Dissemination and Characteristics of High-Level Erythromycin-Resistant Enterococcus Faecalis From Bulk Tank Milk of Dairy Companies in Korea

Background: Enterococci are environmental pathogens that can cause bovine mastitis and macrolides are widely used for the treatment of bovine mastitis caused by staphylococci and streptococci/enterococci. The aim of this study was performed to compare the phenotypic and genotypic characteristics of high-level erythromycin-resistant (HLER) Enterococcus faecalis (E. faecalis) collected from bulk tank milk of four dairy companies (A, B, C, and D) in Korea. Results: Although isolates from company D showed the highest prevalence of E. faecalis, the prevalence of HLER E. faecalis in company A (73.1%) and C (57.0%) was signicantly higher than company D (33.9%) (P < 0.05). A total of 149 HLER E. faecalis isolates showed high rates of resistance to tetracycline (93.3%), followed by doxycycline (70.0%), and chloramphenicol (48.3%). In the distribution of macrolides resistance genes, 147 (98.7%) isolates carried ermB gene alone, and two isolates carried both ermA and ermB genes. No isolates carried ermC, msrA, msrC, or mef genes. In the distribution of other resistance genes, 72 (48.3%) and 60 (40.3%) isolates carried both tetM and tetL genes, and tetM gene alone, respectively, and 38 (25.5%) isolates carried optrA gene. For aminoglycosides resistance genes, the prevalence of both aac(6′)Ie-aph(2″)-la and ant(6′)-Ia genes (43.0%) was the highest. Moreover, 104 (70.0%) isolates harbored Int-Tn gene carrying the Tn916/1545-like transposon. Although the distribution of ermB gene showed no signicant difference between the dairy companies, the prevalence of other resistance genes and transposons showed a signicant difference between the dairy companies (P < 0.05). Virulence genes,

(2018) [15] reported that enterococci could easily acquire resistance genes against many antimicrobials, and Chajęcka-Wierzchowska et al., (2019) [16] also reported that enterococci could possess highly effective gene transfer mechanisms, such as conjugative transposons. Thus, the aim of this study was performed to compare the phenotypic and genotypic characteristics of high-level ERY-resistant (HLER) Enterococcus faecalis (E. faecalis) isolates from bulk tank milk of dairy companies to assess its threat to public health.

Results
Distribution of HLER E. faecalis isolates and antimicrobial resistance Comparative antimicrobial resistance of the 149 HLER E. faecalis isolates from bulk tank milk of four dairy companies is shown in Table 1. Although isolates from company D showed the highest prevalence of E. faecalis, the prevalence of HLER E. faecalis in company A (73.1%) and C (57.0%) was signi cantly higher than company D (33.9%) (P < 0.05). All HLER E. faecalis isolates showed high rates of resistance to TET (93.3%), followed by DOX (70.0%), and CHL (48.3%). In particular, the resistance against these three antimicrobials showed signi cant differences between dairy companies (P < 0.05). Resistance against AMP, CIP, PEN, RIF, and VAN was only 0.7-5.4% without signi cant differences between dairy companies. Table 1 Antimicrobial resistance of 149 high-level erythromycin-resistant E. faecalis from bulk tank milk in dairy companies

Distribution Of Mdr Patterns
The prevalence of MDR in the 149 HLER E. faecalis isolates is shown in Fig. 1

Distribution Of Antimicrobial Resistance Genes And Transposon Genes
Distribution of antimicrobial resistance genes and transposon genes The distribution of resistance genes and transposons in the 149 HLER E. faecalis isolates is shown in Table 2. In prevalence of macrolides resistance genes, 147 (98.7%) isolates carried ermB gene alone, and two isolates from company C carried both ermA and ermB genes. No isolates carried ermC, msrA, msrC, or mef genes. In the distribution of other resistance genes, 72 (48.3%) and 60 (40.3%) isolates carried both tetM and tetL genes, and tetM gene alone, respectively, which are related with tetracyclines resistance, and 38 (25.5%) isolates carried optrA gene, which is related with phenicols resistance. Of the aminoglycosides resistance genes, the prevalence of both aac(6′)Ie-aph(2″)-la and ant(6′)-Ia genes (43.0%) was the highest. Moreover, 104 (70.0%) isolates harbored Int-Tn gene carrying the Tn916/1545-like transposon. Although the distribution of ermB gene showed no signi cant differences between dairy companies, the prevalence of other resistance genes and transposons showed a signi cant difference between the dairy companies (P < 0.05).  ermC Aminoglycosides

Discussion
Macrolides are one of the most commonly used clinically important antibiotics and have been used to treat infections by Gram-positive bacteria, including enterococci [17]. Additionally, they also widely used in foodproducing livestock in Korea [9]. Although ERY, which is a rst-generation macrolide, is rarely used by dairy companies because it is rapidly inactivated in the stomach due to its extreme acid sensitivity [18], tylosin, which is also one of the macrolides, has been continually used for the treatment of infections caused by staphylococci and streptococci/enterococci. However, pathogen resistance to ERY in food-producing livestock has been conferred by cross-resistance to macrolides, including tylosin [19].
In this study, 149 of the 301 E. faecalis isolates from bulk tank milk samples from four dairy companies revealed HLER, with an MIC ≥ 128 µg/mL to ERY. Generally, two principal mechanisms of macrolides resistance have been identi ed: the methylation of bacteria ribosomal 23S rRNA encoded by a series of structurally related erm genes [6,13], and an e ux pump system mediated by the membrane-bound protein encoded by the macrolide e ux genetic assembly (mef) [20] and macrolide-and streptogramin B-resistant (msr) genes [21]. In this study, all 149 HLER E. faecalis isolates carried ermB gene, and two isolates carried both ermA and ermB genes. Kim et al.,  [13,24,25]. In this study, 132 (88.6%) isolates carried tetM gene, including the combination of both tetL and tetM genes. Moreover, Int-Tn gene, the Tn916/1545-like family, was also carried by 104 (70.0%) isolates. In particular, all HLER E. faecalis isolates from company D harbored both ermB and tetM genes, and 90.5% of isolates from company D also carried Int-Tn gene. Additionally, the dissemination of MDR E. faecalis isolates is expected to increase in company D in the future because target modi cation encoded by ermB gene can also confer cross-resistance to macrolide, lincosamide, and streptogramin B antimicrobials [19].
In this study, 72 (48.3%) isolates and 101 (67.8%) isolates carried aac(6′)Ie-aph(2″)-la and ant(6′)-Ia gene, respectively, and both aac(6′)Ie-aph(2″)-la and ant(6′)-Ia genes also showed in 64 (43.0%) of these isolates. The aac(6′)Ie-aph(2″)-la and ant(6′)-Ia gene, which are associated with plasmid-mediated aminoglycoside-modifying enzymes (AMEs), are responsible for resistance to gentamicin and streptomycin, respectively, in enterococci [26, 27], and this study results demonstrated that erm, tet, and AME genes are commonly disseminated in HLER E. faecalis isolates from bulk tank milk in Korea. In this study, 38 (25.5%) isolates carried the phenicols resistance gene, optrA, which encodes an ATP-binding cassette transporter [28] and no isolates carried cfr or fexA genes. Phenicols resistance genes in milk and dairy products have not been reported in Korea until now, therefore, the presence of optrA gene in E. faecalis isolates from bulk tank milk may signi cantly accelerate the emergence of novel MDR bacteria.
Virulence genes, such as ace (99.3%), cad1 and efaA (each 98.7%), and gelE (83.9%) were also highly conserved in the 149 HLER E. faecalis isolates. Generally, possessed and expressed virulence factors cause a more severe infection than strains that lacking virulence factors [29,30]. Moreover, virulence factors accompanied by resistance genes are of pathogenic importance [29][30][31]. Therefore, the dissemination of virulence factors in bacteria from bulk tank milk can also lead to public health concern. Our results indicate that although HLER E. faecalis isolates from bulk tank milk showed signi cant differences in phenotypic and genotypic characteristics between dairy companies, there is a high prevalence of isolates that carry a variety of antimicrobial resistance genes and virulence factors. Therefore, a structured management protocol by dairy companies and constant monitoring are necessary to minimize public health hazards.

Conclusion
In this comprehensive research on HLER E. faecalis from bulk tank milk of four dairy companies in Korea, the distribution of antimicrobial resistance and the phenotypic and genotypic characteristics of HLER E. faecalis showed a signi cant difference between the dairy companies. Therefore, our results indicate that a structured management protocol by companies and constant monitoring are necessary to minimize publid health hazards.

HLER E. faecalis isolates
A total of 1,584 batches of bulk tank milk, which were sampled twice each in the summer and winter seasons, were collected from 396 dairy farms managed by four dairy companies in Korea. The isolation and identi cation of Enterococcus spp. were performed following the standard microbiological protocols published by the Ministry of Food and Drug Safety (Korea) [32]. Brie y, one mL of the milk sample was cultured in nine mL of buffered peptone water (BPW; BD Biosciences, San Jose, CA, USA). Then, the pre-enriched BPW was transferred to Enterococcosel broth (BD Biosciences) at a 1:10 ratio and steaked onto Enterococcosel agar (BD Biosciences) after incubation at 37°C for 18-24 h. E. faecalis isolates were nally con rmed using polymerase chain reaction (PCR) with a speci c primer for ddl gene as previously described [33]. Although a total of 301 E. faecalis were isolated in this study, 149 HLER E. faecalis strains with a minimum inhibitory concentration (MIC) ≥ 128 µg/mL against ERY by the standard agar dilution method according to the guidelines of the Clinical and Laboratory Standards Institute (CLSI) as previously described [34,35]. E. faecalis ATCC 29212 was used as a standard strain for the MIC tests.

Competing interests
The authors declare that they have no competing interests.

Funding
There was no nancial support for this research.