Antimicrobial resistance genes
A total of 325 ARG subtypes belonging to 21 ARG types were identified. The resistance genes for multidrug (40%), aminoglycoside (8.9%), polymyxin (7.4%), aminocoumarin (7.4%), and tetracycline (6.9%) were the 5 predominant ARG types (Supplementary Figure S1). The detected resistance genes represented all major resistance mechanisms including antimicrobial deactivation, efflux pumps and cellular protection. The total coverage found in the control group ranged from 2506-6224 ×/Gb, indicating that the chicken feces even though without antimicrobial treatment is the hotspot of ARGs (Supplementary Table S2). The coverage found in the four antimicrobial-treated groups was significantly higher than control group (P=0.00, n=60) (Figure 2). Tetracycline resistance gene significantly increased on T15 compared with T5 in chlortetracycline group (P=0.031, n=12), and polymyxin resistance gene significantly increased on T5 compared with T0 in amoxicillin group (P=0.039, n=12). Both florfenicol and chlortetracycline significantly increased the abundance of aminoglycoside resistance gene (P =0.013, n=12). Out of these detected ARG types, we found that 8 ARG types including bacitracin, β-lactam, fosfomycin, glycopeptide, macrolide-lincosamide streptogramin, streptothricin, triclosan resistance genes did not occur any significant change under either of the above antimicrobials administration. We further analyzed the pearson relationship between the abundance of 21 ARG types and the total coverage (Supplementary Table S3). As the most predominant ARG type in the feces, the high correlation (r2 =0.81-0.98, P<0.05) of multidrug resistance gene with total coverage indicated the important role of multidrug resistance gene under the antimicrobial administration (Figure 2).
We further analyzed the ARG subtype variations to quantitatively compare the effects of the antimicrobials on the fecal resistome (Figure 2, Supplementary Table S4). A total of 325 ARG subtypes were detected among the chicken feces. The tetracycline resistance gene tetA was the most abundant ARG subtype in the florfenicol, chlortetracycline, mixed group on T15, while sulfonamide resistance gene sul2 was the predominant gene in amoxicillin group. The multidrug resistance gene acrB, acrF, mdtA, mdtK and CRP only occurred significant increase in amoxicillin group (P<0.05). Beside amoxicillin group, mdfE occurred significant increase in chlortetracycline group, while mdfM occurred significant decrease in florfenicol group (P<0.05). FloR significantly increased in florfenicol group (P=0.03, n=12). The tetracycline resistance gene occurred significant change in abundance only under chlortetracycline treatment. At subtype level, the tetracycline resistance gene tetC significantly increased from T0 to T15 (P=0.013, n=12). Importantly, the mcr-1 gene occurred as high frequencies and significantly increased on T15 compared with T0 (P=0.012, n=12) in mixed group. Antimicrobial pressure also led some emergence of ARG subtype. The gene resistance to multidrug, β-lactam, chloramphenicol, tetracycline accounted for 60% (44 of 74) of the total newly detected ARG subtype during the administration period (Supplementary Table S5). However, there was no specificity in the emergence induced by the antimicrobial treatment. The above antimicrobials could not only result in the emergence of the respective antimicrobial resistance gene, but also the other types of resistance gene. For example, the antimicrobial pressure caused by florfenicol generated some chloramphenicol resistance genes (catB10) and the ARG subtypes belong to the rest of the detected ARGs.
The change of ARGs is correlated with the structure and composition of the bacterial community . We analyzed bacterial community structure using metagenomic sequence data. The four predominant taxonomic phyla in fecal microbiota of broiler chickens were Proteobacteria (70%), Bacteroidetes (15%), Firmicutes (11%), Actinobacteria (0.030%) (Figure 3, Supplementary Table S6).
The antimicrobials had profound influence on the bacterial community structure of the broiler chicken feces. This result was confirmed by the PCA in which PC1 accounted for 81% of the variations between samples (Supplementary Figure S2). We found that the significant compositional shifts of the two primary predominant taxonomic phyla Proteobacteria and Bacteroidetes in these antimicrobial-treated groups occurred after the second time of antimicrobial administration (Figure 3). Specific changes in the microbial community under amoxicillin, chlortetracycline, florfenicol treatment included the decreases (from 51%, 18%, 15% on T0 to 4.2%, 1.4%, 4.9% on T10, respectively) in the abundance of Bacteroidetes compared with the control group (P = 0.015, 0.026, 0.020, n= 33, respectively). On the contrary, the above antimicrobials led significant increases (from 37%, 67%, 60% on T0 to 84%, 83%, 86% on T10) in abundance of Proteobacteria compared with the control group (P = 0.022, 0.027, 0.009, n= 33, respectively). Due to the compound effect, doubtlessly, the mixed group led the Proteobacteria significantly increase from 50% on T0 to 90% on T10 (P= 0.009, n = 33), while the Bacteroidetes significantly decrease from 30% on T0 to 1.4% on T10 (P = 0.015, n = 33) (Figure 3). Furthermore, we characterized the difference of fecal microbiota between groups and discovered indicator taxa by using LDA Effect Size (LEfSe) (Figure 4). The predominant taxa Proteobacteria and Bacteroidetes were the main responders which confirmed by the LDA score (LDA>4, P<0.05). This indicated that a few key members of the community could be the drivers of community dynamics.
We further analyzed the microbial communities on genus level. Escherichia (68%) and Bacteroides (15%) were the two predominant genera in the chicken feces (Supplementary Table S7 and Figure S3). Although amoxicillin, chlortetracycline and florfenicol are against both Gram-positive and Gram-negative bacteria, we found that there were some common points and different points in the effects caused by these antimicrobials on the genus community. On the common side, the variable trend of the two predominant genera mediated by the antimicrobials was the major contributor to the changes of taxonomic phyla of Proteobacteria and Bacteroidetes in the antimicrobial-treated groups observed above. As the predominant genus of Proteobacteria, Escherichia significantly increased in amoxicillin group (from 36% on T0 to 83% on T10, P = 0.019, n= 12), chlortetracycline group (from 45% on T5 to 80% on T10, P = 0.013, n=12), florfenicol group (from 60% on T5 to 85% on T10, P = 0.013, n = 12), mixed group (from 49% on T0 to 88% on T10, P = 0.013, n=12) (Supplementary Figure S4). In contrast with Escherichia, Bacteroides, the predominant genus of Bacteroidetes, significantly decreased in amoxicillin group (from 51% on T0 to 4.0% on T10, P = 0.013, n= 12), chlortetracycline group (from 40% on T0 to 1.4% on T10, P = 0.013, n=12), florfenicol group (from 15% on T0 to 5.0% on T10, P = 0.013, n = 12), mixed group (from 30% on T0 to 1.4% on T10, P = 0.013, n=12) (Figure 5). On the different side, we found that genus which accounted for more than 1% responded quite different to the antimicrobials. For example, Klebsiella significantly decreased from 2.7% on T0 to 0.15% on T10 in florfenicol group (P=0.028, n=12), while significantly increased from 0.67% on T0 to 3.7% on T15 in chlortetracycline group (P=0.028, n=12). However, amoxicillin did not lead significant change of Klebsiella. Furthermore, the distances and variations of genus among all the samples were visualized by principal component analysis (PCA) which PC1 accounted for 80% of the variations between samples, demonstrating similar microbial community composition (Figure 5). The antimicrobial-treated groups clustered clearly apart from the control group, indicating that therapeutic dose of the antimicrobials played important roles in shaping genus compositions in chicken feces.
Bacterial hosts of antimicrobial resistance genes
We annotated 10272 ORFs as ARG-like ORFs that were located in 7234 contigs (Supplementary Table S8). Understanding the composition of the bacterial host of ARGs is conducive to assess the impact induced by the antimicrobial on the chicken feces. Throughout our experiment, the genera harboring most of the ARGs detected were Escherichia (84%), followed by Klebsiella (5.1%), Bacteroides (3.9%), Shigella (2.9%), and Clostridium (1.7%) (Figure 6 and Supplementary Figure S5). Especially for Escherichia, the proportion of it in the antimicrobial resistance bacteria was 84%, indicating that Escherichia carried more resistance genes than other bacterial hosts. Escherichia, Bacteroides, Shigella harbored nearly all kinds of detected ARGs, including the genes resistant to multidrug, tetracycline, aminoglycoside, macrolide lincosamide streptogramin, β-lactam, and sulfonamides (Supplementary Figure S5). Especially for Escherichia, 93% of multidrug resistance genes, 91% of polymyxin resistance genes, 78% of tetracycline resistance genes, 61% of aminoglycoside resistance genes, 58% of β-lactam resistance genes, 86% of aminocoumarin resistance genes resided in Escherichia. During the experimental duration, Escherichia was always the major host for the multidrug resistance genes in all the antimicrobial-treated groups (Figure 6). We also found that some less abundant ARGs such as triclosan resistance genes mainly resided in Pseudomonas (100%) (Supplementary Table S9).
We found the results of ARG-harboring host also match the results in bacterial community. The major change under the antimicrobial administration pressure occurred at T10. In short, the ARG-harboring Escherichia increased at T10 in all the antimicrobial-treated groups, while Bacteroides decreased. These results can be explained by the ARGs they harbored. In amoxicillin group, the β-lactam resistance genes harbored by Escherichia increased from 22% on T0 to 77% on T10. In chlortetracycline group, the tetracycline resistance genes harbored by Escherichia increased from 6.0% on T5 to 97% on T10. In florfenicol group, the chloramphenicol resistance gene harbored by Escherichia increased from 0% on T0 to 100%. Interestingly, the host of chloramphenicol resistance genes changed from Klebsiella (100%) to Escherichia (100%) (Figure 6). In contrast, the β-lactam resistance genes, chloramphenicol resistance genes and the tetracycline resistance genes harbored by Bacteroides decreased after the second antimicrobial administration (Supplementary Table S10). Interestingly, we found that change in the mixed group led by the alternating treatment of the three antimicrobials seems like a combined result of antimicrobial-treated alone groups. For example, the major host for β-lactam resistance genes changed from Bacteroides on T0 into Escherichia on T10 when the mixed group had been feed with amoxicillin. The result found in the mixed group indicated effect caused by antimicrobials has accumulated consequence in shaping the ARG-harboring host composition.