In this study, metagenomic analysis revealed that the abundance of ARGs in the rumen of beef steers were predominant by tetracycline (77%) and followed by MLS (17%), and aminoglycoside (4%) resistance. These findings are similar to those reported in fecal samples of beef cattle fed with antibiotics (ionophores, chlortetracycline, or tylosin), where tetracycline resistance was most prevalent (82%), followed by macrolide (14%), and aminoglycoside ). In addition, tetracycline, MLS, and aminoglycoside classes of resistance were also predominant in fecal samples of feedlot cattle raised without antibiotic , suggesting that the profiles of ARG are consistent in different locations of digestive tract of ruminants. We detected broader ARG profiles (12 classes and 62 individual ARGs) in the rumen of beef cattle not administered antimicrobials used in human medicine than previously reported by Thomas et al.  who studied the beef cattle supplemented with tylosin. Based on the analysis of 5 ruminal samples in cattle supplemented with monensin and tylosin, Thomas et al.  did not detect aminoglycoside or β-lactam resistance genes in any sample. While Auffet et al.  detected a wide range of genes resistant to macrolide, chloramphenicol, β-lactam, and aminoglycoside in the rumen of antimicrobial-free beef cattle under similar feeding condition to our study, however, genes resistant to vancomycin were not detected in their study. The variation could be due to difference in animal, environment and diet. In addition, bioinformatic resources/tools available for resistome analysis may also contribute to the difference in ARG profiles in the rumen among studies. Presently, there are at least 47 bioinformatic resources/tools, but no a ‘standard’ pipeline has been developed specifically to characterize the resistome. In this regard, the results are heavily dependent on the analysis methods (assembly-based or read-based) or reference database . In this study, the ARG-OAP (v2) pipeline was applied, which uses a custom database with a hybrid UBLAST and BLASTX algorithm, reflecting the critical need for a comprehensive database combined with lower identity matching for antimicrobial resistance gene annotation of metagenomic data . However, as there is no inclusive ARG database or one specifically customized for the rumen microbiome, more efforts are needed to construct ‘standardized’ pipeline for to characterize the rumen resistome and resistomes in other habitats (e.g. soil, water, gastrointestinal tract).
Plasmids are mobile genetic elements found in high abundance in the bacterial populations of bovine rumen , which play a major role in the spread of antimicrobial resistance through horizontal gene transfer . Metagenomic approaches have been used to characterize plasmid encoded ARGs in several non-biological habitats such as activated sludge [38, 39] as well as the human gut . We found that aadA and tetW were the most abundant plasmid-associated ARG in the rumen of beef steers. In addition, the expression of tetW was highest among all plasmid-associated ARG transcripts. It has been reported that many of the tetracycline resistant genes are associated with mobile plasmids , among which tetW has been proven to be transmissible among the ruminal bacteria, Butyrivibrio fibrosolvens, Selemonas ruminitanium, and Mitsuokella multiacidus . However, the profiles and expressions of plasmid-associated ARGs have not been examined in food-producing animals including ruminants. Considering that the expression of mobile genetic elements such as integrons is a robust strategy of genetic interchange and one of the main drivers of bacterial evolution , we speculate that the expression of plasmid-associated ARGs has functions other than transferring antimicrobial resistance in the rumen. Recently, a wide range of bacterial hosts of plasmids in wastewater samples has been revealed by analyzing Hi-C and shotgun metagenomic data . Those approaches can also be applied to investigate plasmid-associated ARG as well as their bacterial host in cattle, which may help understand the contribution of plasmids to the transmission of AMR determinants in the rumen.
Considering that the presence of a gene does not directly correlate with the activity of the gene a certain environment, direct measurements of transcripts based on metatranscriptomics may be an important complementary approach to metagenomics. Our results indicated that the expression of ARGs is also not directly linked to the presence of ARGs as previously shown in environmental microbiome . We found that about only 20.96% (13/64) of ARGs were expressed, suggesting that around 80% of ARGs were not functional in the rumen of these steers at the time of sampling. Among the 13 ARGs expressed, the prevalence of tet40, tetM, tetO, tetW, mefA was 100%, while that of aadA, tetO, and vatB was 77.1%, 70.8%, and 58.3%, respectively. This suggests that these eight ARGs may constitute the ‘core’ active resistome in the rumen of the steers studied in our study. In particular, the average abundance (ppm) of tetW, mefA, tetQ, and tet40 was 19.84, 13.61, 7.64, and 6.38, respectively, which were the predominant ARG transcripts in our study. The mechanisms of action of these resistant genes have been well characterized. Both tet40  and mefA  encode for efflux pumps which render antimicrobials ineffective by pumping them out of the cell, while tetQ  and tetW  encode for tetracycline ribosomal protection proteins. Among these ARGs, the expression of tet40 has been detected in Clostridium species in human  and swine  gut. In our previous study, we observed active Clostridium genus (the relative abundance averaged 0.15%) across three breeds of beef cattle , and we thus speculate that it may contain certain Clostridium species that carry tet40 gene. To our knowledge, our study reported for the first time the presence and active ARGs simultaneously for food-producing animals in vivo with a large dataset. Although Sabino et al.  analyzed the expression of rumen ARGs, only 15 metatranscriptomic samples were used (5 dairy cattle, 5 beef cattle, and 5 sheep) and their aim was to confirm the expression of ARGs found in 435 reference genomes of ruminal bacteria and archaea in silico, but not to link the expression of ARGs back to the presence of those ARGs using metagenomic data. More recently, the resistome in chicken and pig gut were analyzed using both metagenomic and metatranscriptomic data, but only 6 fecal samples were used as representative of gut samples for each species . In this regard, more efforts are needed to detect and validate our findings based on both metagenomic and metatranscriptomic analysis. We speculate that besides acting against antimicrobial present in the environment, the detected ARGs in the rumen may have functions in addition to antimicrobial resistance, which deserves further investigation.
It has been reported that the prevalence and abundance of ARGs in the gut of cattle is affected by diet. For example, dietary transition from milk replacer to starter led to alternation in the fecal resistome of dairy calf . In addition, the diversity and abundance of total ARGs were higher in the rumen of beef cattle fed high concentrate than those fed high forage diet, with chloramphenicol and aminoglycoside resistance genes being predominant in forage- and concentrate-fed cattle, respectively . A recent study also suggested that the dietary supplementation of tulathromycin, a macrolide antimicrobial drug used as metaphylaxis, significantly affected the temporal development of fecal microbiota and associated resistome in feedlot cattle . To our knowledge, there is no study reporting how host genetic factors affect the active gut resistome in mammalian species. In food-producing animals such as beef cattle, understanding the ‘host-resistome’ association may be a prerequisite to select breeds with high feed efficiency and low risk of ARG transmission to the environment, as the gut microbiome that harbor ARGs has been proved to be largely host-driven [21, 52–54]. In this study, all beef steers were raised under the same dietary and environmental conditions, suggesting that the prevalence and expressions of ARGs were driven by host genetic factors such as breed and feed efficiency. On the contrary to the findings by Auffret et al. , who didn’t observe breed effect on the abundance of rumen microbiota and abundance of ARGs in beef cattle, we not only observed a significant difference in both prevalence and abundance of ARGs, but also the prevalence of ARG transcripts among three breeds. Specifically, we detected less type of ARG transcripts, especially tetracycline resistant gene transcripts, in the rumen of crossbred (KC) compared with purebred (AN), which may be explained by less copy number of total active bacteria in the rumen of KC than AN animals. Besides, our previous study also indicated that the active phylum Bacteroidetes, which account for a high proportion of the microbial genomes (e.g. species belonging to Prevotella and Bacteroides) that harbor resistance genes in the rumen , was less abundant in KC compared with the other two breeds . The significant correlation between total active bacterial population and the abundance of ARG transcripts observed for KC cattle only further support that the expression of resistome in the rumen may be host breed specific and driven by ruminal microbiota. However, it is not clear why copy number of total active bacteria is negatively correlated with the abundance of multiple tetracycline and macrolide ARG transcripts. In this regard, the active bacterial host of those ARG transcripts deserves further investigations using the pure cultures.
It has been proved that rumen microbiome differs in beef cattle with high and low feed efficiency , which may explain the difference in the prevalence (e.g. ARGs belonging to vanconmycin, aminoglycoside, and MLS) and abundance of several ARGs (e.g. tetX, vatE, and lnuA) between H-RFI and L-RFI beef cattle based on metagenomic data. However, H- and L-RFI steers share a similar ARG transcript profiles, suggesting that ruminal fermentation capacity may not be a main factor driving the expression of ARGs. Our results also showed that breed × feed efficiency interactions only affect the abundance of ARGs, but not ARG transcripts. Taken together, the lack of feed efficiency and interaction effect suggest that host breed is the main drive of rumen resistome of beef steers.