ARG profile in manure and core resistance genes
In manure samples from 29 farms, 20 types of ARGs comprising 626 subtypes were detected; 545 in broiler manure, 427 in swine manure, 419 in layer manure, 288 in dairy cow manure, and 232 in cattle manure. Moreover, 86.58% of the ARGs belonged to the top five subtypes, i.e., beta-lactam, multidrug, macrolide, aminoglycoside, and tetracycline resistance genes; these subtypes accounted for 74.29% of the total ARG abundance (Fig. 1a). The sulfonamide resistance genes (SRGs) comprised three subtypes; however, they accounted for 6.68% of the total ARG abundance. In contrast, the beta-lactam resistance genes accounted for 55.11% of the total ARGs, but they only accounted for 3.47% of the total ARG abundance. The average number (311) and relative abundance (1.59 per 16S rRNA gene) of total ARGs in broiler manure revealed the highest values in manure samples.
The PCoA-based relative abundances of the ARG subtypes revealed that the profile of ARGs in broiler manure was more similar to that in swine manure (Fig. 1b). As the dairy cow manure had the lowest antibiotic residue in all samples (Dataset 2), the profiles of ARGs in dairy cow manure differed from those in manure from other animals.
Manure core-resistome The 201 subtypes of ARGs, mainly comprising multidrug (46), M-L-S (30), tetracycline (28), beta-lactam (26), and aminoglycoside (25) resistance genes, were shared among different animal manures (Fig. 2). Moreover, these subtypes accounted for majority of the total ARG relative abundance in manure samples; layer manure (98–99%), broiler (89–99%), swine (86–99%), and beef and dairy cow (> 99%). The chloramphenicol exporter genes exhibited the highest average relative abundances (0.046 copies per 16S rRNA gene) in all samples, whereas the highest relative abundance (0.25 copies per 16S rRNA gene) of ARG type was found in sulfonamide resistance genes sul1 in a broiler manure sample (Fig. 2b). Furthermore, genes such as tetW (0.0017–0.085 copies per 16S rRNA gene) and tetM (0.001–0.082 copies per 16S rRNA gene) that encode the ribosomal protection proteins and the aminoglycoside inactivation gene (aadE, 0.0022–0.093 copies per 16S rRNA gene) in all manure samples (Fig.2 group A) were the most widely presented ARG subtypes. Aminoglycoside nucleotidyltransferase gene (aadA), streptomycin phosphotransferase genes (AAC(6’)-Ie/APH(2’)-Ia, aph(3_)−I, aph(3)−I, and aph(6)−I), and the M-L-S resistance genes (ermB and lnuA) were dominant in swine and chicken manure samples (group B). The broiler samples exhibited markedly higher relative abundances of multidrug efflux pump genes, such as mdfA, mdtD, mdtE, mdtF mdtG, mdtK, mdtL, mdtM, mdtN, mdtP, and mexX (group C) than the other samples. In contrast, some tetracycline resistance genes (tetO, tetQ, tet32, and tet40) and beta-lactamase gene (cfxA2) were specific to beef cow manure (group D).
Changes in the manure core-resistome after composting
Composting reduced the relative abundance of the main ARGs in the manure core-resistome; the total relative abundance (0.938 copies per 16S rRNA gene) of ARGs in manure was significantly higher (p=0.0016) than that (0.405 copies per 16S rRNA gene) in the composts (Table S2). The relative abundances of 10/19 ARG types decreased significantly in composts than in the manure samples; however, six other ARG types increased significantly after composting, but they all exhibited low relative abundances (Table S2). Moreover, composting did not have any significant effect on three types (multidrug, sulfonamide, and trimethoprim resistance genes). The relative abundances of most main ARG types, such as aminoglycoside, chloramphenicol, M-L-S, tetracycline, and beta-lactam resistance genes, were significantly decreased after composting (Fig. 2a), whereas that of vancomycin resistance genes increased significantly.
Furthermore, the relative abundances of 123 ARG subtypes significantly decreased after composting, whereas those of other 32 ARGs significantly increased among the 201 manure core-resistome ARGs. The relative abundances of the top20 ARGs subtypes decreased significantly after composting, except for two SRGs: sul1 and sul2 and one aminoglycoside aadA (Fig. 2b). Among all the ARG subtypes with average relative abundance >0.001, only eight ARGs, including vanR, mutigrug_ABC_transporter, and fosB, increased significantly in the compost samples(Fig. S3). PCoA revealed that the composition of ARGs in most composts differed from that in the manure samples (Fig. 3c).
Changes in relationship between antibiotic residue and ARGs after composting
Nineteen antibiotics were detected in the manure samples, and they were classified into five categories, i.e., tetracyclines (TCs), quinolones (QNs), macrolides (MLs), sulfonamides (SAs), amphenicols (AMs), and two others (lincomycin and ceftiofur) (Dataset 2). The average total concentrations of antibiotic residues were 5321 μg/kg (layer manure), 96,937 μg/kg (broiler manure), 50,248 μg/kg (swine manure), 858μg/kg (Beef cow manure) and 295 μg/kg (Dairy cow manure). TCs were the dominant antibiotics in swine and broiler manure, and they contributed 96.28% and 93.77% of the total concentration of antibiotics, respectively. Moreover, TCs and QNs accounted for 55.99% and 24.3% of the total concentrations in the layer manure. QNs were the dominant antibiotics in beef cow manure, contributing 89.77% of the total concentration of antibiotics.
We evaluated the correlations between ARGs and antibiotics in the manure samples. Significant positive correlations (p < 0.05, r > 0.5) were observed between the 12 antibiotics and six ARGs (Fig. 4a). The network revealed that most antibiotic residues were related to multiple ARG types. The dominant antibiotics (TCs) in manure exhibited a significant positive correlation with all six ARG types, indicating that numerous antibiotic residues will directly lead to the development of antibiotic resistance in animal manure. Some low concentrations of antibiotics were also correlated with several ARGs. CEFT is a therapeutic antibiotic, which has a low concentration in manure (0–561.42 μg/kg); however, it revealed a significant correlation with 37 ARG subtypes. Procrustes analysis confirmed that manure resistomes were significantly correlated with antibiotic residues (Fig. 4b); however, they were not correlated with antibiotics after composting (Fig. 4c). These results indicate that composting can reduce the antibiotic residues and decrease the correlation between antibiotics and resistance genes in composts.
Changes in bacterial hosts of ARGs
In total, 1202 nonredundant ARCs were assembled from all metagenomes, including 224 ARG subtypes. Proteobacteria and Firmicutes were the dominant hosts, accounting for 50.08% and 37.77% of the ARCs, respectively, whereas Bacteroidetes and Actinobacteria accounted for 6.49% and 5.24% of the ARCs, respectively (Table S3). The host bacteria include pathogens, such as Escherichia, Enterococcus, Klebsiella, Staphylococcus, Acinetobacter, Pseudomonas, Clostridium, Citrobacter, Streptococcus, and Enterobacter (Fig. 5). Enterococcus and Escherichia were the dominant hosts, accounting for 10.4% and 10.23% of the total 1202 ARCs, respectively, whereas Enterococcus and Escherichia carried 17.4% and 33.04% of all detected 224 ARG subtypes, respectively (Table S4). Escherichia carried the most diverse ARGs and it exhibited 74 ARG subtypes classified into 15 types of ARGs (Table S4). Although the relative abundance (cpm) of all the host ARCs in manure decreased after composting, the hosts of the top list ARGs in manure and compost differed (Fig. S4). In the composts, Pseudomonas, not Enterococcus, was the most abundant host; Microbacterium and Riemerella were included in the top ten list in the compost, but Lactobacillus and Streptococcus were not.
Moreover, based on the cpm of ARCs at genus level, the major hosts of the main ARG types, such as aminoglycoside, chloramphenicol, M-L-S, tetracycline, multidrug, and sulfonamide resistance genes were investigated (Fig. S5). The major hosts of aminoglycoside resistance genes were Enterococcus, Streptococcus, and Enterobacter, whereas those of TRGs were Pseudomonas, Lactobacillus, and Streptococcus. Compared to other samples, the cpm of hosts for the six major types of ARGs in broiler and swine manure were significantly higher. Escherichia was the dominant host for multidrug-resistance genes. The richness of ARG host in compost at genus level was significant lower than that in manure samples (Fig S6), while the Shannon and Simpson indices of ARG hosts did not exhibit significant difference between manure and composts.
To observe the changes in the composition of the ARG hosts in manure after composting, the percentage of hosts was calculated based on the relative abundance (cpm) (Fig. S7b). According to the results of taxon classification on ARCs, we found that the hosts could change associated with different ARG types at the phylum level. The aminoglycoside resistance genes (AMRGs) were mainly distributed in Firmicutes and Proteobacteria; however, the two phyla carrying AMRGs were affected by composting; the average percentage of Firmicutes and Proteobacteria with AMRGs decreased from 14.58% to 5.48% and increased from 14.73% to 18.55% after composting, respectively. TRGs were widely distributed in all four phyla. Additionally, the percentage of the other three phylum hosts with TRGs decreased after composting; however, those of Actinobacteria carrying TRGs did not. Firmicutes mainly carried the M-L-S resistance genes, whereas Proteobacteria carried the sulfonamide, multidrug, and trimethoprim resistance genes. Notably, the percentage of Proteobacteria carrying SRGs increased from 6.41% in manure to 17.28% in compost.
Based on the percentage of ARCs at the genus level, the dominant ARGs in animal manure and compost are illustrated in Fig. S8. The percentage of Enterococcus carrying AMRGs accounted for more than 6% in layer, broiler, swine, and dairy cow manure; however, after composting, this percentage decreased to 2.27%. Staphylococcus was the dominant ARG host in beef cow and dairy cow manure. it carried AMRGs accounting for 10.18% of all hosts in dairy cow manure and M-L-S resistance genes accounting for 16.32% and 13.47% in beef cow manure and dairy cow manure, respectively. Pseudomonas carried sulfonamide, accounting for 9.40% of the total ARG hosts in compost. Moreover, Streptococcus with TRGs was dominant, accounting for 9.79% of all hosts in swine manure. Furthermore, the dominant TRG hosts were Lactobacillus in layer (6.49%) and broiler (6.11%) manure, thereby exhibiting high concentration of tetracycline residue, similar to that in swine manure. The results indicated that veterinary antibiotics could cause antibiotic resistance in animal guts; however, the ARGs in different animal guts may have their own dominant hosts.
Distribution of ARGs in mobile gene elements
In general, 531 (44.17%) of 1202 ARCs carried MEGs, including plasmids (31.49%), transposase (14.53%), integron (7.52%), and recombinase (2.73%); the detailed co-occurrence patterns of ARGs and MGEs are summarized in Table S6. Moreover, 525 ARCs were located on plasmids or choromosomes, carrying 181 ARG subtypes classified into 20 ARG types. In our samples, ARGs were more prevalent in the plasmids than in chromosomes, and all the top 20 high relative abundance ARG subtypes (Fig. 3) were carried by plasmids except for bacA (Fig. 6). Of the 181 ARGs, 44.2% were shared between plasmids and chromosomes. The 37.02% ARG subtypes comprising aminoglycoside, beta-lactam, tetracycline, and M-L-S resistance genes were only found in the plasmids, whereas 18.78% subtypes including multidrug, tetracycline, and M-L-S resistance genes were only found on the chromosomes. The TRGs encoded in chromosomes mainly comprised ribosomal protection protein genes and tetracycline efflux proteins. The circos diagram revealed that the plasmids had a higher relative abundance (cpm) of ARCs (Fig. 7a) than the chromosomes. AMRGs and TRGs were dominant in both plasmids and chromosomes. The chromosomes possessed more multidrug resistance genes, whereas plasmids carried more chloramphenicol and M-L-S resistance genes.
The heatmap reveals the changes in the relative abundance (cpm) of plasmid-associated or chromosome-associated ARCs in manure and compost (Fig. S9). The plasmid-only ARCs had higher relative abundance in composts and multiple ARGs carrying contigs than the chromosome-only ARCs (Fig. S9A). The dominant ARG subtypes in composts were mostly carried by the plasmids and chromosome-shared ARCs (Fig. S9B), including TRGs (tetL and tetM), SRGs (sul1 and sul2), chloramphenicol resistance genes (chloramphenicol exporter), and AMRGs (aadA, aad(9), aadE, aph(3), and aph(6)). The chromosome-carrying multidrug resistance genes (Fig. S9C), which had a high relative abundance in meat animal (broiler and swine) manure, could be efficiently reduced by composting.
Of the 107 ARG subtypes, 85 subtypes were linked with transposase, 34 with integron, and 22 with recombinase (Fig. S10). Sul1 and aadA, which had high relative abundance in all manure samples and did not significantly reduce after composting, were linked with all three MGEs in our samples. This result indicated that compared with “single” ARGs, the ARGs connected with MGEs could exhibit higher chance of survival during composting. The heatmaps based on relative abundance (cpm) revealed that integron-carried ARCs exhibited more multiple ARGs (30/100) than the transposase-carried (21/182) and recombinase-carried ARCs (4/37); furthermore, multiple ARGs revealed the highest relative abundance of integron-carried ARCs in composts (Figs. S14–S16). The AMRGs were dominant in all three MGEs carrying ARGs (Fig. 7b). The distribution of ARGs connected to the three MGEs was distinct. Transponase was associated with the highest number and relative abundance of ARGs in all three MGEs (Fig. S10 and S11b); moreover, the MLS-resistance genes, multidrug-resistance genes, and TRGs were more easily found in transponase-related ARCs. The chloramphenicol resistance genes and trimethoprim resistance genes were two dominant ARGs in the integron-related ARCs. Among all the recombinase-carrying ARGs, SRGs had the highest relative abundance. The taxon circos diagram depicts that the potential hosts of transponsase-related ARCs were included with all four phyla, and 98.9% of the integron-carrying ARCs belonged to Proteobacteria (Fig. S11b).