The effects associated to the rising of antimicrobial resistant microorganisms are currently of main concern. Most of the zoonotic processes are caused by pathogens that can affect both animals and humans, but antibiotics are frequently used to battle secondary infections in both human and animal health. For instance, during the COVID-19 pandemic, the WHO warned about how the misuse of antibiotics in COVID-19 mild cases could worsen the situation regarding AMR and encouraged to use them only under clear signs of bacterial infections [18]. The WHO has been advising since 2017 that veterinarians or practitioners should stop using antibiotics in healthy livestock animals to prevent the spread of antibiotic resistance. Thus, it is important to implement surveillance mechanisms to be aware of the ARGs reservoirs that can be present in livestock.
Not all the ARGs present in the rumen spread to the environment, as this depends on the shedding of the ARGs-carrying bacteria from the gastro intestinal tract which will then pass to the faeces, then to the soil if we use these as manure, then to plants and finally to the food chain. ARGs are rarely obtained by selective pressure of antibiotics. These genes are most likely obtained by HGT between environment bacteria [19] or by the transition of the animal from preruminant to ruminant, which is the key moment when the cow ruminal microbiota is first settled [20]. In order to get a deeper insight on how the AMR reach into the rumen, it is necessary to have all information on the antibiotic usage in each farm with individualized records in different stages of the animal life, mainly from its earliest stages, which is usually difficult to obtain under commercial conditions.
We found that the RAs of the most abundant ARGs was similar among farms, showing that the resistance to those ATBs are consolidated in the farms in northern Spain. Even though we found 998 ARGs making up to 1% of the total number of reads, only 69 of them had a RA > 0.005%, similar results have been shown in other studies [21].
The most abundant resistance family found was the efflux pump, which also happened in other studies [22]. Around 25% of the most prevalent ARGs belonged to this category in which the bacterium must use energy to eliminate the antibiotic. Processes and mechanisms mediated by ATP usually require a selective pressure to be maintained, as evolution tends to remove processes that consume energy if they are not important for the survival of the microorganism [23]. The second most abundant family was the tetracycline resistance proteins. This is not surprising as the use of tetracycline is of high importance in the most relevant diseases in dairy cattle and livestock (i.e. metritis and lameness) [15]. The third most abundant family of resistance was the one conferring resistance to vancomycin (van), a glycopeptide antibiotic with forbidden use in livestock. Resistance to this antibiotic is of main concern, as it is used as a last-line defence in life-threatening infections mediated by Gram-positive bacteria. Vancomycin is used against methicillin-resistant Staphylococci and Enterococci. If methicillin-resistant bacteria also gain resistance against vancomycin, there are very few alternatives to treat the infection. Staphylococci are already multi-resistance carriers, posing a serious threat to human and animal health. This family of AMR can be also explained by the use of avoparcin, an analogous of vancomycin as a feed additive in dairy cattle [24]. Resistance to some of these antibiotics have also been described in other studies such as Hui-Zeng Sun’s [25].
Macrolides, the antibiotic class associated to macB, are treatments of single administration to reduce the animal handling and facilitate its dosing [12]. The prevalence of msbA can be explained as nitroimidazoles were widely used as growth-promoters in the past decades, supporting the idea that these genes were not inherited independently in bacteria. We speculate that the resistance to an antibiotic that is no longer administered is only conserved if it there are other co-selection mechanisms present, as evolution tends to remove anything that causes an energetic cost if there is not a selective pressure on it [23]. When bacteriophages (mainly the caudovirales order) infect bacteria, virus-mediated HGT is likely to occur [26].
We classified the genes according to the categorisation of antibiotics made by the EMA for use in animals. Antibiotics belonging to category D should be first-choice treatment and those within category A must be restricted to last-line treatments in human health. Ideally, most of the ARGs detected in our samples should belong to Category C or D, because these categories include first-line treatments with alternatives in human medicine [17]. However, a large proportion of AMR from category A and B was observed in the rumen resistome. It must be emphasised that cephalosporins 3rd and 4th generation (category B) are used to treat respiratory infections, lameness and mastitis mainly due to the reduced withdrawal period in milk. Tetracyclines (category D) are a first-choice [15] treatment in a wide range of nature of dairy cattle and livestock diseases. Carbapenems (category A) are not used in cattle as their use is restricted to human health. The resistance to tetracyclines can be explained by two factors: tetracyclines are used via intrauterine to treat postpartum metritis as they are an effective and safe antibiotic with relatively low milk withdrawal period [13]. They also pose a risk in the environmental dissemination of ARGs due to discharges; the other one is the use of tetracyclines to treat some types of mastitis, one of the most common diseases in dairy cattle and the environmental contamination due to antibiotic residues in waste. The relatively large amount of resistance genes to antibiotics of the category A is worrisome as these antibiotics are strictly reserved to humans and companion animals under exceptional circumstances. Antibiotics are rarely given orally to dairy cattle. In most cases, they are administered by an intramammary or parenteral injection [13]. Many antibiotics are extracted from bacteria that are usually part of the environment. The resistance can be obtained as a normal response of a bacterium to the toxin of another one and not only as a response to the use of the antibiotic. This could be the case of carbapenems, which are last-resort antibiotic [27] used in human medicine but extracted from Enterococci species and Escherichia coli.
In this study, 43% of the analysed ARGs confer resistance to antibiotics of category A and B antibiotic, whereas 57% of them are included in category C and D. It must be pointed out that a bacterium could first develop resistance to a category D antibiotic, but the same gene may also confer resistance to antibiotics belonging to other categories due to chemical similarities between the molecules or the same mechanism of resistance in a process known as cross-resistance. This is the case of the identified adeJ gene, which confers resistance to carbapenem, rifamycin, diaminopyrimidine, tetracycline, phenicol, penem, macrolide, lincosamide, cephalosporin and fluoroquinolone. This gene might have been gained originally as a defence mechanism against tetracycline antibiotics (category D), but it also provides resistance to carbapenems. Furthermore, category C and D antibiotics are also usually administered topically, so these antibiotics might get into the rumen by contaminated feed or water or by licking the treated zone, although the topical application of medicine is not common in dairy cattle, limited mainly to wound care, and direct intramuscular injection is rather preferred [28]. The high correlations between ARGs suggest that there could be multi-resistant plasmids that usually carry those genes together. Most of these genes, that have also been described in other studies such as Ming-Yuan Xue’s [22], are involved in mechanisms of resistance of efflux pumps and target alteration. Among these, macB, parY, rpoB2 and TaeA are related to the treatment of mastitis except TaeA. Although msbA is not present in this cluster, it was highly correlated with macB. This suggests that these ARGs are inherited or transmitted together. The high phenotypic correlation of the bacteriophages with the ARGs might open a new door to the use of these viruses to modulate the abundance of ARGs-carrying bacteria in the dairy cattle rumen. The current study shows that the role of the bacteriophages may be of interest as an indicator of ARG modulations, especially in early stages of the cow’s development. The RA of the bacteriophages partially depends on the genetic background of the animals, and the presence of the ARGs could increase in each generation of cows favouring HGT to happen.
Our results showed that the host genetics exert some control on the RA of ARGs, and bacteriophages, with low to moderate heritabilities.
The heritability of the RA of the ARGs needs to be interpreted as how the genetics of the cow control the variability of the relative abundance of these ARGs. There are certain physiological conditions (folds in the rumen, pH, feeding behavior, feed transit, etc.) that can promote the growth of certain microorganism strains that are related to a reservoir of ARGs. We observed that, even if these values are not as high as the ones for bacteriophages, they are good enough to be considered in future breeding programmes to reduce HGT and the amount of ARGs in the farms.
Besides, genetic correlation with other important traits must be estimated. The main phyla carrying these ARG also showed low to moderate heritability. The high heritability of phyla with a small presence in the ruminal ecosystem such as Elusimicrobia (h2 = 0.39), whose role is not well understood yet [29], can be explained by a strong genetic effect of the host over this phylum. These phyla are not the most prevalent in the ruminal microbiota, as described in another analyses performed using the same data set [30]. However, they made up the core reservoir of ARGs in the rumen and it would be necessary to understand how these microorganisms with such a low RA interact with the environment and how they move across animals to disseminate ARGs.
Further studies are needed to understand why those genes with lower heritability also showed the higher correlations with the bacteriophages, and whether these are causal relationships.
The AMR core present in livestock and human population is one of the concerns for the One Health initiative. These resistances can jump to humans by indirect transmission from faeces used as manure for crops which could lead to the resistances passing to the plants and then to other animals or to humans. Direct transmissions through contact with the animals or with the intake of some of their products is also of concern. Reducing or, at least controlling, the antimicrobial resistances in livestock is also critical to human medicine.