Antimicrobial Resistance Profiles of Enterococcus faecium and Enterococcus faecalis Isolated from Healthy Dogs and Cats in South Korea

Enterococcus spp. are typically found in the gastrointestinal tracts of humans and animals. However, they have the potential to produce opportunistic infections that can be transmitted to humans or other animals, along with acquired antibiotic resistance. In this study, we aimed to investigate the antimicrobial resistance profiles of Enterococcus faecium and Enterococcus faecalis isolates obtained from companion animal dogs and cats in Korea during 2020–2022. The resistance rates in E. faecalis towards most of the tested antimicrobials were relatively higher than those in E. faecium isolated from dogs and cats. We found relatively higher resistance rates to tetracycline (65.2% vs. 75.2%) and erythromycin (39.5% vs. 49.6%) in E. faecalis isolated from cats compared to those from dogs. However, in E. faecium, the resistance rates towards tetracycline (35.6% vs. 31.5%) and erythromycin (40.3% vs. 35.2%) were comparatively higher for dog isolates than cats. No or very few E. faecium and E. faecalis isolates were found to be resistant to daptomycin, florfenicol, tigecycline, and quinupristin/dalfopristin. Multidrug resistance (MDR) was higher in E. faecalis recovered from cats (44%) and dogs (33.9%) than in E. faecium isolated from cats (24.1%) and dogs (20.5%). Moreover, MDR patterns in E. faecalis isolates from dogs (27.2%) and cats (35.2%) were shown to encompass five or more antimicrobials. However, E. faecium isolates from dogs (at 13.4%) and cats (at 14.8%) were resistant to five or more antimicrobials. Taken together, the prevalence of antimicrobial-resistant enterococci in companion animals presents a potential public health concern.


Introduction
Enterococcus spp.are Gram-positive opportunistic bacteria generally present in the gastrointestinal tract of humans and animals.They can cause invasive infections in humans, including endocarditis, sepsis, bacteremia, and nosocomial infections of the urinary system and genital tract [1].Moreover, as commensal bacteria, Enterococcus spp.represent a reservoir for host antimicrobial resistance and are considered a good indicator of the effects of selective pressure on antimicrobial use [2].They can transmit antibiotic resistance to other pathogenic bacterial species through the horizontal transmission of mobile genetic elements [3].Additionally, the regular use of antibiotics in humans and animals favors the emergence of enterococci resistance, which is difficult to treat [4].
Enterococcus faecium and Enterococcus faecalis are the most common members, representing more than 90% of the identified species [5].Moreover, E. faecium and E. faecalis are known to have high levels of multidrug resistance (MDR), and it has been claimed that they can spread directly from companion animals to humans [6,7].A previous study found that companion animals treated with antibiotics in an intensive care unit were a source for the zoonotic transmission of MDR Enterococcus [8].The likelihood of the horizontal transmission of zoonotic infections from animals to their owners is increased because companion animals, such as dogs and cats, live in close proximity to their owners.Preventing the spread of MDR Enterococcus strains between animals or from animals to humans is crucial for both public health and veterinary medicine.Therefore, research into the characteristics of Enterococcus spp.and the potential for the spread of antibiotic resistance is required.
Understanding the antimicrobial resistance profiles of Enterococcus spp.isolated from companion animals provides helpful insight into the extent of resistance in the gut microbiota and into antimicrobial selection for treating infections in humans and animals.The antimicrobial resistance profiles of Enterococcus spp.isolated from dogs and cats have been described in numerous studies from across the globe [9][10][11][12][13].The resistance characteristics of E. faecium and E. faecalis isolates from companion animals have also been studied in various investigations conducted in South Korea [14][15][16][17][18][19].However, most of these previous studies were conducted to investigate the antimicrobial resistance of Enterococcus isolates obtained from diseased animals in a few parts of the country over a very short period of time.Thus, we aimed to conduct this nationwide investigation to determine the antimicrobial resistance profiles and patterns of E. faecium and E. faecalis isolated from healthy dogs and cats in Korea between 2020 and 2022.

Bacterial Isolates
Enterococcus spp.were isolated from feces samples of healthy (with no clinical signs and symptoms of illness) dogs and cats.Sample processing and enterococcal isolation were carried out as described previously [20] using buffered peptone water and Enterococcus agar media (Becton, Dickinson, Sparks, MD, USA).Polymerase chain reaction (PCR) and matrix-assisted laser desorption ionization time-of-flight mass spectroscopy (MALDI-TOF, Biomerieux, Marcy-I'Etoile, France) were used to identify Enterococcus species [21].A total of 352 E. faecium isolates (298 isolates from dogs and 54 from cats) and 573 E. faecalis isolates (448 isolates from dogs and 125 from cats) were collected from eight laboratories/centers that took part in the Korean Veterinary Antimicrobial Resistance Monitoring System (KVARMS) during 2020-2022 (Table S1).In general, the isolates were collected in proportion to the number of different cities in South Korea (Table S2).However, the authors do not have information about the history of antimicrobial use in dogs and cats and the number of samples considered for this study.

Statistical Analysis
The rates of antimicrobial resistance and Pearson correlation were analyzed using Excel (Microsoft-Excel, 2016, Microsoft Corporation, Redmond, WA, USA) and Rex Software (Version 3.0.3,ResSoft Inc., Seoul, Republic of Korea), in which the independent samples t-test, Fisher's exact test, and chi-square test were incorporated.p values < 0.05 were considered statistically significant.

Antimicrobial Resistance
The resistance rates of E. faecium and E. faecalis isolates to the tested antimicrobials are displayed in Tables 1, S7 and S8.Among them, the highest tetracycline resistance (>65%) was observed in the E. faecalis isolates recovered from dogs and cats.Moreover, resistance to tetracycline (34.9 and 67.4%) and erythromycin (39.5 and 41.7%) was high in both E. faecium and E. faecalis, although the resistance rates differed by bacterial species.Resistance to ciprofloxacin (41.8%) and kanamycin (32.5%) was high in E. faecium and E. faecalis, respectively.Ampicillin resistance in E. faecium was observed in both dogs and cats, with levels >15%, but no ampicillin resistance was observed for E. faecalis.Moreover, no or only low resistance to daptomycin, linezolid, tigecycline, and vancomycin was observed in both E. faecium and E. faecalis.
The overall resistance rates toward the tested antimicrobials in both E. faecium and E. faecalis isolated from dogs and cats were not significantly different, except for the rate for chloramphenicol.Resistance to chloramphenicol was markedly higher in cats (31.2%) than in dogs (16.7%) (p < 0.05).In addition, ciprofloxacin, gentamicin, and kanamycin resistance rates were slightly high in both E. faecium and E. faecalis in cats.The resistance rates of erythromycin (40.3% vs. 35.2%)and tetracycline (35.6% vs. 31.5%) in E. faecium were higher in dog isolates than in cat isolates.It has been observed that E. faecium isolates exhibit relatively low resistance to chloramphenicol (0.0-2.3%), daptomycin (1.7-3.7%), and florfenicol (0.0-1.3%).Indeed, compared to cat isolates, dog isolates showed higher rates of chloramphenicol (0.0% vs. 2.3%) and florfenicol (0.0% vs. 1.3%) resistance.The MIC 50 and MIC 90 values of the investigated antimicrobials against E. faecium isolates obtained from dogs and cats are presented in Supplementary Tables S3 and S4.
The antimicrobial resistance among the E. faecium and E. faecalis isolates from dogs and cats varied with their ages (Tables 2 and 3).Overall, the resistance rate in E. faecium from geriatric dogs (≥11 years) was higher compared with those rates obtained from the puppy and juvenile, mature adult, and senior age groups.It was found that 50% of the E. faecium isolates from elderly dogs were resistant to ciprofloxacin.Moreover, the resistance rates toward kanamycin (26.2%), streptomycin (28.6%), and tylosin (26.2%) were significantly higher than those toward other antimicrobials in this age group compared to other age groups.In cats, about 25% of the E. feacium isolates were shown to be resistant to the tested antimicrobials.Overall resistance was similar in the kitten and junior as well as adult groups, although resistance to ampicillin and ciprofloxacin was slightly higher in the junior and adult group (Table 2).In E. faecalis, there was no difference between the age groups of the dogs.Resistance to erythromycin, tetracycline, and tylosin was high in all age groups.In cats, resistance to ciprofloxacin (11.3% vs. 0-5%), florfenicol (15.1% vs. 0-5.0%),and streptomycin (30.2% vs. 16.7-20.0%)in kittens was higher than in other age groups.Furthermore, resistance to chloramphenicol, gentamicin, kanamycin, and tetracycline was much higher in the senior group, although the number of resistant isolates was lower (Table 3).

Antimicrobial Resistance Patterns
Most E. faecium (76.4%) and E. faecalis (69.8%) isolates were resistant to at least one of the tested antimicrobials (Tables 2 and 3).In particular, 36.1% of the E. faecalis and 21% of the E. faecium isolates exhibited MDR (Table 1).MDR was higher in E. faecalis recovered from cats (44%) and dogs (33.9%) than in E. faecium isolated from cats (24.1%) and dogs (20.5%).Additionally, E. faecalis isolates from dogs (27.2%) and cats (35.2%) were observed to be resistant to five or more antimicrobials.However, E. faecium from dogs (13.4%) and cats (14.8%) were resistant to five or more antimicrobials.

Discussion
In this investigation, significant proportions of the E. faecium and E. faecalis isolates recovered from healthy dogs and cats were resistant to one or more antimicrobials.This result could provide a potential strategy for combatting antimicrobial resistance in veterinary and human clinical practice settings.
Enterococcus spp.are frequently shown to be resistant to antimicrobials in companion animals worldwide [26,27].In this study, a considerable portion of enterococci isolates demonstrated resistance to erythromycin, tetracycline, and tylosin.High erythromycin resistance in E. faecium and E. faecalis isolated from dogs and cats was reported in Korea [15][16][17], China [28], Turkey [29], Belgium [30], Italy [31], and the USA [32], constituting results that concur with our findings.In our study, we found high tetracycline resistance rates in E. faecium (34.9%) and E. faecalis (67.4%) isolates from dogs and cats.A previous study in Denmark showed tetracycline resistance was high in E. faecalis (65%) and E. faecium (60%) isolates from dogs [33].Moreover, consistent with this investigation, high tylosin resistance (41.4%) was described in E. faecalis isolated from companion animals in Belgium [26] and the USA [34].Additionally, the ciprofloxacin resistance (41.8%) in E. faecium isolates from dogs and cats was similar to that published in investigations concerning South Korea [18] and Portugal [35].
In this investigation, E. faecium specimens isolated from dogs (2%) and cats (7.7%) exhibited relatively low resistance rates to chloramphenicol and gentamicin.However, the chloramphenicol and gentamicin resistance in E. faecalis specimens isolated from dogs (16.7 and 17.9%) and cats (31.2 and 28%) was higher but still differed from previously published reports.Jackson et al. [34] reported higher chloramphenicol (85%) and gentamicin (79%) resistance in E. faecalis from dog isolates.Nonetheless, the chloramphenicol (5%) and gentamicin (8%) resistance rates were lower in E. faecalis specimens from cat isolates reported in the study by De Graef et al. [36].Moreover, variable levels of chloramphenicol resistance were found in E. faecium (17-61%) and E. faecalis (9-85%) isolated from dogs and cats in several studies conducted in Asia [16,37] and Europe [38,39].Despite the fact that chloramphenicol is no longer permitted for use in veterinary practice, the rise of chloramphenicol-resistant Enterococcus may be linked to the use of other phenicols, such as florfenicol, or to co-selection with unrelated antimicrobials [40].Moreover, the recurrent use of gentamicin in companion animals might contribute to the resistance of this antimicrobial [41].
E. faecium isolates obtained from dogs and cats exhibited comparatively low resistance to the tested aminoglycosides (kanamycin and streptomycin).However, the resistance rates of these antimicrobials were found to be high in E. faecalis isolated from dogs and cats.The E. faecalis isolates from dogs (31.5 and 18.8%) and cats (36 and 24%) were resistant to kanamycin and streptomycin, respectively.Highly varied kanamycin (30-87%) and streptomycin (25-95%) resistance rates have been observed in E. faecium and E. faecalis isolated from companion animals in previous investigations conducted in Korea [17], Portugal [35], Belgium [30], and South Africa [13].Notwithstanding the fact that enterococci have clinically relevant concentrations, they are the antimicrobials of choice for human enterococcal infections when used with synergic antimicrobials.Consequently, resistance to one or more aminoglycoside antimicrobials might be conferred by aminoglycosidemodifying enzymes acting on various aminoglycoside antimicrobials [42].
Penicillin resistance may limit the range of available treatment options for enterococcal infections [43].Although no ampicillin resistance was detected in E. faecalis, 17.3% of the E. faecium specimens isolated from cats and dogs showed resistance to this antimicrobial.This result is consistent with previous findings that E. faecium from dog isolates exhibited resistance in the UK (23%) [44] and Egypt (14.3%) [45].On the contrary, it has been found that E. faecium from companion animals in Denmark (76%) [46] and Italy (56.5%) [39] demonstrated relatively high ampicillin resistance.Ampicillin is one of the most effective β-lactams against Enterococcus, preventing peptidoglycan production [47].Moreover, the intrinsic tolerance of β-lactamase action in E. faecalis may be linked to specific penicillin-binding proteins such as pbp5, which has a low affinity for ampicillin [48].Likewise, it has been demonstrated that ampicillin resistance in E. faecalis is conferred by point mutations in the penicillin-binding protein (pbp4) and the overexpression of β-lactamase [49].
We observed high erythromycin (39.5 and 41.7%) and tylosin (14.5 and 41.4%) resistance in E. faecium and E. faecalis isolates from both dogs and cats.Moreover, it was found that there was a gradually increasing tendency toward erythromycin resistance in both of the enterococci strains isolated from dogs and cats throughout the study period.This result is supported by the fact that a significant portion of E. faecium and E. faecalis isolates from companion animals, particularly dogs and cats, in many countries [7,13,30,39,50] showed resistance to erythromycin (26-96%) and tylosin (21-42%).The increased proportion of isolates resistant to macrolides may also be caused by cross-resistance between erythromycin and tylosin.Additionally, previous studies showed that multidrug-resistant E. faecalis from companion animals possesses erm(B) and tlrA genes, suggesting the spread of macrolide resistance genes among the enterococci isolates [39,51].
In this study, the resistance rates toward quinupristin/dalfopristin in E. faecium isolates from dogs and cats were 7.4% and 9.3%, respectively.The incidence of quinupristin/dalfopristin resistance in E. faecium isolated from dogs was much lower than that previously reported in China (38.04%) [28], Italy (78.4%) [52], and the USA (28.6%) [53] but was consistent with that published in Korea (5%) [16].Although the administration of quinupristin/dalfopristin to animals is not permitted in Korea, the use of streptogramin and virginiamycin that exhibit quinupristin/dalfopristin cross-resistance may be connected to the development of quinupristin/dalfopristin resistance in E. faecium [54].The incidence of quinupristin/dalfopristin resistance Enterococcus in companion animals has the potential to contribute to the occurrence of human infections through frequent and direct interactions.
In our study, we also tested the antibiotic susceptibility of four antimicrobials: daptomycin, linezolid, tigecycline, and vancomycin.These antimicrobials belong to four distinct classes and are not currently approved for veterinary use in Korea [20].In accordance with previous studies conducted in Korea [17] and other countries [55,56], the prevalence of daptomycin resistance is low among the enterococci isolates obtained from companion animals.The infrequency of daptomycin resistance among enterococci may be attributed to the emergence of resistance to spontaneous mutations in EF0631 genes [57].Numerous previous studies have documented tigecycline, vancomycin, and salinomycin resistance in E. faecium and E. faecalis strains obtained from companion animals [52,53,56,58]; however, in our present investigation, all the enterococcal isolates exhibited susceptibility to these antimicrobials.In general, enterococcal isolates demonstrating resistance to clinically significant antimicrobials have the potential to be transmitted directly or indirectly to humans, posing a critical clinical challenge for the treatment of multidrug-resistant infections.
The prevalence of antimicrobial-resistant E. faecium and E. faecalis isolates among dogs and cats with respect to age variation was assessed to elucidate the emergence of antimicrobial resistance, which could help veterinarians in identifying infections and developing treatment strategies and preventive measures for different age groups of companion animals.In our study, it was observed that most of the antimicrobial resistance in the E. faecium isolates from dogs and cats increased with age.The prevalence of resistance in the E. faecium isolates obtained from geriatric dogs (aged ≥11 years) was higher than that isolated from the puppy and juvenile, mature adult, and senior age groups.Additionally, the isolates from the mature group of cats (aged 7-10 years) showed significantly higher rates of resistance to erythromycin, gentamicin, and tetracycline than those from other age groups.The resistance rate toward the tested antimicrobials in E. faecalis was higher in the puppy and juvenile groups compared to the mature adult, senior, and geriatric groups of dogs.Furthermore, it was found that the rate of resistance to the antimicrobials examined was significantly higher in the senior group (aged ≥11 years) of cats compared to the kitten, junior, and mature groups.Consistent with the findings of a previous study, this investigation demonstrated a high prevalence of antimicrobial resistance in isolates obtained from relatively older companion animals [59].This propensity for antimicrobial resistance in relatively older companion animals might be due to their exposure to more treatment medications throughout their lives [60].However, previously published reports still showed mixed antimicrobial resistance dynamics in isolates obtained from animals of varying ages [16].
Most of the E. faecium and E. faecalis isolates exhibited resistance to at least one antimicrobial, with several resistance patterns observed in both species.The rates of multidrug resistance (MDR) in the E. faecium (21%) and E. faecalis (36.1%) strains from dogs and cats were found in this study.A previous report showed a similar MDR phenotype observed in E. faecium (33.3%) isolated from companion animals in the USA [53].However, MDR in E. faecalis isolates obtained from dogs and cats was reported to be higher in China (52.9%) [55] and Korea (71%) [17].Moreover, MDR in E. faecium (97.9%) was found to be much higher in cats and dogs in a previous study conducted Ma et al. [55].
In accordance with the findings reported by Miranda et al. [61], it was seen that MDR patterns often encompass the presence of erythromycin, tetracycline, and ciprofloxacin.Moreover, it was found that E. faecalis isolates obtained from dogs (43.3%) and cats (81.5%) exhibited resistance to five or more antimicrobials, consistent with previously published reports from Korea [16], Turkey [41], and the USA [53].The tendency of Enterococcus spp. to participate in different types of conjugation may be linked to the presence of MDR, leading to the extensive spread of resistance determinants via plasmids [62].Moreover, the survivability of MDR enterococci may also trigger their widespread clonal and zoonotic transmission, leaving limited options for treating bacterial infections in humans and animals [63].

Conclusions
Our study revealed that enterococcal isolates recovered from healthy dogs and cats demonstrated resistance to multiple antimicrobial agents, including those deemed crucial for human health.The prevalence of multidrug-resistant E. faecium and E. faecalis in companion animals is concerning, particularly considering the limited availability of effective antimicrobials for treating enterococcal infections.This resistance can potentially be transmitted to humans as companion animals share a common habitat with humans and are treated for infections using similar medications.Hence, the judicious selection of antimicrobials and the continuous surveillance of antimicrobial resistance in companion animals may play pivotal roles in mitigating the human health risk associated with Enterococcus.In addition, resistance mechanisms and whole-genome sequencing analysis of E. faecium and E. faecalis isolates also remain to be further conducted to address the risk of transmission from dogs and cats to humans.

Supplementary Materials:
The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/microorganisms11122991/s1,Table S1: Enterococcus faecium and Enterococcus faecalis isolates recovered from healthy dogs and cats during 2020-2022 in South Korea; Table S2: Enterococcus faecium and Enterococcus faecalis isolate collection cities in South Korea; Table S3: The MIC 50 and MIC 90 of the tested antimicrobials against E. faecium (n = 298) isolated from healthy dogs during 2020-2022 in South Korea; Table S4: The MIC 50 and MIC 90 of the tested antimicrobials against E. faecium (n = 54) isolated from healthy cats during 2020-2022 in South Korea; Table S5: The MIC 50 and MIC 90 of the tested antimicrobials against E. faecalis (n = 448) isolated from healthy dogs during 2020-2022 in South Korea; Table S6: The MIC 50 and MIC 90 of the tested antimicrobials against E. faecalis (n =125) isolated from healthy cats during 2020-2022 in South Korea; Table S7: Antimicrobial resistance rate in Enterococcus faecium isolated from healthy dogs (n = 298) and cats (n = 54) during 2020-2022 in South Korea; Table S8: Antimicrobial resistance rate in Enterococcus faecalis isolated from healthy dogs (n = 448) and cats (n = 125) during 2020-2022 in South Korea;

Table 2 .
Distribution of antimicrobial-resistant Enterococcus faecium among different age groups of healthy dogs and cats isolated during 2020-2022 in South Korea.p < 0.05 compared with resistance rates in mature adult dogs; # p < 0.05 compared with resistance rates in geriatric dogs; † p < 0.05 compared with resistance rates in junior and adult cats; ∆ p < 0.05 compared with resistance rates in mature cats.MDR, multidrug resistance. *

Table 3 .
Distribution of antimicrobial-resistant Enterococcus faecalis isolates among different age groups of healthy dogs and cats isolated during 2020-2022 in South Korea.
p < 0.05 compared with resistance rates in junior and adult cats; # p < 0.05 compared with resistance rates in mature cats.† p < 0.05 compared with resistance rates in senior cats.MDR, multidrug resistance; ND, none-defined. *

Table 4 .
Frequent resistance patterns of Enterococcus faecium specimens isolated from healthy dogs and cats during 2020-2022 in South Korea.

Table 5 .
Frequent resistance patterns of Enterococcus faecalis isolated from healthy dogs and cats during 2020-2022 in South Korea.