Public Health Weekly Report 2025; 18(3): 105-120
Published online December 13, 2024
https://doi.org/10.56786/PHWR.2025.18.3.1
© The Korea Disease Control and Prevention Agency
SeongJae Joo , Min Kyeong Kim
, Ae Kyung Park
, Junyoung Kim
, Jaeil Yoo *
Division of Bacterial Diseases, Department of Laboratory Diagnosis and analysis, Korea Disease Control and Prevention Agency, Cheongju, Korea
*Corresponding author: Jaeil Yoo, Tel: +82-43-719-8110, E-mail: knihyoo@korea.kr
This is an Open Access aritcle distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/) which permits unrestricted distribution, and reproduction in any medium, provided the original work is properly cited.
Vancomycin-resistant enterococci (VRE) are a leading cause of healthcare-associated infections, with limited options for antimicrobial treatment. Resistance genes and their pathogenicity can be transmitted via transposons, making infection control crucial. In this study, we analyzed the antibiotic resistance trends and pathogenic characteristics of 179 Enterococcus faecium strains, which accounted for 78.5% of the 228 VRE strains collected from the epidemic intelligence division integrated system between 2018 and 2023. The analysis revealed that all strains were resistant to vancomycin, levofloxacin, and ciprofloxacin, but susceptible to linezolid and chloramphenicol. All 179 strains possessed the vanA gene. Among these, 55 strains (30.7%) exhibited the VanB phenotype and showed intermediate resistance or susceptibility to teicoplanin. As a result of virulence factor analysis, 155 strains (86.6%) were confirmed to possess both esp and hyl genes. Multilocus sequence typing analysis revealed ST17 (48.1%) and ST192 (11.1%) as the predominant sequence types, consistent with the global distribution patterns of vancomycin-resistant E. faecium (VREfm). This study provides critical insights into the antibiotic resistance trends and genetic characteristics of VREfm in the Republic of Korea, offering a foundation for antibiotic resistance prevention and infection control efforts.
Key words Vancomycin-resistant enterococci; Enterococcus faecium; Antimicrobial resistance
VRE was first isolated in the Republic of Korea in 1992, and its infection rates have rapidly increased. Between 2005–2006, 20–30% of Enterococcus faecium isolates from secondary and tertiary hospitals were identified as vancomycin-resistant strains.
The vancomycin-resistant faecium strains identified in this study exhibited resistance to more than four antibiotics. Multilocus sequence typing analysis revealed ST17 and ST192 as the main sequence types.
The results of this study would serve as a valuable basis for analyzing and managing future mutation trends of antibiotic-resistant strains.
Antibiotic-resistant bacteria pose a serious threat to public health worldwide. This is largely due to the overuse and inappropriate prescription of antibiotics. A combination of factors, including poor infection control, insufficient awareness regarding the proper use of antibiotics, and the extensive application of antibiotics in agriculture, exacerbate this issue. Vancomycin-resistant enterococci (VRE), initially reported in France and the United Kingdom in 1986, is a notable example of this issue and has become a growing global concern [1,2].
Considering the critical threat posed by antibiotic resistance, the World Health Organization (WHO) designated it as one of its top 10 global health priorities in 2019. The WHO has recommended that countries strengthen surveillance and implement robust infection control systems to combat antibiotic-resistant bacteria. Although vancomycin is considered the last-resort antibiotic for the treatment of multidrug-resistant bacteria, the emergence of VRE has become a substantial challenge for healthcare systems. In the United States, VRE is implicated in thousands of deaths annually. In 2013, the Centers for Disease Control and Prevention classified VRE as a “serious threat” and is continuously monitoring and implementing strategies to prevent and control its spread. In the Republic of Korea (ROK), the incidence of VRE infections has sharply increased since the first reported case in 1992. Data from Korea Global Antimicrobial Resistance Surveillance System (Kor-GLASS) and Korean Antimicrobial Resistance Monitoring System (KARMS) indicated that the VRE resistance rates increased from approximately 30% in the early 2010s to 40.9% in 2019, followed by slight declines to 38.6% and 37.7% in 2020 and 2021, respectively [3].
The primary species of enterococci are Enterococcus faecium and Enterococcus faecalis, with E. faecium accounting for approximately 90% of vancomycin resistance cases. By contrast, infections caused by E. faecalis are relatively less common [4]. Although VRE predominantly causes urinary tract and wound infections, it can also lead to bacteremia in immunocompromised patients, particularly older adults with underlying medical conditions or those who experienced prolonged hospitalization. Such infections can cause severe illness or mortality in vulnerable populations, underscoring the importance of effective infection prevention and control measures.
Vancomycin is an antibiotic that targets peptidoglycan in the bacterial cell wall. Resistance to vancomycin occurs when the terminal D-alanyl D-alanine motif of the peptidoglycan is replaced by D-alanyl D-lactate or D-alanyl D-serine. To date, nine resistance genes have been identified in VRE, with the vanA and vanB genes being the most prevalent distributed [5]. The VanA phenotype is resistant to high concentrations of both vancomycin and teicoplanin, whereas the VanB phenotype exhibits resistance to vancomycin alone but retains susceptibility to teicoplanin [6]. When the strain has the vanA gene but is susceptible or moderately resistant to teicoplanin, the phenotype is classified as the VanB phenotype-vanA genotype, a phenomenon for which the underlying mechanism remains unclear [7]. Van genes are carried by mobile genetic elements such as transposons, which can be transferred horizontally to other bacteria. Consequently, vancomycin resistance genes can be transmitted to pathogenic Gram-positive bacteria, further underscoring the importance of controlling VRE infections. In particular, enterococci possess virulence factors that facilitate adherence to host tissues, a critical step in the infection process. This ability, combined with the acquisition of multidrug resistance, may contribute to the rising incidence of VRE infection [8,9]. The reported virulence factors of enterococci include enterococcal surface protein (esp), aggregation substance (agg and asa1), gelatinase (gelE), cytolysin activator (cylA), and hyaluronidase (hyl) [10].
This study aimed to assess the antibiotic resistance patterns and molecular genetic characteristics of 179 vancomycin-resistant E. faecium (VREfm) isolates, accounting for 78.5% of the 228 VRE isolates submitted to the Korea Disease Control and Prevention Agency (KDCA) through the epidemic intelligence division (EID) integrated system over the past 5 years. The study findings will provide fundamental data for the management and control of VRE infections in ROK.
Of the 228 VRE pathogens referred to the EID integrated system for diagnosis from 2018 to 2023, 179 isolates identified as VREfm were analyzed. VREfm confirmation tests were performed according to the KDCA standard procedures for culture tests for VRE (KDCA-S-CT-VRE-18-01).
To assess the antibiotic susceptibility of VREfm isolates, the broth microdilution method was performed using Sensititre GPALL1F Plates (Thermo Fisher Scientific). These plates contained one of the following 15 antibiotics: chloramphenicol (CHL), streptomycin (STH), gentamicin (GEH), erythromycin (ERY), daptomycin (DAP), penicillin (PEN), ampicillin (AMP), levofloxacin (LVX), ciprofloxacin (CIP), tetracycline (TCY), rifampin (RIF), quinupristin and dalfopristin (QDA), nitrofurantoin (NIT), linezolid (LZD), and vancomycin (VAN). Antibiotic susceptibility was determined by measuring the minimum inhibitory concentration (MIC) in accordance with the Clinical and Laboratory Standards Institute guidelines (M100-33RD ED, 2023).
To identify the Van phenotype, the MIC of teicoplanin was determined using Etest (bioMérieux) to distinguish the Van phenotype for each strain.
Polymerase chain reaction (PCR) was used to detect the vancomycin-resistant genes (vanA and vanB) and seven virulence factors (collagen-binding protein [ace], aggregation substance [asa1], cylA, cell wall-associated protein involved in immune evasion [efaA], esp, gelE, glycoside-hydrolase [hyl]). Each PCR product was subsequently confirmed by sequencing.
If two or more VREfm isolates were identified in the same medical institution within the same time, an outbreak was considered. One to three isolates were selected according to their scale, resulting in the inclusion of 54 isolates in the multilocus sequence typing (MLST) analysis. The seven housekeeping genes used in the analysis were the ATP synthase alpha subunit, D-alanine-D-alanine ligase, glucose-6-phosphate dehydrogenase, phosphoribosylaminoimidazole carboxylase ATPase subunit, glyceraldehyde-3-phosphate dehydrogenase, phosphate ATP-binding cassette transporter, and adenylate kinase. Each gene was subjected to PCR and then subsequently to sequencing. The results were synthesized to identify the sequence type (ST) and clonal complex (CC). The MLST data were analyzed using the PubMLST website (http://pubmlst.org).
The analysis of 179 VREfm isolates for susceptibility to 15 antibiotics revealed universal resistance to VAN, LVX, and CIP. Additionally, 178 isolates (99.4%) were resistant to PEN, whereas 177 isolates (98.9%) were resistant to AMP and ERY. A total of 174 (97.2%) and 168 (93.9%) isolates were resistant to NIT and RIF, respectively, whereas 108 (60.3%) and 70 (39.1%) isolates were resistant to teicoplanin and GEH, respectively. In addition, 16 (8.9%), 14 (7.8%), 6 (3.4%), and 1 (0.6%) isolates were resistant to QDA, TCY, STH, and DAP, respectively, whereas none of the examined isolates showed resistance to CHL and LZD (Table 1).
Antibiotic class | Antimicirobial agenta) | No. of resistant isolatesc) (%) |
---|---|---|
Phenicols | Chloramphenicol | 0 (0.0) |
Aminoglycosides | Streptomycin high | 6 (3.4) |
Aminoglycosides | Gentamicin high | 70 (39.1) |
Macrolides | Erythromycin | 177 (98.9) |
Lipopetides | Daptomycin | 1 (0.6) |
Penicillins | Penicillin | 178 (99.4) |
Penicillins | Ampicillin | 177 (98.9) |
Fluoroquinolone | Levofloxacin | 179 (100.0) |
Fluoroquinolone | Ciprofloxacin | 179 (100.0) |
Tetracyclines | Tetracycline | 14 (7.8) |
Ansamycins | Rifampin | 168 (93.9) |
Streptogramins | Quinupristin/dalfopristin | 16 (8.9) |
Nitrofurans | Nitrofurantoin | 174 (97.2) |
Oxazolidinones | Linezolid | 0 (0.0) |
Glycopeptides | Vancomycin | 179 (100.0) |
Glycopeptides | Teicoplaninb) | 108 (60.3) |
VREfm=vancomycin-resistant Enterococcus faecium. a)Antimicrobial susceptibility test using GPALL1F (Thermo Fisher Scientific). b)Antimicrobial susceptibility test using Etest film (BioMérieux). c)A total of VREfm 179 isolates.
An analysis of antibiotic resistance types revealed that all 179 isolates were resistant to 4 or more antibiotic classes. Among them, 54 isolates (30.2%) were resistant to 6 of 15 antibiotic classes (ERY-(AMP-PEN)-RIF-(VAN-TEC)-(LVX-CIP)-NIT). Furthermore, 37 isolates (20.7%) were resistant to 7 antibiotic classes, whereas 31 isolates (17.3%) were resistant to 6 antibiotic classes (Table 2).
No. of antibiotic classa) | Resistance profile | No. of Enterococcus faecium with pattern (%) |
---|---|---|
R4 | ERY, (AMP, PEN), (VAN, TEC) (LVX, CIP) | 1 (0.6) |
R5 | (AMP, PEN), RIF, VAN, (LVX, CIP), NIT | 2 (1.1) |
ERY, (AMP, PEN), VAN, (LVX, CIP), NIT | 3 (1.7) | |
ERY, (AMP, PEN), (VAN, TEC) (LVX, CIP), NIT | 4 (2.2) | |
ERY, (AMP, PEN), RIF, (VAN, TEC) (LVX, CIP) | 1 (0.6) | |
R6 | ERY, (AMP, PEN), VAN, (LVX, CIP), QDA, NIT | 1 (0.6) |
ERY, (AMP, PEN), RIF, VAN, (LVX, CIP), NIT | 31 (17.3) | |
ERY, GEH, (AMP, PEN), VAN, (LVX, CIP), NIT | 1 (0.6) | |
GEH, (AMP, PEN), RIF, (VAN, TEC) (LVX, CIP), NIT | 1 (0.6) | |
ERY, (AMP, PEN), RIF, (VAN, TEC) (LVX, CIP), TCY | 1 (0.6) | |
ERY, (AMP, PEN), RIF, (VAN, TEC) (LVX, CIP), NIT | 54 (30.2) | |
ERY, GEH, (AMP, PEN), (VAN, TEC) (LVX, CIP), NIT | 2 (1.1) | |
ERY, GEH, (AMP, PEN), RIF, (VAN, TEC) (LVX, CIP) | 2 (1.1) | |
R7 | ERY, GEH, RIF, (VAN, TEC) (LVX, CIP), QDA, NIT | 1 (0.6) |
ERY, GEH, (AMP, PEN), RIF, VAN, (LVX, CIP), NIT | 11 (6.1) | |
ERY, (AMP, PEN), RIF, (VAN, TEC) (LVX, CIP), NIT, TCY | 5 (2.8) | |
ERY, (AMP, PEN), RIF, (VAN, TEC) (LVX, CIP), QDA NIT | 2 (1.1) | |
ERY, GEH, (AMP, PEN), RIF, (VAN, TEC) (LVX, CIP), NIT | 37 (20.7) | |
ERY, STH, (AMP, PEN), RIF, (VAN, TEC) (LVX, CIP), TCY | 1 (0.6) | |
R8 | ERY, GEH, (AMP, PEN), RIF, VAN, (LVX, CIP), NIT, TCY | 1 (0.6) |
ERY, GEH, (AMP, PEN), RIF, VAN, (LVX, CIP), QDA, NIT | 2 (1.1) | |
ERY, STH, (AMP, PEN), RIF, VAN, (LVX, CIP), NIT, TCY | 1 (0.6) | |
ERY, GEH, (AMP, PEN), RIF, (VAN, TEC) (LVX, CIP), NIT, TCY | 3 (1.7) | |
ERY, GEH, (AMP, PEN), RIF, (VAN, TEC) (LVX, CIP), QDA, NIT | 6 (3.4) | |
ERY, STH, (AMP, PEN), RIF, (VAN, TEC) (LVX, CIP), QDA, NIT | 1 (0.6) | |
ERY, (STH, GEH), (AMP, PEN), RIF, VAN, (LVX, CIP), NIT, TCY | 1 (0.6) | |
ERY, (STH, GEH), (AMP, PEN), RIF, VAN, (LVX, CIP), QDA, NIT | 1 (0.6) | |
R9 | ERY, (STH, GEH), (AMP, PEN), RIF, (VAN, TEC) (LVX, CIP), QDA, NIT, TCY | 1 (0.6) |
ERY, DAP, (STH, GEH), (AMP, PEN), RIF, (VAN, TEC) (LVX, CIP), QDA, NIT | 1 (0.6) | |
Total | 179 (100.0) |
VREfm=vancomycin-resistant Enterococcus faecium; R=resistance; ERY=erythromycin; DAP=daptomycin; GEH=gentamicin-high; STH= steptomycin-high; AMP=ampicillin; PEN=penicillin; RIF=rifampin; VAN=vancomycin; TEC=teicoplanin; LVX=levofloxacin; CIP= ciprofloxacin; QDA=quinupristin/dalfopristin; NIT=nitrofurantoin; TCY=tetracyline. a)Number of antibiotics classes resistant.
All 179 VREfm isolates were found to contain the vanA gene, whereas vanB gene was not detected. Notably, 55 isolates (30.7%) of VREfm carried the vanA gene but exhibited a VanB phenotype that showed susceptibility or moderate resistance to teicoplanin.
The analysis of seven virulence factors (ace, asa1, cylA, efaA, esp, gelE, and hyl) revealed the presence of esp and hyl genes only. Among the isolates, 155 (86.6%) carried both the esp and hyl genes, 13 (7.3%) carried the esp gene only, and 6 (3.4%) carried the hyl gene only. In addition, five (2.8%) isolates lacked all seven virulence factors (Table 3).
Pheno-geno type | No. of isolates (%) | Total | |||
---|---|---|---|---|---|
- | esp | hyl | esp+hyl | ||
VanA-vanA | 4 (2.2) | 10 (5.6) | 3 (1.7) | 107 (59.8) | 124 (69.3) |
VanB-vanA | 1 (0.6) | 3 (1.7) | 3 (1.7) | 48 (26.8) | 55 (30.7) |
Total | 5 (2.8) | 13 (7.3) | 6 (3.4) | 155 (86.6) | 179 (100.0) |
VREfm=vancomycin-resistant Enterococcus faecium; esp=enterococcal surface protein; hyl=glycoside-hydrolase; -=not available.
MLST analysis of the 54 selected isolates identified 12 STs, with the majority belonging to clonal complex 17 (CC17). ST17 was the predominant ST, comprising 26 isolates (48.1%), followed by ST192 with 6 isolates (11.1%) and ST262 and ST80 with 5 isolates (9.3%) each. Other identified STs included ST19, ST81, ST89, and ST90. Notably, ST17 is the primary member of CC17, with other STs included within this clonal complex (Figure 1).
This study analyzed 179 VREfm isolates referred for diagnosis through the EID integrated system from referral institutions and the Research Institute of Public Health and Environment from 2018 to 2023. It provided valuable insights into the characteristics of VREfm pathogens in ROK based on the antibiotic resistance patterns, vancomycin resistance genotypes, virulence factors, and MLST analysis results. The analysis revealed an overall high level of antibiotic resistance among VREfm isolates in ROK, with all isolates exhibiting resistance to VAN, LVX, and CIP. Notably, 150 isolates (83.6%) were resistant to 6 to 7 antibiotic classes, highlighting significant challenges in the selection of effective therapeutics for the treatment of VREfm infections in the country. The observed high levels of antibiotic resistance in VREfm isolates are consistent with the findings from both Korean and international studies. These findings suggest that the misuse and inappropriate use of antibiotics in healthcare settings may contribute to high resistance levels [11,12]. This trend has raised a serious concern about infection control in medical institutions in ROK.
All 179 domestic isolates of VREfm were found to possess the vanA gene. However, despite carrying the vanA genotype, 55 isolates (30.7%) exhibited phenotypic features resembling those of the VanB phenotype, characterized by high resistance to vancomycin but susceptibility or moderate resistance to teicoplanin. Such results have been reported in several Asian countries, including Japan, Taiwan, and ROK, suggesting that the VanB phenotype may be associated with regional characteristics [13]. Although the VanB phenotype is believed to be caused by mutations in the vanS gene or rearrangements in the vanX, vanY, and vanZ genes, the exact mechanisms remain unclear, requiring further research [7]. Genotyping offers valuable insights into the mechanisms of vancomycin resistance in VREfm and provides a basis for enhancing surveillance and developing management strategies for controlling VRE infections.
The analysis of VREfm virulence factors revealed that all domestic isolates harbored the esp and hyl genes. The esp gene plays an important role in enhancing the virulence and infection capacity of VREfm. This gene is highly prevalent in the United States and Europe [14]. As the transmission of virulence factors contributes to the spread of infection, continuous surveillance and effective infection control measures are critical. As the prevalence of esp gene is increasing in ROK, a strategic approach is necessary for the prevention and management of VREfm infections.
Genotyping of 54 VREfm isolates associated with outbreaks in ROK over the past 5 years identified 12 distinct STs. Of these STs, ST17 was the predominant type, with 26 isolates (48.1%). Other STs, including ST17, ST192, ST262, ST80, ST18, ST817, ST761, ST981, ST789, ST230, and ST117, were also identified, and all but ST981 belonged to the CC17, which is commonly found in globally reported VRE strains. ST17 is the predominant clone in the globally prevalent VREfm strains. The predominance of CC17 has also been observed in Taiwan, the United States, and Europe. CC17 clones typically exhibit resistance to AMP and quinolone antibiotics, and most of them harbor the esp gene [15]. These findings suggest that the domestic VREfm isolates are closely related to the global clonal complex and contribute valuable insights into the antibiotic resistance profiles and virulence factors of VREfm [16].
This study provides important foundational data on the genetic diversity and resistance mechanisms of VREfm in ROK by investigating its antibiotic resistance characteristics and molecular genetics. Since the initial report of VRE in ROK in 1992, its prevalence reached 2.9% in 1997. Since then, the prevalence of VRE has increased significantly, with resistance rates rising to 15.1% in 1998 and 40.9% in 2019 [3]. These rates are similar to or slightly higher than those observed for vancomycin resistance in enterococci in the United States and some parts of Europe (Lithuania, Serbia, etc.) [17,18]. Considering the increasing global incidence of VRE, strengthening surveillance and control efforts remains crucial. In particular, findings from the analysis of resistance gene distribution, the role of virulence factors, and MLST will provide scientific evidence for developing effective VRE infection control and prevention strategies. Future investigations should further elucidate the genetic variation and antibiotic resistance mechanisms of VRE, enhancing understanding of its pathogenic properties in ROK. This research will provide valuable insights for developing more refined infection control and prevention strategies.
Ethics Statement: Not applicable.
Funding Source: None.
Acknowledgments: None.
Conflict of Interest: The authors have no conflicts of interest to declare.
Author Contributions: Data curation: SJJ, MKK. Formal analysis: SJJ, MKK. Investigation: SJJ, MKK. Visualization: SJJ, AKP. Supervision: AKP, JYK, JIY. Writing – original draft: SJJ. Writing – review & editing: AKP, JYK, JIY.
Public Health Weekly Report 2025; 18(3): 105-120
Published online January 16, 2025 https://doi.org/10.56786/PHWR.2025.18.3.1
Copyright © The Korea Disease Control and Prevention Agency.
SeongJae Joo , Min Kyeong Kim
, Ae Kyung Park
, Junyoung Kim
, Jaeil Yoo *
Division of Bacterial Diseases, Department of Laboratory Diagnosis and analysis, Korea Disease Control and Prevention Agency, Cheongju, Korea
Correspondence to:*Corresponding author: Jaeil Yoo, Tel: +82-43-719-8110, E-mail: knihyoo@korea.kr
This is an Open Access aritcle distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/) which permits unrestricted distribution, and reproduction in any medium, provided the original work is properly cited.
Vancomycin-resistant enterococci (VRE) are a leading cause of healthcare-associated infections, with limited options for antimicrobial treatment. Resistance genes and their pathogenicity can be transmitted via transposons, making infection control crucial. In this study, we analyzed the antibiotic resistance trends and pathogenic characteristics of 179 Enterococcus faecium strains, which accounted for 78.5% of the 228 VRE strains collected from the epidemic intelligence division integrated system between 2018 and 2023. The analysis revealed that all strains were resistant to vancomycin, levofloxacin, and ciprofloxacin, but susceptible to linezolid and chloramphenicol. All 179 strains possessed the vanA gene. Among these, 55 strains (30.7%) exhibited the VanB phenotype and showed intermediate resistance or susceptibility to teicoplanin. As a result of virulence factor analysis, 155 strains (86.6%) were confirmed to possess both esp and hyl genes. Multilocus sequence typing analysis revealed ST17 (48.1%) and ST192 (11.1%) as the predominant sequence types, consistent with the global distribution patterns of vancomycin-resistant E. faecium (VREfm). This study provides critical insights into the antibiotic resistance trends and genetic characteristics of VREfm in the Republic of Korea, offering a foundation for antibiotic resistance prevention and infection control efforts.
Keywords: Vancomycin-resistant enterococci, Enterococcus faecium, Antimicrobial resistance
VRE was first isolated in the Republic of Korea in 1992, and its infection rates have rapidly increased. Between 2005–2006, 20–30% of Enterococcus faecium isolates from secondary and tertiary hospitals were identified as vancomycin-resistant strains.
The vancomycin-resistant faecium strains identified in this study exhibited resistance to more than four antibiotics. Multilocus sequence typing analysis revealed ST17 and ST192 as the main sequence types.
The results of this study would serve as a valuable basis for analyzing and managing future mutation trends of antibiotic-resistant strains.
Antibiotic-resistant bacteria pose a serious threat to public health worldwide. This is largely due to the overuse and inappropriate prescription of antibiotics. A combination of factors, including poor infection control, insufficient awareness regarding the proper use of antibiotics, and the extensive application of antibiotics in agriculture, exacerbate this issue. Vancomycin-resistant enterococci (VRE), initially reported in France and the United Kingdom in 1986, is a notable example of this issue and has become a growing global concern [1,2].
Considering the critical threat posed by antibiotic resistance, the World Health Organization (WHO) designated it as one of its top 10 global health priorities in 2019. The WHO has recommended that countries strengthen surveillance and implement robust infection control systems to combat antibiotic-resistant bacteria. Although vancomycin is considered the last-resort antibiotic for the treatment of multidrug-resistant bacteria, the emergence of VRE has become a substantial challenge for healthcare systems. In the United States, VRE is implicated in thousands of deaths annually. In 2013, the Centers for Disease Control and Prevention classified VRE as a “serious threat” and is continuously monitoring and implementing strategies to prevent and control its spread. In the Republic of Korea (ROK), the incidence of VRE infections has sharply increased since the first reported case in 1992. Data from Korea Global Antimicrobial Resistance Surveillance System (Kor-GLASS) and Korean Antimicrobial Resistance Monitoring System (KARMS) indicated that the VRE resistance rates increased from approximately 30% in the early 2010s to 40.9% in 2019, followed by slight declines to 38.6% and 37.7% in 2020 and 2021, respectively [3].
The primary species of enterococci are Enterococcus faecium and Enterococcus faecalis, with E. faecium accounting for approximately 90% of vancomycin resistance cases. By contrast, infections caused by E. faecalis are relatively less common [4]. Although VRE predominantly causes urinary tract and wound infections, it can also lead to bacteremia in immunocompromised patients, particularly older adults with underlying medical conditions or those who experienced prolonged hospitalization. Such infections can cause severe illness or mortality in vulnerable populations, underscoring the importance of effective infection prevention and control measures.
Vancomycin is an antibiotic that targets peptidoglycan in the bacterial cell wall. Resistance to vancomycin occurs when the terminal D-alanyl D-alanine motif of the peptidoglycan is replaced by D-alanyl D-lactate or D-alanyl D-serine. To date, nine resistance genes have been identified in VRE, with the vanA and vanB genes being the most prevalent distributed [5]. The VanA phenotype is resistant to high concentrations of both vancomycin and teicoplanin, whereas the VanB phenotype exhibits resistance to vancomycin alone but retains susceptibility to teicoplanin [6]. When the strain has the vanA gene but is susceptible or moderately resistant to teicoplanin, the phenotype is classified as the VanB phenotype-vanA genotype, a phenomenon for which the underlying mechanism remains unclear [7]. Van genes are carried by mobile genetic elements such as transposons, which can be transferred horizontally to other bacteria. Consequently, vancomycin resistance genes can be transmitted to pathogenic Gram-positive bacteria, further underscoring the importance of controlling VRE infections. In particular, enterococci possess virulence factors that facilitate adherence to host tissues, a critical step in the infection process. This ability, combined with the acquisition of multidrug resistance, may contribute to the rising incidence of VRE infection [8,9]. The reported virulence factors of enterococci include enterococcal surface protein (esp), aggregation substance (agg and asa1), gelatinase (gelE), cytolysin activator (cylA), and hyaluronidase (hyl) [10].
This study aimed to assess the antibiotic resistance patterns and molecular genetic characteristics of 179 vancomycin-resistant E. faecium (VREfm) isolates, accounting for 78.5% of the 228 VRE isolates submitted to the Korea Disease Control and Prevention Agency (KDCA) through the epidemic intelligence division (EID) integrated system over the past 5 years. The study findings will provide fundamental data for the management and control of VRE infections in ROK.
Of the 228 VRE pathogens referred to the EID integrated system for diagnosis from 2018 to 2023, 179 isolates identified as VREfm were analyzed. VREfm confirmation tests were performed according to the KDCA standard procedures for culture tests for VRE (KDCA-S-CT-VRE-18-01).
To assess the antibiotic susceptibility of VREfm isolates, the broth microdilution method was performed using Sensititre GPALL1F Plates (Thermo Fisher Scientific). These plates contained one of the following 15 antibiotics: chloramphenicol (CHL), streptomycin (STH), gentamicin (GEH), erythromycin (ERY), daptomycin (DAP), penicillin (PEN), ampicillin (AMP), levofloxacin (LVX), ciprofloxacin (CIP), tetracycline (TCY), rifampin (RIF), quinupristin and dalfopristin (QDA), nitrofurantoin (NIT), linezolid (LZD), and vancomycin (VAN). Antibiotic susceptibility was determined by measuring the minimum inhibitory concentration (MIC) in accordance with the Clinical and Laboratory Standards Institute guidelines (M100-33RD ED, 2023).
To identify the Van phenotype, the MIC of teicoplanin was determined using Etest (bioMérieux) to distinguish the Van phenotype for each strain.
Polymerase chain reaction (PCR) was used to detect the vancomycin-resistant genes (vanA and vanB) and seven virulence factors (collagen-binding protein [ace], aggregation substance [asa1], cylA, cell wall-associated protein involved in immune evasion [efaA], esp, gelE, glycoside-hydrolase [hyl]). Each PCR product was subsequently confirmed by sequencing.
If two or more VREfm isolates were identified in the same medical institution within the same time, an outbreak was considered. One to three isolates were selected according to their scale, resulting in the inclusion of 54 isolates in the multilocus sequence typing (MLST) analysis. The seven housekeeping genes used in the analysis were the ATP synthase alpha subunit, D-alanine-D-alanine ligase, glucose-6-phosphate dehydrogenase, phosphoribosylaminoimidazole carboxylase ATPase subunit, glyceraldehyde-3-phosphate dehydrogenase, phosphate ATP-binding cassette transporter, and adenylate kinase. Each gene was subjected to PCR and then subsequently to sequencing. The results were synthesized to identify the sequence type (ST) and clonal complex (CC). The MLST data were analyzed using the PubMLST website (http://pubmlst.org).
The analysis of 179 VREfm isolates for susceptibility to 15 antibiotics revealed universal resistance to VAN, LVX, and CIP. Additionally, 178 isolates (99.4%) were resistant to PEN, whereas 177 isolates (98.9%) were resistant to AMP and ERY. A total of 174 (97.2%) and 168 (93.9%) isolates were resistant to NIT and RIF, respectively, whereas 108 (60.3%) and 70 (39.1%) isolates were resistant to teicoplanin and GEH, respectively. In addition, 16 (8.9%), 14 (7.8%), 6 (3.4%), and 1 (0.6%) isolates were resistant to QDA, TCY, STH, and DAP, respectively, whereas none of the examined isolates showed resistance to CHL and LZD (Table 1).
Antibiotic class | Antimicirobial agenta) | No. of resistant isolatesc) (%) |
---|---|---|
Phenicols | Chloramphenicol | 0 (0.0) |
Aminoglycosides | Streptomycin high | 6 (3.4) |
Aminoglycosides | Gentamicin high | 70 (39.1) |
Macrolides | Erythromycin | 177 (98.9) |
Lipopetides | Daptomycin | 1 (0.6) |
Penicillins | Penicillin | 178 (99.4) |
Penicillins | Ampicillin | 177 (98.9) |
Fluoroquinolone | Levofloxacin | 179 (100.0) |
Fluoroquinolone | Ciprofloxacin | 179 (100.0) |
Tetracyclines | Tetracycline | 14 (7.8) |
Ansamycins | Rifampin | 168 (93.9) |
Streptogramins | Quinupristin/dalfopristin | 16 (8.9) |
Nitrofurans | Nitrofurantoin | 174 (97.2) |
Oxazolidinones | Linezolid | 0 (0.0) |
Glycopeptides | Vancomycin | 179 (100.0) |
Glycopeptides | Teicoplaninb) | 108 (60.3) |
VREfm=vancomycin-resistant Enterococcus faecium. a)Antimicrobial susceptibility test using GPALL1F (Thermo Fisher Scientific). b)Antimicrobial susceptibility test using Etest film (BioMérieux). c)A total of VREfm 179 isolates..
An analysis of antibiotic resistance types revealed that all 179 isolates were resistant to 4 or more antibiotic classes. Among them, 54 isolates (30.2%) were resistant to 6 of 15 antibiotic classes (ERY-(AMP-PEN)-RIF-(VAN-TEC)-(LVX-CIP)-NIT). Furthermore, 37 isolates (20.7%) were resistant to 7 antibiotic classes, whereas 31 isolates (17.3%) were resistant to 6 antibiotic classes (Table 2).
No. of antibiotic classa) | Resistance profile | No. of Enterococcus faecium with pattern (%) |
---|---|---|
R4 | ERY, (AMP, PEN), (VAN, TEC) (LVX, CIP) | 1 (0.6) |
R5 | (AMP, PEN), RIF, VAN, (LVX, CIP), NIT | 2 (1.1) |
ERY, (AMP, PEN), VAN, (LVX, CIP), NIT | 3 (1.7) | |
ERY, (AMP, PEN), (VAN, TEC) (LVX, CIP), NIT | 4 (2.2) | |
ERY, (AMP, PEN), RIF, (VAN, TEC) (LVX, CIP) | 1 (0.6) | |
R6 | ERY, (AMP, PEN), VAN, (LVX, CIP), QDA, NIT | 1 (0.6) |
ERY, (AMP, PEN), RIF, VAN, (LVX, CIP), NIT | 31 (17.3) | |
ERY, GEH, (AMP, PEN), VAN, (LVX, CIP), NIT | 1 (0.6) | |
GEH, (AMP, PEN), RIF, (VAN, TEC) (LVX, CIP), NIT | 1 (0.6) | |
ERY, (AMP, PEN), RIF, (VAN, TEC) (LVX, CIP), TCY | 1 (0.6) | |
ERY, (AMP, PEN), RIF, (VAN, TEC) (LVX, CIP), NIT | 54 (30.2) | |
ERY, GEH, (AMP, PEN), (VAN, TEC) (LVX, CIP), NIT | 2 (1.1) | |
ERY, GEH, (AMP, PEN), RIF, (VAN, TEC) (LVX, CIP) | 2 (1.1) | |
R7 | ERY, GEH, RIF, (VAN, TEC) (LVX, CIP), QDA, NIT | 1 (0.6) |
ERY, GEH, (AMP, PEN), RIF, VAN, (LVX, CIP), NIT | 11 (6.1) | |
ERY, (AMP, PEN), RIF, (VAN, TEC) (LVX, CIP), NIT, TCY | 5 (2.8) | |
ERY, (AMP, PEN), RIF, (VAN, TEC) (LVX, CIP), QDA NIT | 2 (1.1) | |
ERY, GEH, (AMP, PEN), RIF, (VAN, TEC) (LVX, CIP), NIT | 37 (20.7) | |
ERY, STH, (AMP, PEN), RIF, (VAN, TEC) (LVX, CIP), TCY | 1 (0.6) | |
R8 | ERY, GEH, (AMP, PEN), RIF, VAN, (LVX, CIP), NIT, TCY | 1 (0.6) |
ERY, GEH, (AMP, PEN), RIF, VAN, (LVX, CIP), QDA, NIT | 2 (1.1) | |
ERY, STH, (AMP, PEN), RIF, VAN, (LVX, CIP), NIT, TCY | 1 (0.6) | |
ERY, GEH, (AMP, PEN), RIF, (VAN, TEC) (LVX, CIP), NIT, TCY | 3 (1.7) | |
ERY, GEH, (AMP, PEN), RIF, (VAN, TEC) (LVX, CIP), QDA, NIT | 6 (3.4) | |
ERY, STH, (AMP, PEN), RIF, (VAN, TEC) (LVX, CIP), QDA, NIT | 1 (0.6) | |
ERY, (STH, GEH), (AMP, PEN), RIF, VAN, (LVX, CIP), NIT, TCY | 1 (0.6) | |
ERY, (STH, GEH), (AMP, PEN), RIF, VAN, (LVX, CIP), QDA, NIT | 1 (0.6) | |
R9 | ERY, (STH, GEH), (AMP, PEN), RIF, (VAN, TEC) (LVX, CIP), QDA, NIT, TCY | 1 (0.6) |
ERY, DAP, (STH, GEH), (AMP, PEN), RIF, (VAN, TEC) (LVX, CIP), QDA, NIT | 1 (0.6) | |
Total | 179 (100.0) |
VREfm=vancomycin-resistant Enterococcus faecium; R=resistance; ERY=erythromycin; DAP=daptomycin; GEH=gentamicin-high; STH= steptomycin-high; AMP=ampicillin; PEN=penicillin; RIF=rifampin; VAN=vancomycin; TEC=teicoplanin; LVX=levofloxacin; CIP= ciprofloxacin; QDA=quinupristin/dalfopristin; NIT=nitrofurantoin; TCY=tetracyline. a)Number of antibiotics classes resistant..
All 179 VREfm isolates were found to contain the vanA gene, whereas vanB gene was not detected. Notably, 55 isolates (30.7%) of VREfm carried the vanA gene but exhibited a VanB phenotype that showed susceptibility or moderate resistance to teicoplanin.
The analysis of seven virulence factors (ace, asa1, cylA, efaA, esp, gelE, and hyl) revealed the presence of esp and hyl genes only. Among the isolates, 155 (86.6%) carried both the esp and hyl genes, 13 (7.3%) carried the esp gene only, and 6 (3.4%) carried the hyl gene only. In addition, five (2.8%) isolates lacked all seven virulence factors (Table 3).
Pheno-geno type | No. of isolates (%) | Total | |||
---|---|---|---|---|---|
- | esp | hyl | esp+hyl | ||
VanA-vanA | 4 (2.2) | 10 (5.6) | 3 (1.7) | 107 (59.8) | 124 (69.3) |
VanB-vanA | 1 (0.6) | 3 (1.7) | 3 (1.7) | 48 (26.8) | 55 (30.7) |
Total | 5 (2.8) | 13 (7.3) | 6 (3.4) | 155 (86.6) | 179 (100.0) |
VREfm=vancomycin-resistant Enterococcus faecium; esp=enterococcal surface protein; hyl=glycoside-hydrolase; -=not available..
MLST analysis of the 54 selected isolates identified 12 STs, with the majority belonging to clonal complex 17 (CC17). ST17 was the predominant ST, comprising 26 isolates (48.1%), followed by ST192 with 6 isolates (11.1%) and ST262 and ST80 with 5 isolates (9.3%) each. Other identified STs included ST19, ST81, ST89, and ST90. Notably, ST17 is the primary member of CC17, with other STs included within this clonal complex (Figure 1).
This study analyzed 179 VREfm isolates referred for diagnosis through the EID integrated system from referral institutions and the Research Institute of Public Health and Environment from 2018 to 2023. It provided valuable insights into the characteristics of VREfm pathogens in ROK based on the antibiotic resistance patterns, vancomycin resistance genotypes, virulence factors, and MLST analysis results. The analysis revealed an overall high level of antibiotic resistance among VREfm isolates in ROK, with all isolates exhibiting resistance to VAN, LVX, and CIP. Notably, 150 isolates (83.6%) were resistant to 6 to 7 antibiotic classes, highlighting significant challenges in the selection of effective therapeutics for the treatment of VREfm infections in the country. The observed high levels of antibiotic resistance in VREfm isolates are consistent with the findings from both Korean and international studies. These findings suggest that the misuse and inappropriate use of antibiotics in healthcare settings may contribute to high resistance levels [11,12]. This trend has raised a serious concern about infection control in medical institutions in ROK.
All 179 domestic isolates of VREfm were found to possess the vanA gene. However, despite carrying the vanA genotype, 55 isolates (30.7%) exhibited phenotypic features resembling those of the VanB phenotype, characterized by high resistance to vancomycin but susceptibility or moderate resistance to teicoplanin. Such results have been reported in several Asian countries, including Japan, Taiwan, and ROK, suggesting that the VanB phenotype may be associated with regional characteristics [13]. Although the VanB phenotype is believed to be caused by mutations in the vanS gene or rearrangements in the vanX, vanY, and vanZ genes, the exact mechanisms remain unclear, requiring further research [7]. Genotyping offers valuable insights into the mechanisms of vancomycin resistance in VREfm and provides a basis for enhancing surveillance and developing management strategies for controlling VRE infections.
The analysis of VREfm virulence factors revealed that all domestic isolates harbored the esp and hyl genes. The esp gene plays an important role in enhancing the virulence and infection capacity of VREfm. This gene is highly prevalent in the United States and Europe [14]. As the transmission of virulence factors contributes to the spread of infection, continuous surveillance and effective infection control measures are critical. As the prevalence of esp gene is increasing in ROK, a strategic approach is necessary for the prevention and management of VREfm infections.
Genotyping of 54 VREfm isolates associated with outbreaks in ROK over the past 5 years identified 12 distinct STs. Of these STs, ST17 was the predominant type, with 26 isolates (48.1%). Other STs, including ST17, ST192, ST262, ST80, ST18, ST817, ST761, ST981, ST789, ST230, and ST117, were also identified, and all but ST981 belonged to the CC17, which is commonly found in globally reported VRE strains. ST17 is the predominant clone in the globally prevalent VREfm strains. The predominance of CC17 has also been observed in Taiwan, the United States, and Europe. CC17 clones typically exhibit resistance to AMP and quinolone antibiotics, and most of them harbor the esp gene [15]. These findings suggest that the domestic VREfm isolates are closely related to the global clonal complex and contribute valuable insights into the antibiotic resistance profiles and virulence factors of VREfm [16].
This study provides important foundational data on the genetic diversity and resistance mechanisms of VREfm in ROK by investigating its antibiotic resistance characteristics and molecular genetics. Since the initial report of VRE in ROK in 1992, its prevalence reached 2.9% in 1997. Since then, the prevalence of VRE has increased significantly, with resistance rates rising to 15.1% in 1998 and 40.9% in 2019 [3]. These rates are similar to or slightly higher than those observed for vancomycin resistance in enterococci in the United States and some parts of Europe (Lithuania, Serbia, etc.) [17,18]. Considering the increasing global incidence of VRE, strengthening surveillance and control efforts remains crucial. In particular, findings from the analysis of resistance gene distribution, the role of virulence factors, and MLST will provide scientific evidence for developing effective VRE infection control and prevention strategies. Future investigations should further elucidate the genetic variation and antibiotic resistance mechanisms of VRE, enhancing understanding of its pathogenic properties in ROK. This research will provide valuable insights for developing more refined infection control and prevention strategies.
Ethics Statement: Not applicable.
Funding Source: None.
Acknowledgments: None.
Conflict of Interest: The authors have no conflicts of interest to declare.
Author Contributions: Data curation: SJJ, MKK. Formal analysis: SJJ, MKK. Investigation: SJJ, MKK. Visualization: SJJ, AKP. Supervision: AKP, JYK, JIY. Writing – original draft: SJJ. Writing – review & editing: AKP, JYK, JIY.
Antibiotic class | Antimicirobial agenta) | No. of resistant isolatesc) (%) |
---|---|---|
Phenicols | Chloramphenicol | 0 (0.0) |
Aminoglycosides | Streptomycin high | 6 (3.4) |
Aminoglycosides | Gentamicin high | 70 (39.1) |
Macrolides | Erythromycin | 177 (98.9) |
Lipopetides | Daptomycin | 1 (0.6) |
Penicillins | Penicillin | 178 (99.4) |
Penicillins | Ampicillin | 177 (98.9) |
Fluoroquinolone | Levofloxacin | 179 (100.0) |
Fluoroquinolone | Ciprofloxacin | 179 (100.0) |
Tetracyclines | Tetracycline | 14 (7.8) |
Ansamycins | Rifampin | 168 (93.9) |
Streptogramins | Quinupristin/dalfopristin | 16 (8.9) |
Nitrofurans | Nitrofurantoin | 174 (97.2) |
Oxazolidinones | Linezolid | 0 (0.0) |
Glycopeptides | Vancomycin | 179 (100.0) |
Glycopeptides | Teicoplaninb) | 108 (60.3) |
VREfm=vancomycin-resistant Enterococcus faecium. a)Antimicrobial susceptibility test using GPALL1F (Thermo Fisher Scientific). b)Antimicrobial susceptibility test using Etest film (BioMérieux). c)A total of VREfm 179 isolates..
No. of antibiotic classa) | Resistance profile | No. of Enterococcus faecium with pattern (%) |
---|---|---|
R4 | ERY, (AMP, PEN), (VAN, TEC) (LVX, CIP) | 1 (0.6) |
R5 | (AMP, PEN), RIF, VAN, (LVX, CIP), NIT | 2 (1.1) |
ERY, (AMP, PEN), VAN, (LVX, CIP), NIT | 3 (1.7) | |
ERY, (AMP, PEN), (VAN, TEC) (LVX, CIP), NIT | 4 (2.2) | |
ERY, (AMP, PEN), RIF, (VAN, TEC) (LVX, CIP) | 1 (0.6) | |
R6 | ERY, (AMP, PEN), VAN, (LVX, CIP), QDA, NIT | 1 (0.6) |
ERY, (AMP, PEN), RIF, VAN, (LVX, CIP), NIT | 31 (17.3) | |
ERY, GEH, (AMP, PEN), VAN, (LVX, CIP), NIT | 1 (0.6) | |
GEH, (AMP, PEN), RIF, (VAN, TEC) (LVX, CIP), NIT | 1 (0.6) | |
ERY, (AMP, PEN), RIF, (VAN, TEC) (LVX, CIP), TCY | 1 (0.6) | |
ERY, (AMP, PEN), RIF, (VAN, TEC) (LVX, CIP), NIT | 54 (30.2) | |
ERY, GEH, (AMP, PEN), (VAN, TEC) (LVX, CIP), NIT | 2 (1.1) | |
ERY, GEH, (AMP, PEN), RIF, (VAN, TEC) (LVX, CIP) | 2 (1.1) | |
R7 | ERY, GEH, RIF, (VAN, TEC) (LVX, CIP), QDA, NIT | 1 (0.6) |
ERY, GEH, (AMP, PEN), RIF, VAN, (LVX, CIP), NIT | 11 (6.1) | |
ERY, (AMP, PEN), RIF, (VAN, TEC) (LVX, CIP), NIT, TCY | 5 (2.8) | |
ERY, (AMP, PEN), RIF, (VAN, TEC) (LVX, CIP), QDA NIT | 2 (1.1) | |
ERY, GEH, (AMP, PEN), RIF, (VAN, TEC) (LVX, CIP), NIT | 37 (20.7) | |
ERY, STH, (AMP, PEN), RIF, (VAN, TEC) (LVX, CIP), TCY | 1 (0.6) | |
R8 | ERY, GEH, (AMP, PEN), RIF, VAN, (LVX, CIP), NIT, TCY | 1 (0.6) |
ERY, GEH, (AMP, PEN), RIF, VAN, (LVX, CIP), QDA, NIT | 2 (1.1) | |
ERY, STH, (AMP, PEN), RIF, VAN, (LVX, CIP), NIT, TCY | 1 (0.6) | |
ERY, GEH, (AMP, PEN), RIF, (VAN, TEC) (LVX, CIP), NIT, TCY | 3 (1.7) | |
ERY, GEH, (AMP, PEN), RIF, (VAN, TEC) (LVX, CIP), QDA, NIT | 6 (3.4) | |
ERY, STH, (AMP, PEN), RIF, (VAN, TEC) (LVX, CIP), QDA, NIT | 1 (0.6) | |
ERY, (STH, GEH), (AMP, PEN), RIF, VAN, (LVX, CIP), NIT, TCY | 1 (0.6) | |
ERY, (STH, GEH), (AMP, PEN), RIF, VAN, (LVX, CIP), QDA, NIT | 1 (0.6) | |
R9 | ERY, (STH, GEH), (AMP, PEN), RIF, (VAN, TEC) (LVX, CIP), QDA, NIT, TCY | 1 (0.6) |
ERY, DAP, (STH, GEH), (AMP, PEN), RIF, (VAN, TEC) (LVX, CIP), QDA, NIT | 1 (0.6) | |
Total | 179 (100.0) |
VREfm=vancomycin-resistant Enterococcus faecium; R=resistance; ERY=erythromycin; DAP=daptomycin; GEH=gentamicin-high; STH= steptomycin-high; AMP=ampicillin; PEN=penicillin; RIF=rifampin; VAN=vancomycin; TEC=teicoplanin; LVX=levofloxacin; CIP= ciprofloxacin; QDA=quinupristin/dalfopristin; NIT=nitrofurantoin; TCY=tetracyline. a)Number of antibiotics classes resistant..
Pheno-geno type | No. of isolates (%) | Total | |||
---|---|---|---|---|---|
- | esp | hyl | esp+hyl | ||
VanA-vanA | 4 (2.2) | 10 (5.6) | 3 (1.7) | 107 (59.8) | 124 (69.3) |
VanB-vanA | 1 (0.6) | 3 (1.7) | 3 (1.7) | 48 (26.8) | 55 (30.7) |
Total | 5 (2.8) | 13 (7.3) | 6 (3.4) | 155 (86.6) | 179 (100.0) |
VREfm=vancomycin-resistant Enterococcus faecium; esp=enterococcal surface protein; hyl=glycoside-hydrolase; -=not available..
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