1. Introduction
The rapidly growing demand for animal products has led to the intensification of animal production and associated with this is the increased use of antimicrobials to maintain animal health and welfare [
1]. Antimicrobials have been widely used in livestock production systems, particularly in intensive feeding operations, for different purposes, including therapeutic, metaphylactic, and prophylactic uses for infectious disease treatment, control, and prevention [
2]. The judicious use of antimicrobials as livestock treatments is vital in the face of few alternatives for specific diseases, while their overuse and/or misuse has led to the development of antimicrobial resistance (AMR). Antimicrobial treatments applied to beef cattle indiscriminately affect pathogenic bacteria present at the site of infection but also may impact the commensal microbiota of the gastrointestinal tract [
3,
4]. This may result in the elimination of susceptible microbial populations, thereby reducing competition from resistant bacteria, which may proliferate under antimicrobial selection pressure [
5].
Enterococci are natural inhabitants of the intestinal tract of different organisms, including cattle [
6]. They are resilient organisms capable of surviving in a broad range of temperatures and pH levels [
7]. In human medicine, the emergence of AMR to Gram-positive and broad-spectrum antimicrobials among enterococcal species such as
Enterococcus faecium has made the treatment of the infections caused by these opportunistic pathogens a real challenge for clinicians [
8]. Resistance to antimicrobial agents can occur through intrinsic mechanisms found universally in the bacterial genome, in spontaneous mutations, or through the acquisition of new genetic material through recombination [
9]. Enterococci are intrinsically resistant to a number of antimicrobial agents, including β-lactams and aminoglycosides [
10]. Among the main enterococcal species,
E. faecalis is most important to public health and is intrinsically resistant to clindamycin (a lincosamide), quinupristin (streptogramin B class), and dalfopristin (streptogramin A class) through activity conferred by the expression of the
lsa gene [
11]. They also have the capacity to acquire new mechanisms of AMR, either by mutation or genetic recombination via plasmids and transposons. In this way, enterococci have acquired AMR to many classes of antimicrobials, including glycopeptides (vancomycin), macrolides, quinolones, streptogramins, and tetracyclines [
12].
The emergence of multidrug-resistant (MDR) strains such as vancomycin-resistant enterococci (VRE) in livestock production and human clinical settings has become a globally significant concern, although VRE are yet to be reported in Australian livestock species [
13]. MDR enterococci are able to survive in the gastrointestinal tract and potentially become the dominant flora under antimicrobial pressure [
8]. Furthermore, gene transfer occurs readily between closely related enterococcal species, but can also occur between different genera [
14]. For example, enterococci may transmit vancomycin resistance to more pathogenic microorganisms such as methicillin-resistant
Staphylococcus aureus in human patients [
15].
The potential transfer of AMR from the enteric bacteria found in animals to humans (or vice versa) is a current global public health concern [
16]. In previous studies, some genetic similarity was reported to exist between enterococci isolated from animals and those causing human infections [
10,
17,
18]. However, more recent studies based on whole genome sequence analysis have found that many enterococci isolated from animals are unrelated to the strains causing infections in humans [
19,
20]. The prevalence of AMR among enterococci isolated from animals and humans varies between geographical location and patterns of antimicrobial use [
21,
22], and the identification of antimicrobial-resistant bacteria in agricultural settings may at times create issues for both producers and consumers [
23]. Australia has strict registration and regulation of antimicrobial use and conducts periodic assessments on the status of antimicrobial-resistant bacteria in healthy livestock at slaughter [
13,
24,
25]. Despite these restrictions and the periodic study, continuous follow-up is needed to understand the colonization dynamics of AMR surveillance indicators and zoonotic genera under various forms of production and antimicrobial selection pressure. Such a situation exists in cattle in Australia when, upon entry to a feedlot, they experience major changes in environment, diet, husbandry/management, and antibiotic treatments. Hence, the aim of this study was to determine whether changes in AMR status occur among
Enterococcus spp. isolated from the same animals on entry into a beef feedlot farm and again at the slaughterhouse.
4. Discussion
Enterococci have the potential to develop resistance to almost all the classes of antimicrobials of importance to human medicine [
38]. Whilst both
E.
faecalis and
E.
faecium are associated with human infections, a higher proportion of VRE belong to
E. faecium [
39,
40]. However, the improved understanding of AMR among enterococci would benefit from the inclusion of additional species in the AMR surveillance programmes [
19]. This study focused on the AMR phenotypes and genotypes identified among enterococci isolated from cattle faeces collected from the same animals at the entry to and the exit from a beef feedlot in Southern Australia. The shift in specific
Enterococcus spp. isolated, with a higher prevalence of
E. faecium identified at the exit compared to the entry (which was dominated by
E. hirae), was a noteworthy finding of the study. Second, among the enterococci species isolated,
E. faecium more frequently expressed an MDR phenotype to several combinations of antimicrobials. This included both ciprofloxacin and quinupristin/dalfopristin resistance (detected only in
E. faecium isolates), with the majority obtained from samples collected at the entry to the feedlot. Third, no ARGs were identified that could account for the moderate to high frequency of daptomycin resistance observed among the enterococci isolates, with
E. hirae significantly higher compared to
E. faecium. Fourth, no ARGs were identified that could account for the nitrofurantoin resistance among the
E. faecium isolates, which increased markedly between the entry and exit samples.
The most prevalent
Enterococcus spp. isolated at the feedlot entry was
E. hirae (86.5%)
, followed by
E. faecium (8.7%), and
E. mundtii 3 (2.9%). This result is in line with previous reports that indicated that
E. hirae and
E. faecium are frequent species detected in the faecal content of healthy animals [
38,
41]. Similarly,
E. hirae was reported elsewhere as the most predominant species detected in beef cattle [
19]. In the present study, a very high proportion of enterococci at the feedlot entry were resistant to lincomycin (60.6%) and daptomycin (25.0%), with lower proportions resistant to nitrofurantoin (8.7%), ciprofloxacin (6.7%), tetracycline (4.8%), tigecycline (non-susceptible 3.9%), and quinupristin/dalfopristin (2.9%). Multidrug resistance was commonly observed. Resistance to daptomycin was more likely to be present among the
E. hirae isolates, whereas resistance to ciprofloxacin and quinupristin/dalfopristin was observed more frequently in
E. faecium. These AMR trends have been reported in previous international studies. For example, in one Canadian study resistance varied between
E. faecium and
E. hirae for tetracycline (45% vs. 59%), nitrofurantoin (45% vs. 16%), macrolides (29% vs 33%), tigecycline (3% vs. 12%), and quinupristin/dalfopristin (3% vs. 1.4%) [
19].
The present study confirmed that cattle arriving at the feedlot may already be colonised with
Enterococcus spp. resistant to critically important antimicrobials that are not used in livestock in Australia. These antimicrobials include daptomycin, ciprofloxacin, nitrofurantoin, quinupristin/dalfopristin, and tigecycline (non-susceptibility), though it is fair to say that the ARGs conferring these resistant phenotypes were only identified in our
E. faecium collection for ciprofloxacin (
efmA) and quinupristin/dalfopristin (
eatAv, msr(C), vat(E)). None of the classes these antimicrobials belong to is registered for use in livestock in Australia apart from virginiamycin, a streptogramin which can be strictly used only for the management of acute rumen acidosis [
42]. As these antimicrobials are all used in human medicine, it is possible that they originate from background environmental sources at the point of origin of the feeder cattle. In this study, the effect of breed was tested and was found insignificant. Whilst AMR can be spread from humans to animals by transfer of the resistant bacteria through direct contact, further interrogation of the
E. faecium genomes is required to determine their origins and transmissibility [
43].
Unlike at the entry,
E. faecium (81.3%) became the most predominant
Enterococcus spp. identified at the exit, followed by
E. hirae (17.4%). The change in diet from grass to a more concentrated energy/protein rich ration is the most likely reason for the observed change in species diversity, although age may also have been a factor in the altered faecal microbial community [
44]. Similarly to our study findings,
E. faecium isolates of animal origin have been found to be resistant to ciprofloxacin, tetracycline, and nitrofurantoin [
45]. Ciprofloxacin resistance is more commonly detected in
E. faecium compared to other enterococci [
19,
46]. The levels of resistance to daptomycin, erythromycin, lincomycin, and tetracycline in this study were also consistent with other Australian studies (abattoir surveys), which focused on both grazing and feedlot cattle [
13,
47], pigs [
25], and poultry [
20].
Antimicrobial resistance, particularly multi-resistance, is common among enterococci because of their ability to acquire ARGs [
48]. In total, 41 isolates (29%) were MDR in the present study, with some isolates resistant to up to five antimicrobial classes. The emergence of new antimicrobial resistance in enterococci is likely associated with their ability to acquire new genetic elements through horizontal gene transfer (HGT) [
49]. Additionally, innate resistance to some antimicrobials also must be considered [
50]. In the present study, deeper interrogation of the genomes will be required to map ARGs to particular mobile genetic elements, the origin of which (human, animal, or environmental) will require further study.
Enterococci are naturally resistant to many classes of antimicrobials, such as aminoglycosides and β-lactams, and can also acquire resistance to other classes, including glycopeptides, quinolones, and tetracyclines [
51]. In this study, the mutated form of the wildtype
eatA ABC-F subfamily protein
eatAv gene, which confers resistance to lincosamides, streptogramin A and pleuromutilins, was observed in 75.8% of the
E. faecium isolates. The antimicrobial efflux pump
efmA gene, important for the removal of macrolide and fluoroquinolone antimicrobials from the intracellular environment of bacterial cells, was observed in 66.7% of the ciprofloxacin-resistant
E. faecium isolates. The prevalence of ciprofloxacin resistance among
E. faecium isolates was higher in the much smaller number of isolates obtained at the entry compared to the exit samples. The dual nature of the resistance imparted by
efmA likely explains the high prevalence of resistance in the absence of fluoroquinolone selection pressure, given that this antimicrobial class has never been registered for use in Australian food-producing animals. Ciprofloxacin resistance occurs through either the chromosomal mutation of DNA gyrase (
gyrA) and topoisomerase IV (
ParC) genes, the active efflux pump (
efmA), target protection (
Qnr-like determinants), or combinations thereof [
52,
53,
54]. In the present study, only the efflux pump gene
efmA was identified and both the
gyrA and the
parC genes did not contain mutations in their quinolone resistance determining regions, confirming that none of the isolates has developed point mutations under previous fluoroquinolone selection pressure [
55].
Daptomycin resistance is reported to be linked with mutations of the genes encoding the cell envelope stress response (
LiaFSR and
YycFGHIJ) and the genes responsible for the metabolism of phospholipids (
gdpD and
cls) [
56,
57]. In this study, the WGS analysis revealed no mutation in these target genes that could account for the isolates with the MICs over the resistance breakpoint. Thus, at this point in time, the molecular mechanism of daptomycin resistance in enterococci is yet to be fully elucidated, and the relative impact of the use of other drug classes in the human vs. animal contexts on its distribution is completely unknown.
Interestingly, in the present study we found a high proportion of
E. faecium isolates obtained at the feedlot exit (compared to the feedlot entry) that were resistant to nitrofurantoin, an antimicrobial used to treat urinary tract infection in humans [
58] that has not been used in livestock worldwide since the early 1990s [
59] and that, to the best of the authors’ knowledge, has never been used in livestock in Australia. High frequencies of nitrofurantoin resistance have also been reported in
E. faecium isolated from feedlot cattle elsewhere (e.g., in Canada 45%) [
19]. However, we hypothesise that nitrofurantoin resistance in this study may be yet to be elucidated or that, possibly, reverse zoonotic transfer has occurred. Nitrofurantoin resistance in human medicine occurs through the development of mutations in
nfsA and/or
nfsB, both of which encode oxygen insensitive nitroreductases [
60]. In addition, the plasmid-mediated efflux genes,
oqxAB, are associated with high-level nitrofurantoin resistance [
61]. However, neither the mutation nor the efflux ARG were detected in the nitrofurantoin-resistant
E. faecium isolates selected for sequencing in the present study. In a recent study of human
E. faecium isolates obtained from urinary tract infections in China, the absence of the nitroreductase-encoding genes
ef0404 and
ef0648 was associated with high-level nitrofurantoin resistance [
62]. However, a detailed scan of our
E. faecium genomes found no correlation between gene absence and nitrofurantoin resistance (data not shown). These findings confirm that the mechanism of nitrofurantoin resistance in beef cattle
E. faecium isolates remains not fully understood at this point in time, and further research is required to determine if it is chromosomally or mobile genetic element-encoded. Resistance outcomes for one antimicrobial class can be linked with resistance to other classes through co-selection [
63].
Although the determination of the resistance phenotype and ARGs present in the commensal enterococci inhabiting healthy feedlot cattle at the entry to and exit from the feedlot has provided valuable insight, this study had some limitations. First, the study was conducted at a single beef feedlot in southern Australia, whereas many of Australia’s largest feedlots are distributed in the sub-tropical zones of Queensland and New South Wales. Second, we were unable to determine the effect of antimicrobial treatment on the development of AMR as only 13 cattle received curative treatment (mostly macrolides) during the 90-day feeding period. Third, faecal samples could not be obtained from the cattle at the feedlot immediately before transport to the abattoir; hence, the exit samples could only be obtained post-slaughter, and microbial population changes may have occurred during transport. Larger scale multi-site longitudinal studies are therefore recommended to fully investigate the bacterial AMR status in Australian feedlot cattle production systems. Future whole genome sequencing studies should also consider the associations between the AMR genes, plasmids, virulence factors, and genetic relatedness of the isolates.