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Journal of Clinical Microbiology, June 2001, p. 2140-2145, Vol. 39, No. 6
Division of Clinical Pathology and
Experimental Pathology, Department of Pathology, Tri-Service General
Hospital and National Defense Medical Center, Taipei, Taiwan,
Republic of China
Received 22 January 2001/Returned for modification 11 March
2001/Accepted 17 March 2001
Thirty-six VanB glycopeptide-resistant Enterococcus
faecium isolates were collected from patients in five different
hospitals in Taiwan. The vancomycin resistance genes were amplified by
the long vanB PCR, which amplifies the 6,373-bp
vanB gene cluster including the
vanRB2, vanSB2,
vanYB2, vanWB2,
vanHB2, vanB2, and vanXB2 genes. The deduced amino acid sequences
were found to be 95 to 98% homologous to those of the
vanB1 gene cluster: VanRB1, 97%;
VanSB1, 97%; VanYB1, 96%; VanHB1,
95%; VanB1, 96%; and VanXB1, 98%. Restriction enzyme
analysis of the long vanB PCR products revealed that all 36 isolates had the same vanB2-specific pattern. DNA sequence
analysis of the vanB2 gene, which is a
D-Ala-D-Lac ligase gene, revealed that none of
the 36 sequences were identical to the previously published
vanB2 sequence. Thirty-one isolates had 1 nucleotide
different from the published vanB2 sequence. The sequences
of the other five isolates differed from the published vanB2 sequence by 2 or 3 nucleotides. Four isolates with a
low or moderate resistance to vancomycin (MIC = 4 to 32 µg/ml)
were found to have the same leucine-to-methionine change at amino acid position 308 of the vanB2 gene. The genomic DNAs of all 36 isolates were digested with SmaI and then typed by
pulsed-field gel electrophoresis (PFGE). Eight different PFGE types (I
to VIII) were observed, and type I was found to be prevalent in all
hospitals examined in this study. This result suggests that intra- and
interhospital dissemination of this E. faecium strain has
occurred in Taiwan.
Glycopeptide resistance in
enterococci is genotypically and phenotypically diverse. The VanA and
VanB types are the two most common classes of acquired glycopeptide
resistance in enterococci. VanA enterococci are resistant to high
levels of vancomycin and teicoplanin due to the presence of the
vanA gene cluster that includes the vanR, vanS, vanH,
vanA, vanX, vanY, and vanZ genes (1). The
vanA gene cluster is usually located on transposon Tn1546 or related elements (2). Strains with
the VanB phenotype have various levels of resistance to vancomycin but
are susceptible to teicoplanin (25). The VanB-type
resistance is mediated by the vanB gene cluster that
includes the vanRB, vanSB,
vanYB, vanW, vanHB,
vanB, and vanXB genes (8). The
vanB gene cluster may reside on Tn1547, which is
flanked by two distantly related insertion sequences, an
IS256-like element and IS16, in direct-repeat
orientation (24). A novel vanB-containing
transposon, Tn5382, was recently described in a
vancomycin-resistant Enterococcus faecium (4).
The vanB gene encodes a D-Ala-D-Lac
ligase and is classified into three different types, vanB1,
vanB2, and vanB3, based on DNA sequence heterogeneity
(11, 22). The vanB1 gene was previously referred to as vanB (6, 10). The
vanB2 gene was first found in Enterococcus
faecalis SF300 (11). The vanB3 gene was
found recently in two E. faecalis isolates
(22). Its nucleotide sequence differs from that of
vanB1 by 5% (22) and from the published vanB2 sequence by 3.6% (22).
We have recently used PCR primers specific for the vanB1
gene (6) to amplify the vancomycin resistance gene from
VanB enterococci in Taiwan. The resulting PCR products were found to be
1,100 bp, not the expected 433 bp. The PCR product of one isolate,
E. faecium TSGH1, was sequenced and found to contain a
portion of the vanHB2 gene, the entire
vanB2 gene, and a portion of the
vanXB2 gene (GenBank accession no. Z83305). The
vanB2 sequence of TSGH1 was found to differ by only 1 nucleotide from that of the published 801-bp vanB2 gene
(22). This result suggests that E. faecium TSGH1 harbors a vanB2 gene cluster. We therefore examined
all VanB E. faecium isolates that we have collected. The
entire vanB gene cluster of each isolate was amplified, and
the vanB ligase gene of each isolate was sequenced.
Pulsed-field gel electrophoresis (PFGE) was performed to identify the
predominant strain of E. faecium in Taiwan.
Bacterial isolates.
Thirty-six clinical isolates of VanB
E. faecium collected from five hospitals (13 isolates from
hospital A, 4 from hospital B, 5 from hospital C, 2 from hospital D,
and 12 from hospital E) in Taiwan were studied. They were not
consecutive vancomycin-resistant enterococcus (VRE) isolates. They were
selected because their stock cultures were available and they were
previously determined to carry the vanB gene
(20). E. faecalis V583 CDC containing vanB1 (26), E. faecium TUH2-18
containing vanB2(7), and E. faecium
VRE45 containing vanB3 (7) were used as
controls in PCR, PFGE, and DNA sequencing.
Antimicrobial susceptibility assay.
VanB E. faecium isolates were assayed for susceptibility to vancomycin and
teicoplanin by the E test (AB Biodisk, Piscataway, N.J.) according to
the manufacturer's instructions. E. faecalis ATCC 29212 was
used as the control strain for the susceptibility test.
PCR amplification.
Bacterial DNA was isolated as previously
described (19). Briefly, the DNA from a loop of cultured
bacteria was extracted by boiling the bacteria with lysis buffer (1%
Triton X-100, 10 mM Tris-HCl [pH 8.0], 1 mM EDTA) for 30 min. After
centrifugation, the DNA in the supernatant was analyzed for the
presence of the van gene cluster by PCR. PCRs were performed
in the GenAmp PCR system (model 2400; Perkin-Elmer, Norwalk, Conn.).
Based on sequence analysis of the vanA and vanB
gene clusters, primers VB211F and VB6545R were designed (Table
1) to amplify the 6,373-bp
vanB2 gene cluster. This PCR method was named the long
vanB PCR. The PCR mixture contained 50 ng of template DNA,
PCR buffer (10 mM Tris-HCl [pH 8.3], 50 mM KCl, 2.5 mM
MgCl2, 0.001% gelatin), 20 pmol of each PCR primer, 0.4 mM
each deoxynucleoside triphosphate, and 2.5 U of TaKaRa Ex
Taq DNA polymerase (Takara Shuzo Co., Ltd., Otsu, Shiga, Japan) in
a total volume of 100 µl. After a 10-min denaturation at 94°C, the
reaction mixture was run through 40 cycles of denaturation for 1 min at
94°C, annealing for 1 min at 55°C, and extension for 6 min at
72°C and then incubated for 10 min at 72°C. After amplification, 5 µl of PCR products was electrophoresed on a 1% agarose gel in TBE
buffer (0.0445 M Tris, 0.0445 M boric acid, 0.001 M EDTA) containing
ethidium bromide (0.5 µg/ml). The PCR products were purified with the
GFX PCR DNA and Gel Band Purification kits (Amersham Pharmacia Biotech,
Inc., Piscataway, N.J.) according to the manufacturer's instructions.
0095-1137/01/$04.00+0 DOI: 10.1128/JCM.39.6.2140-2145.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
High Prevalence of VanB2 Vancomycin-Resistant
Enterococcus faecium in Taiwan
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
TABLE 1.
Primers used to characterize the vanB2 gene
cluster in this study
DNA sequencing. The long vanB PCR products were sequenced by primer walking using primers designed according to the published sequence of the V583 vanB gene cluster or new sequences obtained in this study (Table 1). The vanB2 ligase genes in various isolates were sequenced using primers VB5382F, VB5367R, VB5745F, and VB5808R (Table 1). The BigDye Terminator Cycle Sequencing Ready Reaction kit (Applied Biosystems Inc., Foster City, Calif.) was used for sequencing.
Restriction fragment length polymorphism (RFLP) analysis. Ten microliters of each long vanB PCR product was digested with different restriction enzymes in a total volume of 20 µl. After incubation for 2 h at 37°C, the digested DNA samples were electrophoresed on a 1% agarose gel in TBE buffer containing ethidium bromide (0.5 µg/ml).
Typing of VRE by PFGE. Preparation of bacterial DNA for PFGE was performed as described by Gouby et al. (12). Bacteria were grown overnight on blood agar plates and then suspended in 10 mM Tris-0.1 mM EDTA solution to a concentration of 10 on the McFarland scale. This bacterial suspension was mixed with an equal volume of molten 1.6% low-melting-point agarose and then cast into plugs. The plugs were treated with lysis solution (6 mM Tris-HCl [pH 7.6], 100 mM EDTA [pH 7.5], 1 M NaCl, 0.2% deoxycholate, 0.5% sodium lauroylsarcosine, 1 mg of lysozyme/ml) at 37°C for 24 h. The solution was subsequently replaced with ESP buffer (0.5 M EDTA, 1% sodium lauroylsarcosine, 0.5 mg of proteinase K/ml) and incubated at 37°C for 24 h. The plugs were then washed once in distilled water and four times in 10 mM Tris-0.1 mM EDTA buffer at 37°C for 30 min each time.
The genomic DNA embedded in the plugs was digested overnight with 50 U of SmaI (New England Biolabs, Beverly, Mass.) in a total reaction volume of 0.3 ml in NEB4 buffer (New England Biolabs) at 25°C. The plugs were then washed in 10 mM Tris-0.1 mM EDTA buffer at 37°C for 1 h, loaded into wells of a 1% agarose gel, and then electrophoresed in 0.5× TBE buffer. A plug containing a 50-kb-to-1-Mb lambda DNA ladder (Bio-Rad Laboratories, Hercules, Calif.) was used as the molecular size marker. PFGE was performed with a CHEF Mapper XA system (Bio-Rad Laboratories) at 14°C under the autoalgorithm mode (gradient, 6 V/cm; run time, 27 h 12 min; included angle, 120; initial switch time, 2.91 s; final switch time, 35.38 s; ramping factor, linear). The gel was stained with ethidium bromide and then photographed. The PFGE results were interpreted according to the criteria of Tenover et al. (27).Nucleotide sequence accession numbers. The nucleotide sequences of the whole vanB2 gene cluster of isolate TSGH1 and the vanB2 ligase genes of isolates VRE-1, SLH475, and CG4248 have been deposited in GenBank with accession numbers AF310956, AF310953, AF310954, and AF310957, respectively.
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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All 36 VanB isolates were amplified by the long vanB
PCR (Table 2). The sizes of amplified
products were approximately 6,400 bp. The PCR products were digested
with HaeII or BclI, and all of the products
yielded identical HaeII or BclI digestion
patterns that were characteristic of vanB2 but different
from those of the vanB1 or the vanB3 gene cluster
(Fig. 1).
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The entire 6,400-bp vanB2 gene cluster of isolate TSGH1 was
sequenced. This gene cluster was found to contain the 663-bp
vanRB2, 1,344-bp vanSB2,
807-bp vanYB2, 828-bp
vanWB2, 972-bp
vanHB2, 1,029-bp vanB2, and 609-bp
vanXB2 genes in addition to the 175-bp vanSB-vanYB intergenic region. The
nucleotide sequences of the vanB2 gene cluster were found to
be more similar to those of the vanB1 gene cluster (94 to
96% homology) than to those of the vanA gene cluster (48 to
81% homology) and the vanD gene cluster (33 to 71%
homology). The deduced amino acid sequences of the vanB2 gene cluster also had a higher degree of homology with those of the
vanB1 gene cluster (95 to 97% homology) than to those of
the vanA and vanD gene clusters (14 to 76%
homology) (Table 3).
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The vanB2 ligase genes of all 36 isolates were then sequenced. Four types of vanB2 ligase gene sequences, designated TSGH-1, V4248, VRE-1, and SLH475, were observed. Thirty-one isolates belonged to sequence TSGH-1, three belonged to VRE-1, and one each belonged to CG4248 and SLH475 (Table 2). These four sequences differed from each other by only 5 nucleotides at positions 520, 538, 556, 922, and 967. The nucleotides of TSGH-1 at these positions were T, A, C, C, and A, respectively. Those of V4248 were T, G, C, A, and G. Sequence VRE-1 had T, C, A, A, and A at these positions, and SLH475 had G, G, A, C, and A. These sequences differed from that of the vanB1 ligase gene at 44 to 46 (4.3 to 4.5%) nucleotide positions, resulting in 14 or 15 amino acid changes.
The 1,029-bp vanB2 sequences from the 36 isolates were also compared with the previously published 801-bp vanB2 ligase gene sequence (22), which is missing the first 102 and the last 126 bp. Only 1 to 3 nucleotides were found to be different from the published vanB2 ligase gene sequence. The G-to-C change at nucleotide position 538 of the 31 sequences represented by TSGH-1 resulted in a valine-to-leucine change. The A-to-C and C-to-A changes at positions 556 and 922 of the sequence CG4248 resulted in threonine-to-proline and leucine-to-methionine changes at these positions. The VRE-1 sequence had G-to-C and C-to-A changes at positions 538 and 922, causing valine-to-leucine and leucine-to-methionine changes at these positions. The T-to-G change at position 520 of the sequence SLH475 caused a serine-to-alanine change at that position. Four isolates (CG4248, VRE-1, VRE-3, and VRE-8) were found to have a low or moderate resistance to vancomycin (MIC = 4 to 32 µg/ml) (Table 2). Analysis of the sequences revealed that all four of these isolates had a C-to-A change at position 922, resulting in a leucine-to-methionine change at codon 308 of the vanB2 ligase gene.
Eight different PFGE types, designated types I to VIII, were observed
(Fig. 2). Type I was found to be
predominant (Table 2). Twenty-five (69.4%) isolates belonged to type
I, three isolates were type II, and two isolates each belonged to types
III and VIII. One isolate each belonged to types IV, V, VI, and VII.
Type I was also found to be the predominant type in all of the
hospitals. Types II and III were found only in hospitals A and C. None
of the control strains, one each VanB1 and VanB3 strain from the United
States and one VanB2 strain from Norway, belonged to these eight PFGE
types (Table 2).
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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In this study, we found that all 36 VanB E. faecium isolates examined harbored the vanB2 gene cluster. No VanB1 or VanB3 E. faecium were found. These 36 VanB E. faecium isolates were collected from five different hospitals in Taiwan. To our knowledge, this is the first report that all VanB E. faecium isolates from a certain geographical location harbor the vanB2 gene. Previous studies have shown that VanB2 VRE are uncommon in the United States but are quite common in Europe (6, 7, 21). Dahl et al. (7) have recently examined 17 VanB VRE isolates from hospitalized patients in Europe and the United States and showed that only 2 of 9 U.S. isolates but 7 of 8 European isolates harbored the vanB2 gene. McGregor and Young (21) found that 28 of 32 VanB VRE from hospitalized patients in Scotland harbored vanB2. Clark et al. (6) examined 105 clinical isolates of VRE collected from 31 U.S. hospitals in 14 states and found that all 26 VanB VRE isolates harbored the vanB1 gene.
The results of this study also revealed sequence heterogeneity of the vanB ligase genes in different VRE isolates. The vanB2 sequences obtained in this study had a marked difference from those of vanB1 (4.3 to 4.5% difference) and vanB3 (2.8 to 3% difference). In addition, none of the 36 vanB2 ligase gene sequences determined in this study were identical to that of the previously described vanB2 ligase gene (22). There were 1- to 3-nucleotide differences between that sequence and our vanB2 ligase gene sequences. However, the HaeII and BclI digestion patterns of the long vanB PCR products of all 36 isolates are identical. This observation could provide an alternative means for identification of VanB2 VRE for epidemiological or surveillance studies.
Although there are several types of vanB, no correlation has been observed between vanB subtypes and levels of vancomycin resistance (7). A unique finding in this study is that all four VanB2 VRE isolates with a low or moderate resistance to vancomycin (MIC = 4 to 32 µg/ml) had a leucine-to-methionine change at codon 308 of the vanB2 gene. Whether this change actually results in a low or moderate vancomycin resistance in VanB2 VRE remains to be investigated.
The sequence heterogeneity in the vanB ligase genes is in contrast to the reported DNA sequence homogeneity in the vanA ligase gene (13, 14, 28). The sequences of the vanA ligase genes in different VRE isolates appeared to be identical. However, the Tn1546 or Tn1546-like elements that carry the vanA gene clusters are structurally different (13, 14, 28). The structural diversity in Tn1546 elements is caused by deletions or insertions which may have occurred during recombination events (13, 22, 28). VanA VRE have been isolated from a variety of sources, including animals, animal foodstuffs, and humans (14-17, 28). The food chain has been suggested to be a source of VanA VRE (5, 18). To our knowledge, VanB VRE have only been detected in hospitalized patients. The finding that type I E. faecium harboring the vanB2 gene was prevalent (69.4%) suggests that intra- and interhospital dissemination of this VRE has occurred in Taiwan. Because of the frequent occurrence of oxacillin-resistant Staphylococcus aureus, the use of vancomycin has increased tremendously in Taiwan during the last decade. It is possible that the spread of the type I VanB2 E. faecium in Taiwan is the result of unregulated use of vancomycin in humans.
The vanB gene cluster has been found to be associated with various DNA elements ranging from 60 to 250 kb (3, 4, 23, 29). A 64-kb composite transposon (Tn1547), bounded by IS256-like and IS16 elements, has been shown to transpose the vanB gene cluster from the chromosome to a plasmid (23). A 55-MDa transferable plasmid was found to contain both vanB and a gentamicin resistance gene (29). A 27-kb conjugative transposon (Tn5382) which may be part of a 160-kb plasmid containing both the vanB gene and the pbp5 gene, which encodes high-level ampicillin resistance, has also been described (4). The observation that HaeII and BclI restriction maps of the vanB gene cluster in various E. faecium isolates are identical suggests that the entire vanB2 gene cluster is transferred from one isolate to another. In addition to horizontal transfer of the vancomycin resistance gene due to DNA transposition or plasmid transfer, clonal dissemination of a certain strain is another mode of transmission. The finding that the majority (65%) of VanB isolates examined in this study belong to the same PFGE type (type I) is one example of this type of transmission. Based on the results of this study, it is recommended that both the mobile vancomycin resistance determinants and the PFGE types should be determined for epidemiological studies of VRE infections.
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ACKNOWLEDGMENTS |
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We thank Jiunn-Jong Wu, National Cheng-Kung University Medical College; Tsu-Lan Wu, Chang Gung Memorial Hospital; and Po-Ren Hsueh, National Taiwan University Hospital, for supplying VRE isolates. This study was supported by grants from the National Science Council (NSC88-2314-B-016-053 and NSC89-2320-B-016-033), Taiwan, Republic of China.
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FOOTNOTES |
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* Corresponding author. Mailing address: Molecular Diagnostics Laboratory, Division of Clinical Pathology, Department of Pathology, Tri-Service General Hospital, No. 325, Section 2, Chengkung Rd., Taipei 114, Taiwan, Republic of China. Phone: 886-2-87927227. Fax: 886-2-87927227. E-mail: jjl{at}ndmctsgh.edu.tw.
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Materials and Methods
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Discussion
References
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Linkage of vancomycin and high-level gentamicin resistance genes on the same plasmid in a clinical isolate of Enterococcus faecalis.
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35:179-184 |
Journal of Clinical Microbiology, June 2001, p. 2140-2145, Vol. 39, No. 6
0095-1137/01/$04.00+0 DOI: 10.1128/JCM.39.6.2140-2145.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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