation factor (BAF, b) of carbendazim in the earthworm among treatments. NE-CBD1 and NE-CBD2 represent the earthworm samples in the un-manured soil with 1.0 and 2.0 mg kg⁻¹ CBD, respectively. ME-CBD1 and ME-CBD2 represent the earthworm samples in the manured soil with 1.0 and 2.0 mg kg⁻¹ CBD, respectively

Fig. 2

Composition of microbiota at phylum level (a) and relative abundance of the genera belonging to Actinobacteria (b) with significant differences in the earthworm gut among treatments. NG-CK, NG-CBD1, and NG-CBD2 represent the earthworm gut samples in the un-manured soil with 0, 1.0, and 2.0 mg kg⁻¹ CBD, respectively. MG-CK, MG-CBD1, and MG-CBD2 represent the earthworm gut samples in the manured soil with 0, 1.0, and 2.0 mg kg⁻¹ CBD, respectively

Fig. 3

Comparison of total abundance (a) and diversity (b) of antibiotic resistance genes (ARGs) among different treatments, heatmap of the dominant ARGs based on common logarithmic transformed abundance (c), and bipartite network showed the shared and unique ARGs types (d) among treatments. The nodes and edges were colored according to ARGs types. NG-CK, NG-CBD1, and NG-CBD2 represent the earthworm gut samples in the un-manured soil with 0, 1.0, and 2.0 mg kg⁻¹ CBD, respectively. MG-CK, MG-CBD1, and MG-CBD2 represent the earthworm gut samples in the manured soil with 0, 1.0, and 2.0 mg kg⁻¹ CBD, respectively

Fig. 4

Abundance (a) and absolute abundance (b) of the dominant ARGs (top 10) in the earthworm gut among treatments. NG-CK, NG-CBD1, and NG-CBD2 represent the earthworm gut samples in the un-manured soil with 0, 1.0, and 2.0 mg kg⁻¹ CBD, respectively. MG-CK, MG-CBD1, and MG-CBD2 represent the earthworm gut samples in the manured soil with 0, 1.0, and 2.0 mg kg⁻¹ CBD, respectively

Fig. 5

Procrustes analysis (a) of ARGs and bacterial communities, Spearman’s correlation-based co-occurrence network (b) of the dominant ARGs and genera (> 1%), and networks of ARGs hosts (c) based on the metagenomic assembly analysis in the earthworm gut among treatments. NG-CK, NG-CBD1, and NG-CBD2 represent the earthworm gut samples in the un-manured soil with 0, 1.0, and 2.0 mg kg⁻¹ CBD, respectively. MG-CK, MG-CBD1, and MG-CBD2 represent the earthworm gut samples in the manured soil with 0, 1.0, and 2.0 mg kg⁻¹ CBD, respectively

Fig. 6

The abundance of MGEs (a), linear-regression analysis between the abundance of ARGs and MGEs (b), and the co-occurrence arrangements of ARGs and MGEs on the contigs (c) in the earthworm gut among treatments. NG-CK, NG-CBD1, and NG-CBD2 represent the earthworm gut samples in the un-manured soil with 0, 1.0, and 2.0 mg kg⁻¹ CBD, respectively. MG-CK, MG-CBD1, and MG-CBD2 represent the earthworm gut samples in the manured soil with 0, 1.0, and 2.0 mg kg⁻¹ CBD, respectively