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Keywords:

  • esp;
  • multilocus sequence typing;
  • sequence type;
  • Tn1546;
  • vancomycin

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Aims:  Multilocus sequence typing (MLST) was performed for vancomycin-resistant Enterococcus faecium (VREF) from diverse geographical areas in Korea to obtain insights into the genetic relationships with other molecular profiles. To understand the diversity of lineages, vancomycin-susceptible E. faecium (VSEF) were included.

Methods and Results:  A total of 60 E. faecium isolates were analysed by MLST and esp profile. Molecular typing of Tn1546 of 30 VREF strains was evaluated by overlapping PCR of Tn1546 and DNA sequencing. Seven sequence types (ST) were found among 30 VSEF isolates, and four STs were found among 30 VREF isolates. The types most frequently encountered were ST 78 (26 isolates) and ST 203 (16 isolates). Of the 60 E. faecium isolates, 35 isolates were positive for the esp gene. On molecular typing of Tn1546, all VREF isolates were divided into four main types. Strains with the same ST showed divergence in Tn1546 types and strains with the same Tn1546 type represented different STs.

Conclusions:  An association between Tn1546 typing and MLST was not found.

Significance and Impact of the Study:  These results suggest that the horizontal spread of Tn1546 between strains plays a major role in the dissemination of vancomycin resistance in Korea.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Vancomycin-resistant enterococci (VRE) have emerged as the important nosocomial pathogens worldwide (Woodford 1998; Patel 2003), hence it is crucial to elucidate the underlying mechanism in the spreading of VRE. For one of the proposed mechanisms, the vanA gene cluster has been demonstrated to be transferred as a component of Tn1546-like elements. This may indicate that the horizontal transfer of Tn1546-like elements plays an important role in the spreading of vanA type VRE. Therefore, an investigation of the genetic variation of Tn1546-like elements would be essential to understand the mechanism of VRE spreading, particularly in the case of horizontal gene transfer (Schouten et al. 2001). The majority of variations are composed of an integration of the insertion sequences (IS) with or without a deletion at the insertion site, point mutations and deletions.

The continuing spread of VRE is associated with the development of standardized typing methods for molecular epidemiological purposes. Among these methods, pulsed-field gel electrophoresis (PFGE) has been used for comparing the restriction pattern of the whole genome (Morrison et al. 1999). It is capable of distinguishing between clonal populations of VRE; however, it fails to show the relatedness among epidemiologically unrelated isolates.

Moreover, PFGE and structural analysis of Tn1546 are used for short-term epidemiological studies but are less sufficient for global epidemiology where the relatedness of isolates obtained from different countries needs to be assessed. Long-term epidemiology, therefore, requires the use of variation that accumulates with a relatively slow rate. Multilocus sequence typing (MLST) is a new molecular typing method that is based on the well-tested principles of multilocus enzyme electrophoresis. MLST assigns the alleles at each locus directly by nucleotide sequencing, rather than indirectly from electrophoretic mobilities (Enright and Spratt 1999; Homan et al. 2002; Bonora et al. 2004), hence it is highly reproducible. The MLST scheme is provided in the website (http://www.mlst.net).

Since the first isolation of the VRE in Korea in 1992, the prevalence of VRE has dramatically increased in the last decade (Kim and Song 1998; Lee et al. 2001). Enterococcus faecium was the most prevalent isolate for vancomycin resistance. Nowadays, vancomycin-resistant E. faecium (VREF) has become endemic in many hospitals in Korea (Shin et al. 2003; Yu et al. 2003). In this study, we described the MLST analysis for VREF isolated from diverse geographical areas in Korea to determine the genetic relationships with other molecular profiles. To understand the diversity of lineages, we included vancomycin-susceptible E. faecium (VSEF).

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Bacterial strains

During 2002–2004, 60 clinical isolates of E. faecium were collected from three different university hospitals (AJ, CA and YS) from diverse geographical areas in Korea. PFGE was used to exclude duplicate isolates to focus on strain-independent variability. The isolates genetically unrelated in the PFGE pattern were included in our study. PFGE was performed on a CHEF-DR III apparatus (Bio-Rad Laboratories, Richmond, CA, USA) as described previously (Murray et al. 1990). Previously characterized VRE strain, E. faecium BM4147, was used as the control (Arthur et al. 1993). Organisms were identified using conventional biochemical reactions, the Vitek identification system (BioMérieux, Hazelwood, MO, USA) and API20 Strep system (BioMérieux).

MLST

MLST was performed according to the scheme described previously (Homan et al. 2002). Internal fragments of seven housekeeping genes (adk, atpA, ddl, gdh, gyd, purK and pstS) were amplified by PCR and directly sequenced. The allele number for each gene assigned based on the E. faecium MLST database (http://www.mlst.net). The combination of the allelic sequences for the seven genes yielded the allelic profile [sequence type (ST)] for each isolate.

Esp gene profile

The esp gene encodes the enterococcal surface protein, which was first described for E. faecalis and thought to be involved in the adhesion and colonization of the urinary tract (Shankar et al. 1999). The presence of the esp gene in isolates was determined by PCR using primers esp11 and esp12, as described by Shankar et al. (1999). To determine repeat number variations of esp A and C repeats, two different primer combinations were used; espfs7F–espfm5R and espfm5F–espfs3R respectively (Leavis et al. 2004).

Molecular characterization of Tn1546

Extraction of bacterial DNA was performed by using the Qiagen DNeasy Kit (Qiagen GmbH, Hilden, Germany) according to the manufacturer's instruction. For structural analysis of Tn1546, the overlapping PCR amplification of internal regions of Tn1546 was performed. The purified PCR products were directly sequenced by using an ABI Prism 3100 DNA Sequencer (Applied Biosystems, Foster city, CA, USA). The dnasis for windows v.2·6 (Hitachi Software Engineering, South San Francisco, CA, USA) was used for sequence analysis.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Bacterial isolates

A total of 60 E. faecium isolates were recovered from urine (n = 38), wound (n = 12), blood (n = 7), pleural fluid (n = 2) or peritoneal fluid (n = 1) cultures from hospitalized patients. Among all isolates, 30 isolates were resistant to vancomycin and were vanA genotype, whereas the remaining 30 isolates were susceptible to vancomycin.

MLST typing and esp repeat profile

A total of 60 isolates were analysed by MLST and esp profile. Seven STs were found among 30 VSEF isolates (Fig. 1), and four STs were found among 30 VREF isolates (Fig. 2). ST 203 and ST 205 were unique allelic types in Korea. The types most frequently encountered were ST 78 (26 isolates) and ST 203 (16 isolates). Among VREF isolates from AJ hospital, only two allelic types ST 78 and ST 203 were found. Ten of 12 VREF isolates from AJ hospital were ST 78. However, VREF isolates from CA and YS hospital revealed diverse STs. All VREF and VSEF isolates except one strain shared allele purK1.

image

Figure 1.  Dendrogram (categorical, UPGMA) showing the genetic relatedness among the sequence types (STs) of vancomycin-susceptible Enterococcus faecium. The following data are included: ST, allelic profile; presence of the esp gene with repeat profile. AJS, isolates from AJ hospital; CAS, isolates from CA hospital; YSS, isolates from YS hospital.

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image

Figure 2.  Dendrogram (categorical, UPGMA) showing the genetic relatedness among the sequence types (STs) of vancomycin-resistant Enterococcus faecium. The following data are included: ST, allelic profile; Tn1546 type; presence of the esp gene with repeat profile. AJ, isolates from AJ hospital; CA, isolates from CA hospital; YS, isolates from YS hospital.

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Of the 60 E. faecium isolates, 35 isolates were positive for esp gene (60% in VREF and 57% in VSEF) and the frequency observed in the VREF and VSEF was not different. Among VREF (Fig. 2), 10 of 12 isolates from AJ hospital were esp positive but the incidence of isolates with the esp gene from CA and YS hospitals were lower (42% and 45% respectively). In AJ hospital, isolates with ST 78 uniquely contained the esp gene. The number of A repeats varied from three to six, whereas the number of C repeats varied from three to eight, resulting in five distinct esp-repeat profiles with the majority of the isolates possessing 6A and 7C repeats.

Molecular structural analysis of Tn1546

All isolates were divided into four main types according to the distribution of IS-integrated Tn1546 elements (Fig. 3). Type P was identical to the pattern predicted for the published sequence of Tn1546 (Arthur et al. 1993). Type I was characterized by an IS1542 insertion in the orf2vanR intergenic region. Type II was characterized by an IS1542 insertion in the orf2vanR intergenic region and an IS1216V insertion in the vanXvanY intergenic region. Type III represented two copies of IS1216V at the left end of Tn1546 and in the vanXvanY intergenic region as well as IS1542 in the orf2vanR intergenic region. Type IIIb was the most prevalent among isolates.

image

Figure 3.  Genetic maps of Tn1546 types of Enterococcus faecium isolated from Korean hospitals. The positions of genes, open reading frames (orf1 and orf2) and the direction of transcription are marked by open arrows at the top of the figure. Dotted boxes represent insertion sequence (IS) elements. The position of the first nucleotide upstream and the first nucleotide downstream from the IS insertion sites are depicted. Solid arrows indicate the transcriptional orientation of the inserted IS elements. Deletions are indicated by dotted lines.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

MLST is a new molecular typing method and its application for VREF was well described by Homan et al. (2002) who suggested that epidemic lineages of E. faecium emerged worldwide, while genetic variation in both VREF and VSEF was created by long-term recombination. A specific genetic lineage (C1), associated with nosocomial outbreaks and infections and clearly distinct from lineages composed of human community- and animal-derived isolates, was identified (Homan et al. 2002). According to Homan's scheme, all epidemic VREF strains have belonged to epidemic genetic lineage, C1 and the presence of the esp virulence gene (Homan et al. 2002; Bonora et al. 2004).

In our study, seven STs were identified among all isolates. The most frequent types were ST 78 and ST 203. ST 78 was reported in the epidemic strains isolated from Italy (Bonora et al. 2004). ST 203 and ST 205 were unique allelic types in E. faecium isolated from Korea. VREF isolates belonged to four STs and VSEF isolates belonged to seven STs. VSEF was genetically more diverse than VREF. Ten of 12 VREF isolates from AJ hospital represented ST 78, whereas only four of 10 VSEF isolates from the same hospital were ST 78. These data suggest that the distribution of VREF from AJ hospital resulted from strains carrying the unique allele. In contrast, VREF isolates from CA and YS hospitals were overall genetically diverse than the isolates from AJ hospital. All VREF isolates and 29 of 30 VSEF isolates were found in MLST lineage C1, which shared the purK1. The other isolate of VSEF possessed purK2. These results suggest that the lineage C1 is the most prevalent in both VREF strains and VSEF strains isolated from hospitalized patients in Korea.

Willems et al. (2001) reported that the esp gene was associated with epidemic VREF clones. In this study, the percentage of esp-positive isolates was 58% and the frequency between VREF and VSEF was not different. However, the epidemic lineage isolated from AJ hospital was characterized by the presence of the esp gene. The incidence of esp-positive isolates in AJ hospital was significantly higher, which might be associated with the spread of epidemic strains. The repeat profile of the esp gene was highly polymorphous, with variations in the numbers of A and C repeats, even among isolates of the epidemic lineage. No association was found between the number of A or C repeat units within the esp gene and the ST in esp-positive isolates. This finding suggests that esp repeat profiles are short-term epidemiological tools.

On molecular typing of Tn1546, all VREF isolates were classed into four main types according to the distribution of IS-integrated Tn1546 elements. Five isolates from CA hospital revealed prototype (type P), which is identical to transposon Tn1546, as described by Arthur et al. (1993), whereas no isolates from AJ and YS hospitals represented type P. The distribution of type P in CA hospital could be related to the use of teicoplanin, which has been used instead of vancomycin. Such a trend of glycopeptide use might have introduced elements of resistance – an aspect that warrants future studies. The remaining 25 nonprototype elements harboured IS1542, which is restricted to Europe (Darini et al. 1999; Schouten et al. 2001). The insertion of IS1542 within the orf2vanR intergenic region (nucleotide position 3932) may be an essential event in the genetic rearrangement of VREF in Korea. Type IIIb was the most prevalent isolate, suggesting that the inter-hospital spread of Tn1546 and the specific Tn1546 type may affect their transmissibility.

An association between Tn1546 typing and MLST was not found. Strains with the same ST showed divergence in Tn1546 types and strains with the same Tn1546 type showed different STs. These results suggest that the horizontal spread of Tn1546 between strains is proposed to play a major role in the dissemination of vancomycin resistance in Korea.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

We thank K. Lee and W. S. Shin for providing E. faecium strains. This work was supported by the Korea Research Foundation Grant R04-2004-000-10047-0.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
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