AAC
Home Help [Feedback] [For Subscribers] [Archive] [Search] [Contents]
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Other Versions of this Article:
AAC.00557-06v1
51/2/736    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Sletvold, H.
Right arrow Articles by Nielsen, K. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Sletvold, H.
Right arrow Articles by Nielsen, K. M.

 Previous Article  |  Next Article 

Antimicrobial Agents and Chemotherapy, February 2007, p. 736-739, Vol. 51, No. 2
0066-4804/07/$08.00+0     doi:10.1128/AAC.00557-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Comparative DNA Analysis of Two vanA Plasmids from Enterococcus faecium Strains Isolated from Poultry and a Poultry Farmer in Norway{triangledown}

H. Sletvold,1* P. J. Johnsen,1 G. S. Simonsen,2,3,4 B. Aasnæs,3 A. Sundsfjord,2,3,4 and K. M. Nielsen1,5

Department of Pharmacy,1 Department of Microbiology and Virology, Faculty of Medicine, University of Tromsø,2 Department of Microbiology and Infection Control, University Hospital of North Norway, Tromsø,3 Norwegian Institute of Public Health, Oslo,4 Norwegian Institute of Gene Ecology, Tromsø, Norway5

Received 5 May 2006/ Returned for modification 13 July 2006/ Accepted 8 November 2006


   ABSTRACT
Top
 Abstract
Text
 References
 
The DNA sequences of two plasmids carrying vanA, pVEF1 (39,626 bp) and pVEF2 (39,714 bp), were determined. Forty-three shared coding sequences were identified, and the only nucleotide difference was an 88-bp indel. A postsegregational killing system was identified. This system possibly explains the persistence of the vanA gene cluster in Norwegian poultry farms.


   TEXT
Top
 Abstract
 Text
References
 
Glycopeptide-resistant Enterococcus faecium (GREF) strains in which the resistance is plasmid encoded persist on Norwegian poultry farms, despite the ban on the use of avoparcin in 1995 (11, 18). Glycopeptide R plasmids were isolated from two genomically different GREF strains sampled from a chicken and a farmer to determine the basis for their long-term persistence in glycopeptide-free environments. Plasmids pVEF1 and pVEF2 are vanA-containing plasmids that express high-level glycopeptide resistance (vancomycin MICs, ≥64 mg/liter; teicoplanin MICs, >4 mg/liter). The plasmids were isolated in 1999 from two genomically different E. faecium strains, strains 399/F99/H8 and 399/F99/A9, from a farmer and his poultry, respectively. Both originated from a single Norwegian farm previously exposed to the animal growth promoter avoparcin (11).

Bacterial and plasmid isolation, species identification, and antimicrobial susceptibility testing were performed as previously described (11, 18). The plasmid DNA was randomly sheared and cloned into pCR4Blunt-TOPO. Plasmid DNA from approximately 450 subclones was purified, and the insert was sequenced by using ABI BigDye chemistry. Custom primers were used in the PCRs to fill gaps and to ensure the recovery of double-strand data for the complete sequences of the plasmids. The sequence data were assembled by use of the Staden package (19) and Phrap software (http://www.phrap.org/) and were completed by using Gap4 software (4). The final assemblies were verified by restriction map comparisons with BamHI, ClaI, EcoRI, HindIII, and PstI digests of the plasmids. The sequences were annotated by using the Artemis program (16), and the predicted coding sequences (CDSs) were identified by the use of GLIMMER software (7) and manually by correlation scores of the open reading frames (ORFs) with ≥50 amino acids. Sequence similarity was identified by using the FASTA (14) and the BLASTP (1) programs as well as the Pfam (3) and the ProSite (10) databases for protein domain prediction.

Sequence analysis of plasmids pVEF1 and pVEF2 revealed that they consist of 39,626 and 39,714 bp, respectively. Annotation revealed an identical gene composition expressed through 43 CDSs. The only difference identified between the two plasmids, an 88-bp indel, did not influence the gene composition but caused differences in the last six amino acids of the truncated transposase (CDS22). The functions of 37 CDSs were predicted on the basis of their similarities to previously characterized proteins (Table 1). A circular genetic map of the gene synteny is given in Fig. 1. The vanA genes expressing glycopeptide resistance are present on transposon Tn1546 on both plasmids, and the transposons show 100% identity to the first published sequence (2). Tn1546 is flanked by a truncated streptomycin resistance gene (str) with identity to plasmid pS194 from a clinical strain of Staphylococcus aureus (15). The G+C content of the two plasmids is 36.2%, which is similar to that of enterococcal genomic DNA (13). The G+C contents of individual CDSs ranged from 25.5% to 44.7%, consistent with the hypothesis that pVEF1 and pVEF2 are composed of DNA regions from multiple sources. The flanking sequences of Tn1546 identified in pVEF1 and pVEF2 are identical to the junction sequences (determined by inverse PCR) of the first described GREF plasmid (pIP816) isolated from a clinical strain in France in 1986 (2). The nonconjugative plasmids pVEF1 and pVEF2 have several insertion sequence (IS) elements that are likely facilitators of intra- or interplasmid recombinations (Table 1).


View this table:
[in this window]
[in a new window]

  		TABLE 1. CDSs of the vanA plasmids pVEF1 and pVEF2

 

Figure 1
View larger version (24K):
[in this window]
[in a new window]

  		FIG. 1. Genetic map of pVEF1 and pVEF2. The numbering of the plasmids commences at the first nucleotide of the ATG start codon of CDS1 of pVEF1 and pVEF2, which are predicted to encode replication proteins. Coding regions are represented by arrows indicating the direction of transcription. The coordinates of the indel are indicated on the map by positional marks.

 
Comparisons of the sequences of the vanA plasmids in this report and that of the only other sequenced enterococcal vanA plasmid, pHTß (20), revealed exceptionally low levels of identity, in which the only shared CDS is the pheromone-responsive gene prgN. Plasmid pHTß also has a Tn1546-like fragment, but direct comparative analysis is currently not possible since the pHTß Tn1546-like sequence is not available in the databases.

Genes responsible for stable replication and plasmid persistence were found. The amino acid sequence of CDS1 has a high degree of identity to that of a replication protein found on the draft genome of E. faecium DO, while the sequence of CDS26 is identical to that of a repR gene (orf1) found on pRE25 from Enterococcus faecalis (17). Upstream of repR are two CDSs, prgO and prgP, previously described in pCF10 and pRE25 as putatively involved in plasmid replication (9, 17). Interestingly, several CDSs with high sequence similarity to a plasmid maintenance system previously described in pSM19035 and pRE25 (5, 12, 17) and encoded by the {delta}-{omega}-{varepsilon}-{zeta} genes were identified. Such toxin/antitoxin systems secure the stable inheritance of plasmids during cell division by killing or impairing the growth of cells that have lost the plasmids and are also called postsegregational killing (PSK) systems (8). CDS32 and CDS33 of pVEF1 and pVEF2 are identical to ORF18 ({varepsilon} gene) and ORF19 ({zeta} gene) in pRE25 and have 78% and 91% amino acid sequence identities to the {varepsilon} antitoxin and {zeta} toxin of pSM19035, respectively. The {omega} protein regulates the expression of the {varepsilon} and {zeta} genes (5, 6), and at the amino acid level CDS8 and CDS31 are identical to the {omega} gene of pSM19035 and ORF17 of pRE25, respectively. A {delta} gene is present on both pVEF1 and pVEF2 (CDS7), while a truncated {delta} gene (CDS30) is found upstream of the {omega}-{varepsilon}-{zeta} genes. The high degree of identity of the amino acids to those of the {omega}-{varepsilon}-{zeta} PSK system suggests the killing of plasmid-free cells as a major contributor to the long-term persistence of GREF in glycopeptide-free environments. A genetic linkage of Tn1546 to other known antimicrobial or heavy metal resistance determinants was not found.

Nucleotide sequence accession numbers. Plasmids pVEF1 and pVEF2 have the following GenBank accession numbers: AM296544 and AM410096, respectively.


   ACKNOWLEDGMENTS
 
This work was supported by EC contract QLK2-CT-2002-00843 and the Medical Research Foundation, North Norway.


   FOOTNOTES
 
* Corresponding author. Mailing address: Department of Pharmacy, Faculty of Medicine, University of Tromsø, 9037 Tromsø, Norway. Phone: 47 77646150. Fax: 47 77646151. E-mail: hege.sletvold{at}farmasi.uit.no. Back

{triangledown} Published ahead of print on 20 November 2006. Back


   REFERENCES
Top
 Abstract
 Text
 References
 

  1. Altschul, S. F., W. Gish, W. Miller, E. W. Myers, and D. J. Lipman. 1990. Basic local alignment search tool. J. Mol. Biol. 215:403-410.[CrossRef][Medline]
  2. Arthur, M., C. Molinas, F. Depardieu, and P. Courvalin. 1993. Characterization of Tn1546, a Tn3-related transposon conferring glycopeptide resistance by synthesis of depsipeptide peptidoglycan precursors in Enterococcus faecium BM4147. J. Bacteriol. 175:117-127.[Abstract/Free Full Text]
  3. Bateman, A., E. Birney, L. Cerruti, R. Durbin, L. Etwiller, S. R. Eddy, S. Griffiths-Jones, K. L. Howe, M. Marshall, and E. L. Sonnhammer. 2002. The Pfam protein families database. Nucleic Acids Res. 30:276-280.[Abstract/Free Full Text]
  4. Bonfield, J. K., K. Smith, and R. Staden. 1995. A new DNA sequence assembly program. Nucleic Acids Res. 23:4992-4999.[Abstract/Free Full Text]
  5. de la Hoz, A. B., S. Ayora, I. Sitkiewicz, S. Fernandez, R. Pankiewicz, J. C. Alonso, and P. Ceglowski. 2000. Plasmid copy-number control and better-than-random segregation genes of pSM19035 share a common regulator. Proc. Natl. Acad. Sci. USA 97:728-733.[Abstract/Free Full Text]
  6. de la Hoz, A. B., F. Pratto, R. Misselwitz, C. Speck, W. Weihofen, K. Welfle, W. Saenger, H. Welfle, and J. C. Alonso. 2004. Recognition of DNA by omega protein from the broad-host range Streptococcus pyogenes plasmid pSM19035: analysis of binding to operator DNA with one to four heptad repeats. Nucleic Acids Res. 32:3136-3147.[Abstract/Free Full Text]
  7. Delcher, A. L., D. Harmon, S. Kasif, O. White, and S. L. Salzberg. 1999. Improved microbial gene identification with GLIMMER. Nucleic Acids Res. 27:4636-4641.[Abstract/Free Full Text]
  8. Gerdes, K., S. K. Christensen, and A. Lobner-Olesen. 2005. Prokaryotic toxin-antitoxin stress response loci. Nat. Rev. Microbiol. 3:371-382.[CrossRef][Medline]
  9. Hirt, H., D. A. Manias, E. M. Bryan, J. R. Klein, J. K. Marklund, J. H. Staddon, M. L. Paustian, V. Kapur, and G. M. Dunny. 2005. Characterization of the pheromone response of the Enterococcus faecalis conjugative plasmid pCF10: complete sequence and comparative analysis of the transcriptional and phenotypic responses of pCF10-containing cells to pheromone induction. J. Bacteriol. 187:1044-1054.[Abstract/Free Full Text]
  10. Hulo, N., C. J. Sigrist, V. Le Saux, P. S. Langendijk-Genevaux, L. Bordoli, A. Gattiker, E. De Castro, P. Bucher, and A. Bairoch. 2004. Recent improvements to the PROSITE database. Nucleic Acids Res. 32:D134-D137.[Abstract/Free Full Text]
  11. Johnsen, P. J., J. I. Osterhus, H. Sletvold, M. Sorum, H. Kruse, K. Nielsen, G. S. Simonsen, and A. Sundsfjord. 2005. Persistence of animal and human glycopeptide-resistant enterococci on two Norwegian poultry farms formerly exposed to avoparcin is associated with a widespread plasmid-mediated vanA element within a polyclonal Enterococcus faecium population. Appl. Environ. Microbiol. 71:159-168.[Abstract/Free Full Text]
  12. Meinhart, A., J. C. Alonso, N. Strater, and W. Saenger. 2003. Crystal structure of the plasmid maintenance system epsilon/zeta: functional mechanism of toxin zeta and inactivation by epsilon 2 zeta 2 complex formation. Proc. Natl. Acad. Sci. USA 100:1661-1666.[Abstract/Free Full Text]
  13. Paulsen, I. T., L. Banerjei, G. S. Myers, K. E. Nelson, R. Seshadri, T. D. Read, D. E. Fouts, J. A. Eisen, S. R. Gill, J. F. Heidelberg, H. Tettelin, R. J. Dodson, L. Umayam, L. Brinkac, M. Beanan, S. Daugherty, R. T. DeBoy, S. Durkin, J. Kolonay, R. Madupu, W. Nelson, J. Vamathevan, B. Tran, J. Upton, T. Hansen, J. Shetty, H. Khouri, T. Utterback, D. Radune, K. A. Ketchum, B. A. Dougherty, and C. M. Fraser. 2003. Role of mobile DNA in the evolution of vancomycin-resistant Enterococcus faecalis. Science 299:2071-2074.[Abstract/Free Full Text]
  14. Pearson, W. R., and D. J. Lipman. 1988. Improved tools for biological sequence comparison. Proc. Natl. Acad. Sci. USA 85:2444-2448.[Abstract/Free Full Text]
  15. Projan, S. J., S. Moghazeh, and R. P. Novick. 1988. Nucleotide sequence of pS194, a streptomycin-resistance plasmid from Staphylococcus aureus. Nucleic Acids Res. 16:2179-2187.[Abstract/Free Full Text]
  16. Rutherford, K., J. Parkhill, J. Crook, T. Horsnell, P. Rice, M. A. Rajandream, and B. Barrell. 2000. Artemis: sequence visualization and annotation. Bioinformatics 16:944-945.[Abstract/Free Full Text]
  17. Schwarz, F. V., V. Perreten, and M. Teuber. 2001. Sequence of the 50-kb conjugative multiresistance plasmid pRE25 from Enterococcus faecalis RE25. Plasmid 46:170-187.[CrossRef][Medline]
  18. Sorum, M., P. J. Johnsen, B. Aasnes, T. Rosvoll, H. Kruse, A. Sundsfjord, and G. S. Simonsen. 2006. Prevalence, persistence, and molecular characterization of glycopeptide-resistant enterococci in Norwegian poultry and poultry farmers 3 to 8 years after the ban on avoparcin. Appl. Environ. Microbiol. 72:516-521.[Abstract/Free Full Text]
  19. Staden, R., K. F. Beal, and J. K. Bonfield. 2000. The Staden package, 1998. Methods Mol. Biol. 132:115-130.[Medline]
  20. Tomita, H., and Y. Ike. 2005. Genetic analysis of transfer-related regions of the vancomycin resistance enterococcus conjugative plasmid pHTß: identification of oriT and a putative relaxase gene. J. Bacteriol. 187:7727-7737.[Abstract/Free Full Text]

Antimicrobial Agents and Chemotherapy, February 2007, p. 736-739, Vol. 51, No. 2
0066-4804/07/$08.00+0     doi:10.1128/AAC.00557-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:


This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Other Versions of this Article:
AAC.00557-06v1
51/2/736    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Sletvold, H.
Right arrow Articles by Nielsen, K. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Sletvold, H.
Right arrow Articles by Nielsen, K. M.

Home Help [Feedback] [For Subscribers] [Archive] [Search] [Contents]
Clin. Vaccine Immunol. Clin. Microbiol. Rev.
J. Clin. Microbiol. ALL ASM JOURNALS