1. Introduction
The
Enterococcus spp. are Gram-positive bacteria that are commonly found in the intestinal flora of humans and animals [
1,
2,
3]. They are also important opportunistic pathogens and are among the leading causes of healthcare-associated pathogens in the United States [
4,
5,
6].
Enterococcus faecalis and
Enterococcus faecium are the two clinically important species as they account for most enterococcal infections, which cause a variety of infections, including urinary tract infections, soft tissue infections, bacteremia, and endocarditis in humans [
2]. Some factors that have made the enterococci a successful nosocomial pathogen are their intrinsic resistance to many antimicrobial agents and their ability to acquire resistance via horizontal gene transfer [
2]. The widespread resistance among enterococci has also made them one of the sentinel organisms used to track antimicrobial resistance trends by the National Antimicrobial Resistance Monitoring System (NARMS), a partnership between the Centers for Disease Control and Prevention (CDC), the U.S. Food and Drug Administration (FDA), and the U.S. Department of Agriculture (USDA) [
7]. From 1996 to 2011, the surveillance of bacterial isolates of animal origin was conducted by the USDA Agricultural Research Service (USDA-ARS), and from 2011 to the present, by the USDA Food Safety and Inspection Service (FSIS). Certain enteric bacteria are monitored by NARMS to track trends in antimicrobial resistance in humans, retail meats, and food animals in the United States, and the enterococci are included to monitor antimicrobial resistance trends in Gram-positive bacteria.
Antimicrobials administered to food animals for therapeutic or prophylactic purposes have resulted in the development of antimicrobial resistance in enterococci. While most enterococci are commensals and do not cause illnesses in food animals, enterococci commonly carry plasmids encoding antimicrobial resistance, and the transfer of such plasmids from enterococci to more pathogenic bacteria is cause for concern. Studies have shown that enterococci can disseminate their antimicrobial resistance genes to other bacteria, such as
Staphylococcus aureus, through horizontal gene transfer [
8,
9]. Thus, enterococci in food animals serve as a reservoir of antimicrobial resistance, with the potential to transfer antimicrobial resistance to human pathogenic bacteria and spread to the human population through the consumption of animal products or through human–animal contact. Hence, it is important to study the prevalence of plasmids, especially those associated with antimicrobial resistance, in enterococci in food animals and their potential transfer to other bacteria.
In 2010, a system for the classification of plasmids for Gram-positive bacteria was determined by Jensen et al. [
10]. The system was established by the comparison of sequences of replication-initiating genes (
rep) as well as
rep-like genes from Gram-positive plasmids available from GenBank; 21 replicon families (20
rep-families and a unique sequence) were determined. In this study, the distribution and prevalence of plasmid families from multidrug-resistant (MDR; resistance to three or more antimicrobials)
E. faecalis and
E. faecium from poultry, collected as part of NARMS, were determined using multiplex PCR developed from the analysis. Isolates representative of each plasmid prototype from detected
rep-families were selected and used to determine if the plasmids could transfer to other enterococci using bacterial conjugation. Results from this study will provide information on
rep-family prevalence, diversity, and mobility in MDR
E. faecalis and
E. faecium from poultry.
4. Discussion
Studies characterizing the antimicrobial resistance and plasmid content of bacterial strains of clinical origin are well-represented in the literature; however, knowledge of plasmid distribution for strains isolated from non-clinical sources such as food animals is largely unavailable. The use of antimicrobials in the food supply, including food animal production, coupled with the potential for transfer of antimicrobial-resistant bacteria into the human population, supports the need for additional data from non-clinical sources. As commensal bacteria such as enterococci contain plasmids that can harbor multiple resistances and are often mobilizable, the present study aimed to analyze the plasmid replicon content of
E. faecalis and
E. faecium collected from poultry during an eight-year period, in which the USDA-ARS participated as a part of NARMS. The analysis utilized a PCR-based plasmid replicon typing system designed to define plasmid replicon families from Gram-positive bacteria, including enterococci [
10].
Of the replicon families examined in this study, ten of the
rep-families were found in both
E. faecalis and
E. faecium, indicating a broad range of distribution. This observation was different from that previously described as only four
rep-families were shared between
E. faecalis and
E. faecium based on information from PubMed and GenBank [
10]. Limited information on the distribution of
rep-families in enterococci can be attributed to the low number of strains examined, the limited sources of the strains (i.e., clinical sources), and specific strain characteristics such as certain antimicrobial resistance phenotypes or genotypes [
15]. Although the present study specifically examined
E. faecalis and
E. faecium from poultry, a large collection was analyzed, and the different origins of the isolates allowed for a comparison to the clinical as well as non-clinical sources. Furthermore, biasing due to antimicrobial resistance to one specific antimicrobial was minimized by targeting MDR isolates, which allowed for a greater number of isolates to be included as well as a higher probability of isolates containing at least one plasmid replicon family.
About 20% of the total enterococcal isolates were positive for at least one
rep-family. Previous studies on the plasmid classification of antimicrobial-resistant
Enterococcus have shown that a considerable portion of
Enterococcus from animal and environment sources (approximately 30%) did not harbor any plasmids from the 21
rep-families, while
Enterococcus from human sources were mostly positive for
rep genes [
10,
16,
17,
18,
19,
20]. This suggests that plasmids present in non-human enterococcal isolates are different from those found in enterococcal isolates of human origin and may be comprised of those not included in this classification system. It could also indicate that less antibiotic pressure in non-clinical settings may have resulted in the loss of plasmids.
It is interesting to note that the number of enterococcal isolates positive for
rep genes decreased over time, as did the diversity of
rep-families. About half of the isolates tested were identified to harbor plasmids in 2004 and 2005, while the number of the plasmid-positive isolates decreased to just one isolate in 2009. This was unexpected as the selective pressure of antibiotics would enhance the acquisition and exchange of resistance genes through various mechanisms, including horizontal gene transfer via plasmids. In the absence of antibiotics, plasmids encoding antimicrobial resistance tend to be lost as plasmid maintenance is a burden to the bacterial cell [
21]. However, it is unlikely that selective conditions were absent in the poultry farms during the years the samples were taken, although changes in the class of antibiotics used cannot be ruled out. Antibiotics were still being used in food animals until their use in farm animals for growth promotion purposes was banned in the U.S. in 2017 [
22,
23]. The reason for the decrease in the number of
rep-families in enterococcal isolates may be due to the presence of plasmids that do not belong to the 21
rep-families or due to unknown conditions that led to an instability and the subsequent loss of plasmids.
The predominant
rep-family detected in the strains was
rep9, represented by the pCF10 prototype, one of the pheromone-responsive plasmids in enterococci. As pheromone-responsive plasmids have been found almost exclusively in
E. faecalis [
10,
24], the prevalence of this plasmid in this species was not unexpected. However, the predominance of
rep9 in
E. faecium isolates was surprising and could be due to the insufficient study of a wide variety of enterococci from poultry sources. The small sampling of
rep-families from poultry
E. faecalis and
E. faecium previously reported
rep0,
rep2, and
rep9 in
E. faecalis, with a variety of
rep-families (
rep2,
rep3,
rep4,
rep5,
rep6,
rep7,
rep14,
rep17) found in
E. faecium [
10,
25]. Because of the nature of poultry production, such as the number of birds and environmental conditions pre-processing, it is not unreasonable that the opportunity for the transfer of plasmid
rep-families is far greater in food animal production than clinical medicine, which may also account for the different
rep-families in enterococci from poultry.
Both
E. faecalis and
E. faecium contained
rep-families that were restricted to one or the other species in this study.
Rep7 and
rep17 were found exclusively in
E. faecalis, while
rep5 and
rep8 were only found in
E. faecium. Prototype pUSA02 (
rep7) is characterized as a broad-host range plasmid that has been detected primarily in clinical
E. faecalis [
15,
26,
27], while prototype pRUM (
rep17) is a conjugative, MDR plasmid originally isolated from a clinical
E. faecium [
28]. Surprisingly, none of the
E. faecium in the present study were positive for
rep17, which is a major deviation from results of some previous studies [
10,
29]. Both
rep5 and
rep8 were found only in
E. faecium in this study. Both
rep-families are described as having a narrow-host range, with
rep5 predominating in
S. aureus, while
rep8 is another
rep-family containing pheromone-responsive plasmids primarily found in
E. faecalis [
10].
Rep5 has previously been identified in
E. faecium from chicken [
25] and in a recent study of
S. aureus from retail poultry from the U.S. [
30], suggesting the genetic exchange of plasmid replicons among enterococci and staphylococci in poultry.
As most of the MDR isolates in this study included resistance to tetracycline, this antibiotic phenotype was used to determine the frequency of plasmid replicons associated with tetracycline resistance and was employed as a marker in conjugation studies. Interestingly, only for year 2005 was tetracycline resistance associated with the presence of a plasmid replicon. Although the specific
rep-families associated with tetracycline resistance were not identified, 2005 was also the year with the highest number of different
rep-families detected. The transfer of tetracycline resistance and
rep17 using conjugation indicated a possible linkage between the antibiotic and
rep type in a previous study [
10]. It is also possible that some or more plasmid replicons for that year harbored tetracycline resistance genes. A definitive reason for the association of tetracycline resistance with the
rep-family in this study was not determined.
Characterizing the mobility of plasmids is fundamental to understanding the epidemiology of plasmid-encoded antimicrobial resistance. In the present study, conjugation was performed to see if plasmids present in enterococcal isolates were transmissible which would enable determination of the potential transmissibility of the traits encoded on the enterococcal plasmids from animal sources to human sources. The transfer of plasmid replicons using conjugation, which was conducted using an
E. faecalis recipient, was seen in the
E. faecalis donors but not in
E. faecium, which is indicative of the narrow-host range of some of the
rep-families, and that intraspecies transfer is preferred over interspecies transfer [
31]. Moreover, not all plasmids of the
rep-families tested were transferred, which agrees with the previous analysis that revealed how some of the plasmids are non-transmissible [
32]. Eight
rep-families were successfully transferred from
E. faecalis donors to the
E. faecalis recipient, and some of these
rep-families included
rep9-containing pheromone-responsive plasmids such as pAD1 and pPD1—in addition to pCF10 discussed above—and the unique
rep, which contains pMG1, a pheromone-independent conjugative plasmid. Pheromone-responsive plasmids, well-described in
E. faecalis, transfer at a high frequency using aggregation substances for the clumping of donor and recipient cells; non-pheromone-responding plasmids are also known to transfer efficiently between different
Enterococcus species [
24,
33]. There were four
rep-families that were not transferred from
E. faecalis donors to the
E. faecalis recipient, and these included
rep1, which contains pIP501, pAMβ1, and pSM19035, as well as
rep2, which contains pRE25. These broad-host range conjugative plasmids do not transfer well in broth suspensions and transfer at a lower frequency on solid surfaces as compared to pheromone-responding plasmids and pMG1-related, pheromone-independent conjugative plasmids that transfer well on solid and in liquid, as reflected in our mating results [
24,
33].