Variations of ARGs and intI1
Seven target genes, including six ARGs (tetA, tetQ, tetW, sulI, sulII, and blaTEM-1), and intI1, were detected to evaluate the effect of digested sludge-amendment on the abundances of ARGs in soils. The absolute and relative abundances of ARGs and intI1 in digested sludge were 0.7-2.2 and 0.2-1.6 logs higher than those of raw soil, respectively (Fig. 1a and 1b). After mixed the raw soil and digested sludge (1:2 wt/wt wet basis), the absolute and relative abundances of target genes (i.e., six ARGs and intI1) in the amended soil (blank-0 soil) were 2.8×105-3.3×108 copies/g DS and 2.1×10-4-4.8×10-1 copies/16S rRNA gene copies, respectively (Fig. 1), which were 1.3-20.4 and 1.6-16.2 folds higher than those in the raw soil, respectively (Fig. 1).
After 80-days cultivation, the absolute and relative abundances of various ARGs in the amended soil without earthworms (blank-80 soil) decreased to 1.1×104-7.7×107 copies/g DS and 4.3×10-5-3.0×10-1 copies/16S rRNA gene copies, respectively (Fig. 1a and 1b), which were 4.6%-35.2% and 12.2%-62.5% of those in the blank-0 soil, respectively (Fig. 1). The presence of earthworms further enhanced the reduction of most target genes (except tetW). After 80-day cultivation, the absolute abundances of ARGs in surface wormcast and residual soil (with earthworms) decreased to 1.7×103-2.4×107 and 4.1×103-4.4×107 copies/g DS, respectively. Those were 15.3%-68.5% and 37.2%-88.3% of those in the blank-80 soil (without earthworms), respectively. Furthermore, the absolute abundances of ARGs (except tetW) in surface wormcast were even 6.1%-37.5% of those in the raw soil (without digested sludge amendment) (Fig. 1). For the relative abundances, enhanced reduction of ARGs was also achieved by earthworms (Fig. 1b and 1b). The relative abundances of ARGs (i.e., tetA, tetQ, sulI, sulII, and blaTEM-1) in surface wormcast (with earthworms) were 28.7%-95.4% of those in blank-80 soil (Fig. 1b). In contrast, the absolute and relative abundances of tetW in surface wormcast increased by 51.5% and 184.4%, respectively, compared with those in the blank-80 soil.
The abundances of ARGs and 16S rRNA gene in the gut of earthworms were also determined. After cultivation in the digested sludge-amended soil for 80 days, the absolute abundance of 16S rRNA gene in the gut of earthworms decreased by about 0.8 log (Fig. 1c). The absolute and relative abundances of tetA, sulI, sulII, blaTEM-1, and intI1 in earthworm gut after cultivation increased by 0.8-1.3 and 1.6-2.1 logs, respectively. In contrast, the absolute abundances of tetQ and tetW decreased by 2.1 and 0.9 logs, and their relative abundances decreased by 1.3 and 0.1 logs, respectively (Fig. 1c).
To reveal the similarity of gene pattern among different samples, PCoA analysis was performed based on the relative abundances of target genes. As shown in Fig. 2a, the digested sludge-amended soil samples (i.e., surface wormcast and residual soil) after 80-days cultivation in the presence of earthworms were distinctly separated from the blank-0 soil and blank-80 soil (without earthworms), further demonstrating that earthworms could change the abundance of ARGs and thus alter the gene pattern of soils. Meanwhile, the gene patterns of earthworm gut were greatly altered by digested sludge amendment after 80-days cultivation given that they were distinctly separated from those in the gut of earthworm before cultivation (Fig. 2b).
Composition of Microbial Community
As shown in Fig. 3, the microbial community of digested sludge was different from that of raw soil. Higher abundances of Clostridia and Bacteroidia were found in the digested sludge. After introducing digested sludge, the abundances of anaerobic microbes (e.g., Bacteroidia and Clostridia) increased in the digested sludge-amended soil (blank-0 soil) compared with those in the raw soil (Fig. 3). At the phylum level, the dominated phyla in the blank-0 soil were Proteobacteria (occupying 52.8%), Bacteroidetes (15.9%), Actinobacteria (6.6%), and Firmicutes (6.3%), which occupied 81.6% of the total microbial abundance (Fig. 3a). At the class level, the dominated classes in the blank-0 soil were Gammaproteobacteria (26.6%), Alphaproteobacteria (17.2%), Bacteroidia (15.0%), Deltaproteobacteria (9.0%), and Clostridia (5.5%), occupying 73.3% of the total microbial abundance (Fig. 3b). However, 80-day cultivation greatly decreased the relative abundances of anaerobic microbes and enriched aerobic microbes in soils (Fig. 3a and Fig. 3b). For example, at the class level, the relative abundances of Bacteroidia and Clostridia in the blank-80 soil (without earthworm) decreased from 15.0% to 8.8% and from 5.5% to 1.6% after 80-days cultivation, respectively. Meanwhile, the relative abundances of aerobic microbes in blank-80 soil showed increasing trends. For example, the abundance Acidimicrobiia increased from 2.9% to 4.0%. Under the effect of earthworms, the variation of microbial community was further amplified (Fig. 3a and 3b). At the phylum level, the relative abundance of Actinobacteria in surface wormcast were greatly changed by earthworms, which increased by 175.6% compared to that in the blank-80 soil (without earthworms). However, the abundance of Bacteroidia in surface wormcast decreased by 37.0%.
The variation of the microbial community in the gut of earthworms was also observed (Fig. 3a). At the class level, the relative abundances of Acidimicrobiia in the gut of earthworms decreased by 70.6%. In contrast, the relative abundances of Clostridia and Bacteroidia increased by 409.4% and 79.8% in the gut of earthworms after cultivation, respectively. At the family level, significant attenuation of Saprospiraceae, Sneathiellaceae, and Rhodanobacteraceae in soils as well as Rhodocyclaceae, Microscillaceae, and Nitrosomonadaceae in the gut of earthworms after cultivation were observed (Fig. 3c). The abundances of Sandaracinaceae and Haliangiaceae in soil as well as Rhizobiaceae in the earthworm gut were all enriched after cultivation.
PCoA analysis shows that the microbial community patterns of the amended soils with and without earthworm after 80-days cultivation were separated from that of the blank-0 soil (Fig. 4). In addition, the samples of earthworm guts show a distinct difference from all soil samples. The surface wormcast and residual soil samples were clustered, and they were separated from other samples.
Relationships between ARGs and microbial communities
As shown in Table 1, significant correlations between two target ARGs (i.e., tetA, and sulII) and intI1 were observed based on Spearman’s correlation analysis. The relationships between microbial communities and targeted ARGs were assessed using redundancy analysis (RDA). A total of 93.9% variance of ARGs could be explained by selected variables (Fig. 5). In particular, Three phyla (Firmicutes, Bacteroidetes, and Acidobacteria) exhibited positive relationships with ARGs in the blank-0 soil. Proteobacteria showed a significantly positive correlation with the ARGs abundance in the blank-80 soil and the raw soil. Moreover, Actinobacteria and Chloroflexi were positively related to the surface wormcast samples.
To further investigate the potential host bacteria of the ARGs, the quantitative correlation between the target ARGs and 29 bacterial taxa at the family level were described by the heatmap (Fig. 6). The significant positive correlations between three families (i.e., Rhodanobacteraceae, Saprospiraceae, and Chitinophagaceae) and two ARGs (i.e., tetQ and sulII) were observed (R > 0.8, P < 0.01). The potential hosts for intI1, sulI, and blaTEM-1 were identified as Saprospiraceae, Chitinophagaceae, and unidentified Clostridiales, respectively. In particular, Saprospiraceae and Chitinophagaceae were also identified as potential multi-ARG hosts (Fig. 6).