Molecular Characteristics of Enterococcus faecalis and Enterococcus faecium from Bulk Tank Milk in Korea

Simple Summary Enterococci can be an opportunistic pathogen in milk, which can easily disseminate antimicrobial resistance and virulence genes. The purpose of this study was to characterize and compare the enterococci isolates from samples of bulk tank milk obtained from four dairy companies in Korea to prevent the spread of pathogenic and antimicrobial-resistant enterococci in dairy companies. The results demonstrated various degrees of antimicrobial resistance and virulence-factor distribution in enterococci from bulk tank milk in Korea and support the assessment that pathogens from bulk tank milk can also become a reservoir for dissemination of antimicrobial resistance and virulence factors through cross-contamination processes. Abstract Enterococci are considered to be environmental mastitis-causing pathogens that can easily spread antimicrobial resistance or virulence genes via horizontal transfer. In this study, the molecular characteristics of enterococci from bulk tank milk were investigated to assess the importance of dairy herd management. A total of 338 enterococci (305 Enterococcus faecalis and 33 Enterococcus faecium) were isolated from 1584 batches of bulk tank milk samples from 396 farms affiliated with four dairy companies in Korea, and significant differences (40.6–79.7%) (p < 0.05) in the prevalence of enterococci were observed in the samples from different companies. Enterococci showed the highest resistance to tetracycline (TET) (73.4%), followed by doxycycline (DOX) (49.7%) and erythromycin (ERY) (46.2%), while two enterococci isolates showed resistance to vancomycin (VAN). Among 146 tetracycline (TET) and ERY-resistant enterococci, each 50 (19.4%) enterococci carried combination-resistance and transposon gene types erm(B) + tet(M) + IntTn and erm(B) + tet(L) + tet(M) + IntTn, respectively. The virulence genes such as ace (99.0%), efaA (97.7%), cad1 (95.7%), and gelE (85.9%) were highly conserved in E. faecalis and significantly predominated over E. faecium (p < 0.001). Our results indicate that pathogens from bulk tank milk can also become a reservoir for the dissemination of antimicrobial resistance and virulence factors through cross-contamination processes.


Introduction
Enterococci are normal flora commonly found in the gastrointestinal tracts of humans and animals but are environmental mastitis-causing pathogens. In particular, Enterococcus faecalis (E. faecalis) and Enterococcus faecium (E. faecium) are the major species that account for approximately 90% of enterococci causing inframammary infection and are generally isolated from infected udders and dairy environments [1]. The udders of cattle are the primary reservoir of enterococci, and their pathogens can cause infections among quarters and cows during the milking process. The best way to treat mastitis is through the affected udder compartment, but the efficacy of antimicrobials can be limited due to the emergence of resistance against some antimicrobials [1].
Enterococci often acquire resistance by acquiring new genes with antimicrobial resistance on plasmids or transposons that can cross species and genera. Recently, resistance against tetracycline (TET) and erythromycin (ERY) has been reported in enterococci from many food-producing animals [2,3], and the presence of resistance genes against TET and ERY has been reported to be associated with resistance in general [3,4]. In the last few decades, clinical and subclinical mastitis have been successfully treated with TET and ERY, which are the most widely marketed antimicrobials in Korea [5], but increasing resistance to these antimicrobials from animal isolates continues to be reported [4][5][6][7].
The virulence of enterococci can enhance enterococcal infections and contribute to their potential pathogenesis [8]. Many researchers have reported that several virulence factors in enterococci may be involved in disease severity or exacerbation in humans and animals [8][9][10]. However, the direct relationship between virulence factors and the onset of diseases in cattle, such as clinical mastitis, have not been elucidated. Moreover, relatively little is known about the prevalence of virulence factors in enterococci from dairy products in Korea.
Antimicrobial resistance and virulence genes can be carried on mobile genetic elements [11]. Therefore, enterococci in milk can contribute to the spread of potentially pathogenic, antimicrobial-resistant strains to humans through food. The purpose of the present study was to compare the prevalence and distribution of antimicrobial resistance and virulence factors in enterococci isolates recovered from milk samples obtained from four dairy companies and to prevent the spread of pathogenic, antimicrobial-resistant enterococci in dairy companies.

Sample Collection and Bacterial Isolation
A total of 1584 batches of bulk tank milk samples were collected from 396 farms managed by four dairy companies in Korea. Milk samples were aseptically collected twice each in the summer and winter seasons. The isolation and identification of Enterococcus spp. were performed following the standard microbiological protocols published by the Ministry of Food and Drug Safety (Korea) (2018) [12]. Briefly, 1 mL of milk sample was cultured in 9 mL of buffered peptone water (BPW; BD Biosciences, San Jose, CA, USA). Then, pre-enriched BPW was mixed with enterococcosel broth (BD Biosciences) at a 1:10 ratio and incubated at 37 • C for 18-24 h. Each medium was streaked onto enterococcosel agar (BD Biosciences), and the formation of E. faecalis and E. faecium was confirmed using polymerase chain reaction (PCR) with specific primers such as ddl E. faecalis and ddl E. faecium as previously described [13].

Statistical Analysis
Statistical analyses were performed using SPSS version 25 (IBM Corp., Armonk, NY, USA). Analyses of the differences in enterococci prevalence, antimicrobial resistance, antimicrobial-resistant genes, and virulence genes in milk samples from different companies, were conducted using Chi-square tests. Differences were considered significant at p < 0.05.

Prevalence of Enterococci
The prevalence of enterococci in bulk tank milk samples from four dairy companies is shown in Figure 1. The prevalence of E. faecalis and E. faecium in samples from the four companies varied from 40.6 to 79.7%. Samples from company A had a significantly higher prevalence of E. faecalis and E. faecium than samples from the other companies (p < 0.05).

Distribution of Antimicrobial Resistance
The antimicrobial resistance of 338 isolates of enterococci (305 E. faecalis and 33 E. faecium) taken from samples of bulk tank milk from four dairy companies is shown in Table 1. Enterococci showed high rates of resistance to TET (73.4%), followed by DOX (49.7%) and ERY (46.2%). Moreover, resistance to CHL, CIP, ERY, TET, PEN, and VAN showed significant differences depending on the source company (p < 0.05). In particular, enterococci isolates from samples of bulk tank milk from company C showed significantly higher rates of resistance to C (33 isolates, 56.9%), ERY (41 isolates, 70.7%), and TET (51 isolates, 87.9%) than isolates from the samples from other companies (p < 0.05); two enterococci isolates from samples from company B showed resistance to VAN.

Distribution of MDR Isolates
The distribution of 164 MDR enterococci in samples of bulk tank milk from four dairy companies is shown in Figure 2. The prevalence of MDR was also significantly different depending on the source company (p < 0.05). In particular, MDR (70.4%) in enterococci isolated from samples obtained from company C showed a significantly higher rate than isolates from the samples obtained from other companies (p < 0.05). Although one isolate of enterococci (0.8%) from company A showed MDR against six antimicrobial classes, 45 isolates (34.1%) and 19 isolates (20.0%) from companies A and D, respectively, showed the highest MDR against three classes. Moreover, 14 isolates (26.4%) and 23 isolates (39.7%) from companies B and C, respectively, showed the highest MDR against four classes.

Distribution of Antimicrobial Resistance and Transposon Genes in TET-and ERY-Resistant Enterococci
The distribution of resistance and transposon genes in 146 TET-and ERY-resistant enterococci isolated from samples of bulk tank milk from four dairy companies is shown in Table 2. Although one isolate of TET-and ERY-resistant enterococci showed none of the genes, 145 (99.3%) enterococci isolates carried various types of resistance genes. In particular, the most prevalent types, erm(B) + tet(M) + IntTn (50 isolates, 19.4%), showed significant predominance in samples from companies A and C, while erm(B) + tet(L) + tet(M) + IntTn (50 isolates, 19.4%) showed significant prevalence in samples from company D (p < 0.05). Table 2. Distribution of antimicrobial resistance genes and transposon genes in tetracycline-and erythromycin-resistant Enterococcus faecalis and Enterococcus faecium in bulk tank milk from four dairy companies.

No. (%) of Isolates with Antimicrobial Resistance Gene(s) and Transposon
Gene (

Discussion
Enterococci can be an opportunistic pathogen in milk that leads to clinical or subclinical mastitis [19]. Since enterococci can easily spread antimicrobial-resistance or virulence genes via horizontal transfer, the presence of enterococci in milk can enhance the emergence of MDR strains, which can eventually affect the choice of drug [19]. In this study, the prevalence of E. faecalis and E. faecium differed significantly in bulk tank milk samples from four dairy companies. The presence of environmental pathogens such as enterococci in bulk tank milk is associated with poor hygiene. Good hygienic practices in dairy livestock management are important in reducing udder contamination from microbial sources such as the environment, feces, and slurry [20]. In this study, enterococci showed a high rate of resistance to the tetracyclines TET and DOX and the macrolide ERY. Although TET has been banned as a feed additive to reduce antimicrobial resistance in Korea since 2009, tetracyclines such as chlortetracycline calcium, chlortetracycline HCL, oxytetracycline dihydrate, and oxytetracycline HCL continue to be used for the treatment of bovine mastitis and are reported to be the most heavily used antimicrobial agents in dairy fields in Korea [5]. Although ERY is rarely marketed to the Korean dairy industry, the emergence of resistance to ERY in Korea has been previously reported [5,21] and is likely linked to other macrolides such as tylosin. Tylosin is widely used for the treatment of diseases such as streptococcal mastitis in Korea [19]. Moreover, enterococci in samples from company C showed significantly higher MDR, including resistance to TET and ERY, than enterococci isolates in samples from other companies, although their prevalence was the lowest among the samples from all four dairy companies. The results may reflect the use of antimicrobial treatments by dairy companies. Given that misused and overused antimicrobials can contribute to the emergence of antimicrobial-resistant strains and MDR development in the milk production system, further investigation to clarify the association will be needed.
In this study, one E. faecalis and one E. faecium isolate showed resistance to VAN. Although the appearance of vancomycin-resistant enterococci (VRE) in Korea was first reported from hospital infections in 1992 [22], VRE continues to be reported in environmental samples such as sewage, animal waste, and meat and milk products worldwide [23][24][25][26]. VAN is considered to be one of the last resorts for treatment of gram-positive bacterial infections in humans [27] but can be easily transferred through plasmids or transposons, acquiring resistance genes that enable bacteria to block cell-wall formation [28]. The persistence of VRE in milk products is considered to play an important role in human colonization and infection and should be continuously monitored in the dairy production system. CHL-resistant enterococci usually presented with co-resistance to other antimicrobials such as linezolid or ERY because of resistance genes located adjacent to other antimicrobialresistant genes in the same plasmid [29,30]. In this study, the prevalence of CHL-resistant enterococci was 25.4%; the high rate of ERY resistance (46.2%) may be associated with the genetic environment sharing similar antimicrobial-resistant genes.
In particular, RIF-resistant enterococci were detected in samples from all four companies (71 isolates, 21.0%). RIF is commonly used in combination with other antimicrobials for synergetic efficacy in reducing pathogens that remain during antimicrobial therapy [31]. Thus, RIF should be used with caution as it can limit treatment options for MDR pathogens [31].
Many researchers have also reported that genes associated with ERY and TET resistance are easily transferred via conjugative transposons [2,4,17]. In this study, the presence of TET-resistance genes (tet(L), tet(M), and tet(O)), macrolide-resistance genes (erm(A), erm(B), and mef ), and transposon genes (Int-Tn and tndX) were simultaneously compared. The tet(L) and tet(M) genes were more prevalent than the tet(O) gene, while the erm(B) gene was more prevalent than erm(A). The tet(L), tet(M), and erm(B) genes have been reported to be the genes most frequently found in enterococci isolates from humans and animals [3,18]. However, the distribution of combination gene types was significantly different depending on the company from which the samples were sourced. The combination of erm(B), tet(M), and IntTn genes, which is one of the two most prevalent types, showed significant predominance in samples from companies A and C, while the combination of erm(B), tet(L), tet(M), and IntTn genes, which is other of the two most prevalent types, showed significant prevalence in samples from company D. The simultaneous presence of combined erm(B), tet(M), and IntTn genes and combined tet(L), tet(M), and IntTn genes has already been reported in E. faecalis in chickens in Korea [2,4]. Isolates with transposon family Tn916 (IntTn) have been previously reported; they harbor antimicrobial-resistance genes such as tet and erm on active mobile genetic elements facilitating gene transfer by conjugation, which can lead to multi-antimicrobial resistance [32]. In this study, isolates from samples obtained from three of the four dairy companies showed a significantly high prevalence of the transposon gene, IntTn, and other antimicrobial-resistance genes, which is problematic. Thus, the distribution of antimicrobial-resistance and transposon genes should be simultaneously monitored, and continuous surveillance will be important for the prevention of the emergence of MDR strains in the dairy industry.
The virulence factors of enterococci can contribute to the severity of pathogenesis. In this study, the virulence genes such as ace (99.0%), efaA (97.7%), cad1 (95.7%), gelE (85.9%), and asa1 (53.1%) were highly conserved in E. faecalis. Yang et al. (2019) also reported similar results with E. faecalis isolates from mastitic milk samples in China [3]. The collagen-binding protein, ace, promotes collagen binding in the extracellular matrix. The endocarditis-specific antigen, efaA, is associated with biofilm production. These genes are the virulence factors that promote colonization of enterococci in host tissues. Colonization does not necessarily induce pathogenicity but can be harmful when combined with other virulence factors or antimicrobial-resistance genes [33]. The sex pheromone gene, cad1, is reported to be the gene that facilitates conjugation. The presence of this gene can accelerate gene transfer, which is considered to be a public health risk [9]. The gelatinase gene, gelE, is referred to as one of the determinant virulence factors in enterococci and is capable of hydrolyzing bioactive peptides, collagens, elastin, and gelatin [34]. However, the prevalence of asa1, cylA, and gelE genes varied significantly in samples from the companies in this study. This may result from dissemination of the virulence genes within the same environment through horizontal transfer. E. faecium isolates showed the cad1 (81.8%) to be the most prevalent virulence gene, but none of the genes showed significant differences in samples from the companies studied. In general, E. faecalis is reported to present more virulence genes than E. faecium [35][36][37], and our findings were consistent with previous studies. Despite the low prevalence of virulence genes in E. faecium, the potential for the transfer of virulence and antimicrobial-resistance genes through bacteria can be a public health problem.

Conclusions
In this study, enterococci in non-mastitic bulk tank milk from four different dairy companies showed various degrees of antimicrobial resistance and virulence-factor distribution. The enterococci demonstrated their highest rates of resistance to TET, DOX, and ERY, and the isolates were marked by the highest prevalence of resistance genes to ERY such as erm(B) and TET such as tet(L) and tet(M), together with transposon-gene types such as IntTn. Therefore, our results support the assessment that pathogens from bulk tank milk can also become a reservoir for the dissemination of antimicrobial resistance and virulence factors through cross-contamination processes.