Characteristics of High-Level Aminoglycoside-Resistant Enterococcus faecalis Isolated from Bulk Tank Milk in Korea

Simple Summary Aminoglycosides are used to treat various infections in veterinary and human medicine. However, with the emergence of high-level aminoglycoside resistance in human and food-producing animals, the synergism of aminoglycosides with beta-lactam or glycopeptide is being threatened. Moreover, the environmental mastitis-causing agent, enterococci, has emerged as a cause of nosocomial infection due to its antimicrobial resistance. Therefore, the purpose of this study was to investigate the characteristics of high-level aminoglycoside-resistant Enterococcus faecalis isolated from bulk tank milk in Korea. It showed that 185 (61.5%) isolates out of 301 were high-level aminoglycoside resistant, while 149 isolates were multidrug resistant. Abstract Enterococci, which are considered environmental mastitis-causing pathogens, have easily acquired aminoglycoside-resistant genes that encode various aminoglycoside-modifying enzymes (AME). Therefore, this study was conducted to compare the distribution of high-level aminoglycoside-resistant (HLAR) and multidrug-resistant (MDR) Enterococcus faecalis (E. faecalis) bacteria isolated from bulk tank milk in four dairy companies in Korea. Moreover, it analyzed the characteristics of their antimicrobial resistance genes and virulence factors. Among the 301 E. faecalis bacteria studied, 185 (61.5%) showed HLAR with no significant differences among the dairy companies. Furthermore, 129 (69.7%) of the 185 HLAR E. faecalis showed MDR without significant differences among companies. In contrast, HLAR E. faecalis from companies A, B, and C were significantly higher in resistance to the four classes than those in company D, which had the highest MDR ability against the three antimicrobial classes (p < 0.05). In addition, in the distribution of AME genes, 72 (38.9%) and 36 (19.5%) of the isolates carried both aac(6′)Ie-aph(2″)-la and ant(6)-Ia genes, and the ant (6)-Ia gene alone, respectively, with significant differences among the companies (p < 0.05). In the distribution of virulence genes, the ace (99.5%), efa A (98.9%), and cad 1 (98.4%) genes were significantly prevalent (p < 0.05). Thus, our results support that an advanced management program by companies is required to minimize the dissemination of antimicrobial resistance and virulence factors.


Introduction
Aminoglycosides are antimicrobials, including gentamicin, streptomycin, and kanamycin, that inhibit bacterial protein synthesis [1]. In particular, aminoglycosides are used in the treatment of aerobic Gram-negative bacilli infections, and with broadspectrum beta-lactam for severe infections [2]. However, the emergence of resistance to aminoglycosides has continuously been reported among isolates from humans and foodproducing animals. This resistance has also been associated with exposure to the commonly used agents [3,4].
Enterococci have increasingly emerged as a cause of serious nosocomial infection in humans and have also been considered environmental mastitis-causing pathogens in veterinary medicine [5,6]. Although synergic combinations of penicillin or a glycopeptide with an aminoglycoside have been used for treating such infections, enterococci have easily acquired aminoglycoside-resistant genes that encode various aminoglycoside-modifying enzymes (AME). These acquired genes cause high resistance to aminoglycosides [7]. In particular, high-level resistance to aminoglycosides can abolish the synergic effect between commercially available aminoglycosides and cell-wall active agents, such as beta-lactams or glycopeptides [8].
Although high-level aminoglycoside-resistant (HLAR) enterococci were first reported in the 1980s in humans [9], and have been described in several studies investigating antimicrobial resistance profiles in raw milk or dairy products worldwide [10,11], there have been no comprehensive surveys to date on the characteristics of HLAR enterococci obtained from raw milk or dairy products in Korea. Hence, this study was conducted to compare the distribution of HLAR and multidrug-resistant (MDR) Enterococcus faecalis (E. faecalis) isolated from bulk tank milk in four major dairy companies in Korea. Moreover, it analyzed the characteristics of the antimicrobial resistance genes and virulence factors of the bacterial strains of interest.

Bacterial Isolation
A total of 1584 batches of bulk tank milk samples from 395 farms belonging to four dairy companies in Korea were collected twice during the summer and winter each (July-December 2019). Then, 50 mL of milk samples were aseptically collected from each bulk sample and sent to the laboratory at 4 • C. For the isolation and identification of E. faecalis, 1 mL of the milk sample was cultured in 9 mL buffered peptone water (BPW; BD Biosciences, San Jose, CA, USA); the pre-enriched BPW was then mixed with an Enterococcosel broth (BD Biosciences) at a 1:10 ratio, and incubated at 37 • C for 18-24 h. Furthermore, each medium was streaked onto an Enterococcosel agar (BD Biosciences), and confirmation of E. faecalis was performed using PCR, as described previously [12]. Among the isolates showing the same antimicrobial susceptibility patterns from the same origin, only one isolate was chosen for this study. As a result, E. faecalis isolates were tested.

Detection of HLAR Enterococci
The standard agar dilution method conducted on brain heart infusion agar was used to determine the minimum inhibitory concentration values for G and S, with a concentration range of 256-2048 µg/mL (serial 2-fold dilutions). Moreover, breakpoints for high-level G and S were set at ≥500 and ≥2000 µg/mL, respectively, following the CLSI guidelines (CLSI, 2019) [13].

Statistical Analysis
The Statistical Package for Social Science version 25 (IBM SPSS Statistics for Windows, Armonk, NY, USA) was used for statistical analysis. Further, Pearson's chi-square test and Fisher's exact test with Bonferroni correction were used to compare the prevalence of isolates between companies [25]. A p value < 0.05 was considered statistically significant.

Prevalence of MDR and HLAR E. faecalis
The distribution of MDR and HLAR in E. faecalis from the bulk tank milk of four dairy companies is presented in Table 2. Among the 301 E. faecalis isolates studied, 149 (49.5%) and 185 (61.5%) showed MDR and HLAR, respectively. Moreover, although company D showed the highest prevalence of E. faecalis, the prevalence of MDR E. faecalis was significantly higher in isolates from company A (61.5%) (p < 0.05). However, the prevalence of HLAR E. faecalis showed no significant difference among the four dairy companies.

Antimicrobial Resistance of HLAR E. faecalis
The distribution of resistance against nine antimicrobial agents of 185 HLAR E. faecalis is presented in Table 3. The results showed that the significantly highest resistance was against TE (93.5%), followed by E (71.9%), then DOX (70.8%). In particular, resistance to DOX also showed significant differences among the dairy companies (p < 0.05). However, resistance to AM, CIP, P, RA, and VA was only 0% to 5.9%. Table 3. Antimicrobial resistance of 185 high-level aminoglycoside-resistant Enterococcus faecalis isolates from the bulk tank milk of four dairy companies. Values with different subscript letters ( a-c ) represent significant differences among farms, while superscript letters ( A-E ) represent the total significant difference (p < 0.05).

Distribution of MDR Patterns
The distribution of MDR isolates among 185 HLAR E. faecalis is presented in Figure 1. Although the prevalence of MDR (129 isolates, 69.7%) in HLAR E. faecalis showed no significant differences among the dairy companies, HLAR E. faecalis from company A showed the highest MDR (80.6%), followed by company D (73.2%), C (62.9%), and B (60.7%), respectively. Likewise, all MDR isolates showed resistance against three to five antimicrobial classes. In particular, HLAR E. faecalis from companies A, B, and C were significantly higher in resistance against the four classes than company D, which showed the highest MDR against only three of the antimicrobial classes (p < 0.05). Furthermore, MDR to five classes was observed only in HLAR E. faecalis from companies A (5.6%) and C (3.7%).

Distribution of Antimicrobial Resistance Genes
The distributions of resistance genes in 185 HLAR E. faecalis are presented in Table 4. In the distribution of AME genes, 72 (38.9%) and 36 (19.5%) isolates, respectively, expressed both aac (6 )Ie-aph(2 )-la and ant (6)-Ia genes, and the ant (6)-Ia gene alone, with significant differences among the dairy companies (p < 0.05). Likewise, for the E resistance genes, the erm B gene (71.4%) among the three genes had the highest prevalence (p < 0.05), although no significant difference among the dairy companies was observed. Moreover, in the tetracycline resistance genes, the prevalence of both tet M and tet L genes (46.5%), as well as the tet M gene (36.3%) alone, had the highest prevalence with significant differences among the studied dairy companies (p < 0.05). Furthermore, the cat A and cfr genes related to resistance to C were observed among 27 (14.6%) and two (1.1%) isolates, respectively. In contrast, the optr A and poxt A genes related to resistance to linezolid were observed only in two (1.1%) isolates and one (0.5%) isolate, respectively. Table 4. Distribution of antimicrobial resistance genes of 185 high-level aminoglycoside-resistant Enterococcus faecalis from the bulk tank milk of four dairy companies. Values with different subscript letters ( a,b ) represent significant differences among farms, while superscript letters ( A-D ) represent the total significant difference (p < 0.05).

Discussion
In Korea, five major dairy companies produce 84% of the total milk and dairy products consumed. Confinement housing is used to run most of the farms, which is a primary management system of dairy production [26]. Washburn et al., (2002) [27] reported that confined cows had 1.8 times more clinical mastitis compared with cows on pasture. Therefore, various antimicrobials have been used for treating mastitis every year in Korea [28]. In particular, aminoglycosides, which are used along with cell-wall active agents, are effective for treating serious enterococcus infection [29]. However, enterococci showing high resistance to aminoglycosides has been reported continuously in food-producing animals [30][31][32]. In this study, 61.5% of the 301 E. faecalis isolates from bulk tank milk were HLAR, although no significant difference among the four companies was shown. Chajęcka-Wierzchowska et al., (2020) [10] reported that 30.2% of the ready-to-eat dairy products in Poland showed HLAR to enterococci. Özdemir and Tuncer (2020) [33] also reported that 59 HLAR enterococci were observed in 100 samples of milk and dairy in Turkey. Furthermore, the high prevalence of HLAR enterococci in Korea compared with that in Poland and Turkey is indirect proof of the consistent use of aminoglycosides to treat bacterial infection in Korea.
Interestingly, 69.7% of the HLAR isolates showed MDR in this study. Hegstad et al. (2010) [34] reported that enterococci have a common feature of easily transferring DNA via plasmid or transposons encoding resistance genes. Therefore, the capacity of transference to other bacteria by these plasmids or transposons leads to the spread of various resistance strains, which ultimately results in MDR. The distribution of MDR patterns in HLAR E. faecalis showed significant differences among the companies in this study. Likewise, HLAR E. faecalis from companies A, B, and C showed the highest prevalence in MDR against the four classes. In contrast, isolates from company D showed the highest prevalence in the three classes. MDR of five classes was observed only in HLAR E. faecalis from companies A and C. These results suggest that the critical point for reducing the emergence of resistant bacteria is the management of which and how dairy companies use antimicrobials.
Hollenbeck and Rice (2012) [35] reported that all enterococci possess intrinsic lowlevel resistance to all aminoglycosides by limiting the uptake of drugs, and this resistance originated from their facultative anaerobic metabolism. However, genes encoding diverse AME acquire high-level resistance to aminoglycosides of enterococci. Thus, it eliminates the synergism of aminoglycosides with cell-wall synthesis interfering agents, such as βlactams [36][37][38]. In this study, the combination of both aac (6 )Ie-aph(2 )-la and ant (6)-Ia genes was significantly prevalent (p < 0.05). The ant (6)-Ia gene is responsible for encoding the ANT (O-adenyltransferase) enzyme that the catalyzes ATP-dependent adenylation of a hydroxyl group, and grant resistance to streptomycin without cross-resistance to other aminoglycosides [39]. In contrast, aac (6 )Ie-aph(2 )-la encodes the bifunctional enzymes AAC (N-Acetyltransferase) and APH (O-Phosphotransferase), which are responsible for resistance to all types of aminoglycosides, except streptomycin and spectinomycin [40]. Therefore, the high prevalence of HLAR in this study can be related to the distribution of these genes.
In this study, HLAR E. faecalis showed the highest resistance to TE (93.5%), followed by E (71.9%). The most prevalent of the different types of antimicrobial resistance genes were tet M (82.7%), including a combination of tet M and tet L, which is related to resistance to tetracyclines, and erm B (71.4%), which has shown resistance to macrolides. Although the high distribution of these genes has been continuously reported in enterococci isolated from humans and food-producing animals [41,42], it is important that the erm B and tet (M) genes can also be transferred easily by conjugative transposons, such as the Tn916/1545 and Tn5397 families [43]. Therefore, the horizontal transfer of these genes in enterococci should be continuously monitored in the future. In Korea, tetracyclines as feed additives have been banned since 2009, but a large amount of chlortetracycline calcium, chlortetracycline HCL, oxytetracycline dehydrate, and oxytetracycline HCL have still been used for treating mastitis [44]. Furthermore, E is rarely used in the dairy industry [25,44], but resistance to E is linked to the use of tylosin, which is a macrolide, and is used widely for treating streptococcal mastitis [45]. Therefore, our results support that acquiring resistance genes in food-producing animals was induced by the use of antimicrobials, which also contribute to the burden of growing antimicrobial resistance in humans.
Furthermore, due to the acquisition of virulence genes being directly related to the capability of bacteria to cause illness [46], monitoring virulence genes of enterococci is crucial regarding the public health concerns of dairy products. In this study, HLAR E. faecalis showed a high prevalence of genes such as ace (99.5%), efa A (98.9%), cad 1 (98.4%), gel E (85.9%), and asa 1 (61.6%). This result was in accordance with the high prevalence of virulence genes of E. faecalis from buffalo milk in Brazil [47] and E. faecalis from dairy products in Egypt [48]. Although the presence of virulence genes does not increase pathogenicity, virulence factors promote tissue colonization in the hosts. Moreover, the combination of antimicrobial resistance genes and virulence factors in enterococci, which are potential opportunistic pathogens regarding clinical or subclinical mastitis, could be a public health problem. Thus, in this study, the phenotypic and genotypic characteristics of HLAR E. faecalis showed significant differences among the dairy companies. However, an advanced management protocol by companies is warranted to minimize the dissemination of antimicrobial resistance and virulence factors.

Conclusions
In this comprehensive research on HLAR, E. faecalis isolated from the bulk tank milk of four dairy companies in Korea, the distribution of antimicrobial resistance in these bacteria and the genetic characteristics of HLAR E. faecalis showed a significant difference among the companies. Therefore, our results suggest that advanced management programs by companies are warranted to minimize the emergence of antimicrobial-resistant bacteria and to reduce the dissemination of these resistance genes and virulence factors.

Conflicts of Interest:
The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of the data; in the writing of the manuscript; or in the decision to publish the results.