Department of Medical Microbiology, University and University Hospital of Tromsø, N-9037 Tromsø, Norway1
Norwegian Institute for Gene Ecology, N-9037 Tromsø, Norway2
Author for correspondence: Kristin H. Dahl. Tel: +47 77 64 57 62. Fax: +47 77 64 53 50. e-mail: kristind{at}fagmed.uit.no
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ABSTRACT |
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Abbreviations: IS, insertion sequence; VRE, vancomycin-resistant enterococci
GenBank and GenPept accession numbers are given in Table 3.
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INTRODUCTION |
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Acquired glycopeptide resistance in enterococci is phenotypically and genotypically heterogeneous, with VanA and VanB phenotypes being the most commonly encountered forms (Arthur et al., 1993 ; Quintiliani & Courvalin, 1996
). Their mechanism of resistance involves the synthesis of peptidoglycan precursors that terminate in D-lactate instead of D-alanine, thus reducing the affinity for glycopeptide antibiotics (Evers & Courvalin, 1996
). The vanA gene cluster is located on the non-conjugative transposon Tn1546 or related elements, which can be part of large mobile chromosomal elements as well as non-conjugative and conjugative plasmids (Arthur et al., 1993
; Handwerger & Skoble, 1995
; Handwerger et al., 1995
). Dissemination of the vanB gene cluster appears to result from conjugation of plasmids (Woodford et al., 1995
) or intercellular transfer of chromosomal elements (Carias et al., 1998
; Quintiliani & Courvalin, 1996
) as well as clonal spread. The vanB gene cluster has not been universally linked to a specific mobile genetic element. Recently an approximately 27 kb putative conjugative transposon Tn5382 containing the vanB gene cluster was described. Intercellular transfer of Tn5382 as an integral part of a larger chromosomal element also conferring ampicillin resistance was documented (Carias et al., 1998
), but transposition of Tn5382 itself has not been shown. Intracellular transposition of the vanB gene cluster as part of the composite transposon Tn1547 flanked by insertion sequence elements IS16 and IS256-like to a conjugative plasmid, and interspecies transfer of plasmids or large chromosomal elements containing Tn1547, have also been described (Quintiliani & Courvalin, 1996
). The epidemic potential of conjugative plasmids containing vanB is indicated by the presence of transferable vanB-encoding plasmids in unrelated clinical Enterococcus faecium and E. faecalis strains (Woodford et al., 1995
).
Based on sequence differences the vanB gene cluster can be divided into at least three distinct subtypes: vanB1, vanB2 and vanB3 (Dahl et al., 1999 ; Patel et al., 1998
). In this study we report that the vanB2 subtype gene cluster is an integral part of the putative conjugative transposon Tn5382. Two novel ISs, ISEnfa110 and ISEnfa200, were found in specific vanB2 subtype strains of vancomycin-resistant enterococci (VRE). These IS elements were characterized with respect to their DNA sequence and distribution in enterococci.
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METHODS |
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Preparation of genomic DNA.
DNA for genomic digestion was isolated using the QIAGEN Blood & Cell Culture DNA kit. Bacterial DNA for PCR was prepared with a Dynabeads DNA DIRECT Kit (Dynal) as described by Dahl et al. (1999) or by isolation of total DNA using the GenomicPrep Cells and Tissue DNA Isolation Kit (Pharmacia Biotech) with modification of the lysis step. The bacterial cells were incubated in fresh lysis solution (10 mM Tris/HCl pH 8·0, 1 mM EDTA, 25%, w/v, sucrose, 10 mg lysozyme ml-1 and 0·1 mg RNase A ml-1) at 37 °C for 35 min to achieve optimal lysis.
DNA transfer and hybridization.
Southern transfer of digested genomic DNA to a positively charged nylon membrane (Boehringer Mannheim) was carried out by vacuum blotting (Vacugene XL system, Pharmacia Biotech) according to the manufacturers instructions. Colony blotting was performed as described by Sambrook et al. (1989) with the following modification: the nylon membrane was incubated on a 3 MM paper saturated with fresh lysis solution (10 mM Tris/HCl, 1 mM EDTA, 25%, w/v, sucrose, 10 mg lysozyme ml-1) for 60 min at 37 °C to ensure complete lysis of the enterococci. DNA was fixed to the nylon membrane by UV cross-linking. Probes were labelled using the PCR DIG Probe Synthesis Kit (Boehringer Mannheim) and purified by agarose gel electrophoresis followed by extraction by QIAquick Gel Extraction Kit (QIAGEN). Hybridization was carried out at 68 °C and detection performed using the DIG Luminescent Detection Kit (Boehringer Mannheim). All protocols were performed according to the manufacturers instructions.
Total DNA from the following bacteria was used as templates for probe synthesis: the vanB probe came from E. faecalis V583 (Evers et al., 1994 ), the Tn5382 and pbp5 probes from E. faecium C68 (Carias et al., 1998
), the IS16 and IS256 probes from E. faecalis BM4281 (Quintiliani & Courvalin, 1996
), the ISEnfa110 probe from TUH4-67, the ISEnfa200 probe from TUH7-15, and the 16S rDNA probe from E. faecalis DS16C2 (Franke & Clewell, 1981
). The 16S rRNA genes in E. faecium and E. faecalis have at least 98% homology.
PCR.
PCRs were performed in a GeneAmp PCR System 2400 (Perkin-Elmer) using Perkin-Elmer Standard PCR reaction mix with GeneAmp PCR buffer and Taq DNA polymerase. PCR elongation times were adjusted according to the expected size of PCR amplicons and the alignment temperatures were adjusted according to the specific nucleotide sequence of the primers and hence their melting temperatures.
PCR amplicons and primer sequences used in PCR identification, DNA sequencing and probe syntheses are listed in Table 2. Additional primer sequences and amplicons used in the characterization of ISEnfa110 and ISEnfa200 are described in Fig. 1(a)
and (b
), respectively.
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Computer analysis and sequence accession numbers.
Editing, initial analysis of the DNA sequences and alignments were performed using the Sequence Navigator (Perkin Elmer) software package. Nucleotide sequences were compared to sequences in the GenBank, EMBL, DDBJ and PDB databases and protein sequences to non-redundant GenBank CDS translations, PDB, SWISS-PROT, SPupdate and PIR by using the BLASTN, BLASTP and BLASTX local alignment search tools (Altschul et al., 1990 ). Possible ORFs were found using the ORF finder located at the National Center for Biotechnology Information website. Searches for repeats and palindromes were performed using the GCG package. RNAdraw V1.0 was used to determine potential secondary structures. Novel nucleotide accession numbers and previously published GenBank and GenPept sequences mentioned in the text are shown in Table 3
.
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RESULTS AND DISCUSSION |
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The vanB2 gene cluster is an integral part of Tn5382
A vanB-containing 27 kb putative conjugative transposon, Tn5382, was recently described in E. faecium C68. The presence of Tn5382 was also documented in 15 strains from the USA as well as in one strain from France (Carias et al., 1998 ). In this study, genetic linkage of the specific vanB subtypes to Tn5382 was examined. The VanB strain collection was analysed by a Tn5382-specific PCR internal to the nonintegrase (left) end of Tn5382 (Carias et al., 1998
), Southern hybridization using Tn5382 and vanB amplicons as probes (Table 2
), and DNA sequencing of the vanXBORFC intergenic region and flanking coding sequences in Tn5382 (Fig. 2
). Fourteen of the 23 strains (61%) were Tn5382 PCR positive (Table 1
), which is comparable to the result obtained by Carias et al. (1998)
. All Tn5382 PCR positive strains were of the vanB2 subtype (Dahl et al., 1999
). SmaI-digested total DNA was subjected to PFGE (Fig. 3a
) and Southern hybridization (Fig. 3b
, c
). Co-hybridization of the vanB and Tn5382 probes to the same fragments was detected for all strains (n=14) of the vanB2 subtype, as illustrated in Fig. 3
for strains TUH2-18 (lane 4) and TUH7-15 (lane 7). Strains of the vanB1 or vanB3 subtypes were negative for Tn5382 by PCR and hybridization (Fig. 3
, lanes 2, 3 and 9).
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The DNA sequence of the vanB2 ligase gene seems to be less conserved than the vanB1 ligase gene (Dahl et al., 1999 ; Patel et al., 1998
), indicating that the vanB2 gene cluster might be older in evolutionary terms or evolve more rapidly due to more efficient mechanisms for horizontal spread. As part of Tn5382, the vanB2 gene cluster may be transferable between cells and within the cell to conjugative chromosomal elements or plasmids, thereby promoting a more efficient spread as indicated by the finding of a wide geographical distribution of the vanB2 gene cluster. However, molecular epidemiological studies with regard to the relative distribution of vanB subtypes have not been performed to test this assumption.
Presence of IS16 and IS256-like in VanB-type VRE strains
The vanB gene cluster in E. faecalis BM4281 has been reported to be linked to the mobile genetic elements IS16 and IS256-like as a functional composite transposon, Tn1547. Hybridization studies of clinical VanB-type VRE isolates revealed that the vanB gene cluster was not necessarily linked to IS16 or IS256-like elements (Quintiliani & Courvalin, 1996 ). The vanB gene cluster of Tn1547 (Quintiliani & Courvalin, 1996
) belongs to the vanB1 subtype (this study, Table 3
). In this study the VanB strain collection was examined for prevalence of IS16-like and IS256-like elements and their genetic linkage to vanB gene cluster subtypes.
PCR analyses identified IS16-like sequences in 15 of 23 strains (65%) and IS256-like sequences in 19 of 23 strains (83%) (data not shown). These observations are consistent with the finding of IS16-like sequences in 21 of 32 (66%) and IS256-like sequences in 31 of 32 (97%) clinical VanB-type E. faecium and E. faecalis strains (Quintiliani & Courvalin, 1996 ), and IS256-like sequences in 88 out of 103 (85%) clinical E. faecium and E. faecalis strains (Rice & Thorisdottir, 1994
). PFGE followed by Southern hybridization did not show any universal genetic linkage or co-hybridization of the vanB subtypes to IS16- and IS256-like sequences. Co-hybridization of vanB and both IS16-like and IS256-like sequences was found in only two vanB2 subtype strains, TUH7-15 and 3174. Co-hybridization of vanB and IS16-like sequences was found in two different fragments in TUH1-79, and in one fragment in TUH2-18 and 3568. Co-hybridization of vanB and IS256-like sequences was found in one fragment in V583, TUH7-54 and C68 (data not shown).
Multiple copies of both IS16- and IS256-like sequences were detected in several strains (data not shown). Our results confirm that IS16- and IS256-like sequences are common in enterococci, but that these elements are not generally linked to the vanB1 gene cluster as observed for the composite transposon Tn1547 in E. faecalis BM4281. Multiple copies of these IS elements raise possibilities for IS-based composite transposons and genomic rearrangements. IS256 is capable of forming novel mobile elements in enterococci as illustrated by the detection of Tn5281 (Hodel-Christian & Murray, 1991 ), conferring high level resistance to aminoglycosides, and Tn5384, encoding macrolide resistance and high level gentamicin resistance (Rice et al., 1995
).
Characterization of ISEnfa110, a novel member of the IS110 family
The prevalence of Tn5382 in the VanB strain collection was determined by a Tn5382-specific PCR and hybridization using a 311 bp sequence starting 237 bp downstream of the left inverted repeat of Tn5382 in a nonintegrase sequence (Carias et al., 1998 ). Tn5382 PCR of TUH4-67 DNA showed an enlargement of this region. To identify the exact sequence of this enlargement, the complementary strand of the fragment internal to the nonintegrase (left) end of Tn5382 (Carias et al., 1998
) was sequenced in strain C68 (Table 3
). Sequence analysis of the same region in TUH4-67 revealed a 1611 bp insertion with a putative ORF of 1209 bp transcribed in the opposite direction to the integrase gene of Tn5382 (Figs 1a
and 2
, Table 3
).
No extensive DNA homology was found with known insertion sequences by the BLAST program. However, the deduced 402 amino acid sequence of the ORF within this insert showed low levels (2124%) of identity to about 380 amino acids from putative transposases of IS110, IS2112, IS1110, IS902 and IS901 (Table 3). An alignment of the amino acid sequences of this ORF to transposases of IS elements belonging to the IS110 family (Mahillon & Chandler, 1998
), clearly demonstrated the relatedness between this IS element, hereafter designated ISEnfa110, and IS110 family members. The transposases of the IS110 family show some regions of identity within the N- and C-terminals. Two regions highly conserved in transposases of the IS110 family (Hernandez Perez et al., 1994
) were present in the ISEnfa110 transposase.
Like most of the elements in this family, ISEnfa110 had no terminal inverted repeats. Target sequences of IS110 elements exhibit similarities to the circle junction of the element. Recent in vivo studies of IS110 elements have shown DNA fragments with abutted IS ends only when the transposase gene is intact. These observations are consistent with the model for transposition of IS110 elements by circularization and site-specific recombination (Mahillon & Chandler, 1998 ). A 2 bp sequence (5'-CT-3') was directly repeated at each end of ISEnfa110 after insertion into the integration site. According to the IS110 family criteria these elements do not create target duplications, but a match often occurs between one end of the IS and the target site sequence (Hernandez Perez et al., 1994
). Thus, the CT sequence is probably present both in the attachment site of the circular form of the element and in the target site. ISEnfa110 showed additional sequence homology between the putative circle junction and the site of integration (Fig. 4
).
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Characterization of ISEnfa200: a novel member of the IS200 family
The vanB subtyping strategy revealed an enlargement of the vanSBvanYB intergenic region in strain TUH7-15. Sequence analysis identified a 789 bp insert region (Table 3) with a 471 bp ORF oriented in the same direction as the vanB gene cluster (Figs 1b
and 2
). Searching with the BLAST program in different protein databases revealed sequence similarity to the transposases of the IS200 family, which was strengthened by the preserved structural features of this family (see below). The IS element was designated ISEnfa200.
ISEnfa200 encoded a putative 156 amino acid protein with 70% identity to the first 147 amino acids of the putative 151 amino acid transposase of IS1469 from Clostridium perfringens (Brynestad et al., 1997 ; Table 3
). Amino acid identities above 60% were also detected with putative IS200 transposases of Salmonella typhimurium, S. typhi, Yersinia pestis, Escherichia coli and Actinobacillus actinomycetemcomitans (Table 3
). The IS1469 ORF had sequence homology greater than 60% to ORFs in other IS200-like elements (Brynestad et al., 1997
). In contrast, the ISEnfa200 DNA sequence only showed a significant degree of homology to other IS200 sequences in smaller regions. Thus, ISEnfa200 had a higher level of conservation at the amino acid level compared to the DNA level, suggesting that the coding capacity of the ORF was selectively maintained.
ISEnfa200 possessed general features of IS200 elements. The ISEnfa200 element (787 bp) was comparable in size to IS1469 (789 bp) and Salmonella spp. IS200 (708 bp). The element lacked terminal inverted repeats and had one ORF preceded by several sequences capable of forming hairpin structures (see below). The sequence data indicated that the ISEnfa200 insertion had generated a 2 bp target duplication (5'-TG-3'). However, it is also possible that these 2 bp were part of the ISEnfa200 element or sequences flanking the donor element at the previous target site. ISEnfa200 was inserted in an A+T-rich sequence (68% A+T bp in the neighbouring 22 bp) in a non-coding region upstream of the promoter region in the vanSBvanYB intergenic sequence. Thus, the insertion does not seem to affect expression of the proteins encoded by the vanB gene cluster.
Sequence analysis showed that the primary transcript may form a potential RNA factor independent transcription terminator at the left end of ISEnfa200 [nucleotides 216, G0 -10·9 kcal mol-1 (-45·6kJ mol-1), followed by 4 U residues]. A second potential RNA stemloop structure [nucleotides 119174,
G0 -22.0 kcal mol-1 (-92·0 kJ mol-1)] that might occlude a putative RBS was also present. IS200 in S. typhimurium, S. abortusovis and Vibrio cholerae showed high sequence conservation in the stemloop regions (Beuzón & Casadesús, 1997
). These features still seemed to be preserved in ISEnfa200, although no significant DNA sequence homology was found to other IS200 elements in these regions. The RBS (ACAAAGGAGGN7ATG) upstream of the ISEnfa200 ORF was located at a standard distance from the start codon and displays a strong complementarity (underlined) to the 3' extremity of the B. subtilis 16S rRNA (UCUUUCCUCC) (Moran et al., 1982
). A putative promoter with -35 (CTTACA) and -10 (TATAAT) regions separated by a spacer region of 16 bp was also located upstream of the putative start codon.
Rarity of ISEnfa110 and ISEnfa200 in enterococci
The presence of ISEnfa110- and ISEnfa200-like elements was examined by colony hybridization of 181 Enterococcus strains of geographically diverse origins using internal PCR fragments (Table 2) of the IS elements as probes. All colonies hybridized with the positive control 16S rDNA probe (data not shown). Only TUH4-67 was positive for ISEnfa110, whereas five strains (TUH7-15, C68, 3174, 3332 and 3568), including the original ISEnfa200 strain, were positive for ISEnfa200 (data not shown).
Southern hybridization of total DNA showed hybridization of the ISEnfa110 probe to an approximately 10 kb DraI restriction fragment of strain TUH4-67 (data not shown), indicating the presence of a single copy of ISEnfa110. ISEnfa110 has a G+C content of 51 mol%, which is higher than the G+C range of enterococci (3442 mol%) but in the same range as the target site in Tn5382 (55 mol%). ISEnfa200 hybridization with total DNA revealed a positive 1·5 kb DraI restriction fragment (data not shown) in the five ISEnfa200-positive strains, indicating the presence of a single ISEnfa200 element. The low prevalence of these novel IS elements might reflect recent acquisition or low transposition frequencies of these elements in enterococci. ISEnfa200 has a G+C content of 41 mol%, which is typical of that of Enterococcus species (3442 mol%). The low G+C range of ISEnfa200 reflects to a large extent the preferential use of A and T residues at the third position of codons in the putative ORF.
IS200 elements are mainly found in Gram-negative bacteria, except for IS1469 from C. perfringens and ISEnfa200 in E. faecium. ISEnfa200 had the highest degree of homology to IS1469 (see above). The presence of closely related IS elements in E. faecium and C. perfringens might indicate interspecies gene flux in a common environmental niche. The ISEnfa200 element was inserted in the vanB gene cluster, which has been shown to be transferable among enterococcal strains (Carias et al., 1998 ; Quintiliani & Courvalin, 1996
; Woodford et al., 1995
). Spread of ISEnfa200 will probably be a result of vanB gene cluster transfer rather than transposition of the IS element itself since IS200 elements have extremely low transposition frequencies.
A widespread USA vanB E. faecium strain with a chromosomal Tn5382vanB2ISEnfa200 element genetically linked to pbp5?
Recent reports have shown that the putative conjugative transposon Tn5382 can be located downstream of a pbp5 gene encoding ampicillin resistance in clonally distinct E. faecium strains from different parts of the USA (Carias et al., 1998 ; Hanrahan et al., 1998
). The two resistance determinants were co-transferred during conjugation as parts of a larger genetic element. In this study strains of different geographical origins were analysed for the occurrence of Tn5382 downstream of pbp5, to examine if these resistance elements are linked. The vanB2 positive strains (n=14) were examined for pbp5Tn5382 linkage by PCR-amplifying a 1079 bp region between pbp5 and Tn5382 (Table 3
). Five USA strains (TUH7-15, C68, 3174, 3332 and 3568) revealed a pbp5Tn5382 amplicon of the expected size (data not shown). PFGE analysis of SmaI-digested DNA (Fig. 5
) suggests genetic relatedness between these strains. The ISEnfa200 integration site in the vanSBvanYB intergenic region (Table 3
) was identical in all five strains. Southern blot analyses showed co-hybridization of the pbp5 probe, Tn5382 probe and ISEnfa200 probe (data not shown), confirming the linkage between pbp5 and the Tn5382vanB2ISEnfa200 element.
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ACKNOWLEDGEMENTS |
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This work was supported by grants from the Norwegian Research Council, Scandinavian Society for Antimicrobial Chemotherapy, and the Odd Berg Foundation. We thank the following people for providing strains: P. R. Chadwick of North Manchester General Hospital, Manchester, UK; P. Courvalin, of the Institut Pasteur, Paris, France; S. Harthug and A. Digranes of Haukeland University Hospital, Bergen, Norway; I. Klare of Robert Koch Institute, Wernigerode, Germany; E. B. Myhre of Lund University Hospital, Lund, Sweden; R. Patel of Mayo Clinic and Foundation, MN, USA; M. A. Pfaller and S. A. Marshall of University of Iowa College of Medicine, Iowa City, IA, USA; L. B. Rice, Case Western Reserve University School of Medicine, Cleveland, OH, USA; and A. Voss, University Hospital St Radboud, Nijmegen, the Netherlands. We also thank Marit R. Mikalsen and Mette S. Wesmajervi for excellent technical assistance, and Patrice Courvalin, Gunnar S. Simonsen and Johanna E. Sollid for helpful discussions and critical reading of the manuscript.
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Received 6 December 1999;
revised 18 February 2000;
accepted 8 March 2000.