This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow E-mail this article to a friend
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 Verdier, I.
Right arrow Articles by Vandenesch, F.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Verdier, I.
Right arrow Articles by Vandenesch, F.

 Previous Article  |  Next Article 

Antimicrobial Agents and Chemotherapy, March 2004, p. 1024-1027, Vol. 48, No. 3
0066-4804/04/$08.00+0     DOI: 10.1128/AAC.48.3.1024-1027.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Staphylococcus aureus Isolates with Reduced Susceptibility to Glycopeptides Belong to Accessory Gene Regulator Group I or II

Isabelle Verdier, Marie-Elisabeth Reverdy, Jerome Etienne, Gérard Lina, Michèle Bes, and François Vandenesch*

National Reference Laboratory for Staphylococci, INSERM E0230, IFR62, Faculté de Médecine Laennec, Lyon, France,

Received 2 June 2003/ Returned for modification 11 August 2003/ Accepted 30 October 2003


arrow
ABSTRACT
 
We used multiplex PCR to determine the agr group membership of 18 European glycopeptide heterointermediate and intermediate-resistant Staphylococcus aureus strains. Of the 15 agr group I strains, 13 were resistant and 2 were susceptible to methicillin. The remaining three strains, like the United States and Japanese control strains, belonged to agr group II.


arrow
INTRODUCTION
 
Glycopeptide intermediate-resistant Staphylococcus aureus (GISA) strains have been isolated in many countries since 1997 (2-4, 12, 17, 18, 20, 21, 23). They are defined by vancomycin MICs of 8 to 16 µg/ml (13, 16). Glycopeptide heterointermediate S. aureus (hGISA) strains (11) are susceptible to vancomycin (as determined on the basis of conventional criteria) but contain subpopulations (~10-6) which grow in the presence of vancomycin at concentrations of >=4 µg/ml (11). These two phenotypes of low-level glycopeptide resistance do not involve the van genes that confer vancomycin resistance in enterococci and in some rare S. aureus strains with high-level glycopeptide resistance (5) but seem to be related to antimicrobial sequestration by nonamidated muropeptides within a thickened cell wall (6, 10).

To study whether the expression of this resistance mechanism could be related to global regulatory pathway of S. aureus, Sakoulas et al. analyzed hGISA and GISA strains for variations in the locus of the accessory gene regulator (agr) (19), a global regulon controlling the expression of about a hundred S. aureus genes involved in virulence, metabolism, transport, and degradation pathways (8). Agr comprises a quorum-sensing module producing an autoinducing peptide (AIP) (encoded by agrD) and AgrA-C, a two-component system in which AgrC is a response regulator sensing the AIP and AgrA is the effector. Variability in AIP and AgrC defines four mutually exclusive alleles of the agr system (groups I to IV) (15). Sakoulas et al. found that all hGISA and GISA strains from Japan and the United States belonged to agr group II (19). However, the exclusive representation of the agr group II in these strains reflected the predominance of the agr group II among methicillin-resistant S. aureus (MRSA) isolates from the Boston hospitals studied. In contrast, agr group I MRSA strains could predominate in other settings (22a), raising the question of whether hGISA and GISA strains could belong to agr group I in these parts of the world.

We tested this hypothesis by analyzing 18 clinical hGISA and GISA strains isolated between 1998 and 2002 in France (17 strains, including LIM-2 [17] and MER-23 [2, 18]) or Belgium (1 strain [P1V44]) (7) (Table 1). The reference strains Mu3, Mu50, Michigan, and New Jersey (11, 12, 21) were also studied. The characteristics of these strains were compared to those of 35 non-hGISA and non-GISA MRSA strains recovered during the same period of time and in the same geographical area. Strain S. aureus ATCC29213 (which is susceptible to methicillin) was used as a control.


View this table:
[in this window]
[in a new window]
  		TABLE 1. Methicillin, vancomycin, and teicoplanin MICs for 22 S. aureus strains recovered from the United States, Japan, and Europe

All strains were confirmed to be S. aureus strains by coagulase testing with rabbit plasma (bioMérieux, Marcy-l'Etoile, France) and a Staphyslide agglutination test (bioMérieux). MICs in Mueller-Hinton medium (bioMérieux) were determined using the broth macrodilution method recommended by the National Committee for Clinical Laboratory Standards (NCCLS) (16). Macromethod E-tests (AB BIODISK, Solna, Sweden), a heavy inoculum (adjusted to a McFarland standard of 2), brain-heart infusion agar (bioMérieux), and a 48-h incubation period were used as described by Wooton et al. (23) to detect hGISA. Population analysis was based on the method of Hiramatsu et al. (11).

The results we obtained with previously described strains were compatible with published data: the vancomycin MICs for strains Mu50, LIM-2, MER-23, Michigan, New Jersey, and P1V44 were determined by the NCCLS method to be >=8 µg/ml, and the strains were thus confirmed to be GISA strains. As expected, the vancomycin MIC for Mu3, a hetero-GISA strain, was < 8 µg/ml; however, the examined strain comprised subpopulations able to grow in the presence of vancomycin at concentrations higher than >4 µg/ml (22). The 15 remaining French strains had vancomycin MICs of <8 µg/ml and met the definition of hetero-GISA strains as determined by population analysis (Table 1). E-tests with the enhanced culture conditions described above (23) showed elevated MICs: the vancomycin MICs increased to 8 to 32 µg/ml for GISA strains and to 4 to 16 µg/ml for hGISA strains, and the teicoplanin MICs rose to 12 to 96 µg/ml for GISA strains and to 12 to 64 µg/ml for hGISA strains (Table 1). These differences (detected under NCCLS and enhanced E-test conditions) between strains with respect to levels of susceptibility to vancomycin and teicoplanin appear to be related to the selection of resistant subpopulations by the latter technique (2, 18, 23). The MICs of vancomycin and teicoplanin (as determined by the standard method for the 35 non-hGISA and non-GISA isolates and the reference S. aureus strain) were <=2 µg/ml, and the isolates contained no subpopulations which grew in the presence of >=4 µg of vancomycin/ml (11).

The agr groupings of the complete collection of strains were then determined by multiplex PCR with primers designed using the agr I to IV sequences as described elsewhere (15). Most (15 of 22) hGISA and GISA strains (13 MRSA and 2 methicillin-sensitive S. aureus [MSSA] strains, all from Europe) belonged to agr group I. Seven strains belonged to agr group II. They comprised five previously described (19) MRSA strains isolated in Japan or the United States (Mu3, Mu50, Michigan, and New Jersey) and also one MRSA strain and two MSSA strains from France. None of the strains belonged to agr group III or IV. The non-hGISA and non-GISA MRSA strains belonged mainly to agr group I (31 of 35 strains) and, less frequently, to agr group II (4 of 35). The 22 hGISA and GISA strains and the 35 non-hGISA and non-GISA MRSA strains were then analyzed by pulsed-field gel electrophoresis (PFGE) of SmaI-restricted chromosomal DNA (9). The agr group I hGISA and GISA strains (with the exception of strain PRO, which was distinct from the rest of the group) were closely related to one another. The 31 non-hGISA and non-GISA MRSA strains of agr group I had PFGE patterns closely related to each other (with an index of similarity of >80%), reflecting the clonality of the hospital-acquired MRSA strains spreading in France. Surprisingly, the PFGE patterns of these strains (except for that of the PRO strain) were not related to those of the agr group I hGISA and GISA isolates (Fig. 1). This suggests that (with the exception of strain PRO) French agr group I hGISA and GISA strains have mainly emerged from an agr group I background not detected in our collection.



View larger version (48K):
[in this window]
[in a new window]
  		FIG. 1. PFGE patterns and phylogenetic tree of 22 GISA and hGISA isolates and 35 non-GISA and non-hGISA S. aureus isolates from the United States, Japan, and Europe. Isolate designations in boldface characters indicate the GISA-hGISA phenotype. SmaI macrorestriction patterns were digitized and analyzed with Taxotron software (Institut Pasteur, Paris, France) to calculate Dice coefficients of correlation and to generate a dendrogram by the unweighted pair group method using arithmetic averages (unweighted pair group method with arithmetic mean) clustering. The scale indicates the levels of pattern similarity. The fragment size (in kilobases) of reference strain NCTC8325 is indicated on the bottom lane.

The Japanese and United States strains (Mu3, Mu50, Michigan, and New Jersey), together with strain MER-23, all of which belonged to agr group II, showed distinct but related PFGE patterns (Fig. 1). One of the non-hGISA and non-GISA MRSA strains of agr group II (strain 58-1) had a PFGE pattern closely related to that of strain MER-23 (a GISA MSSA strain), suggesting that the same genetic background independently acquired either the mecA element (for strain 58-1) or the glycopeptide resistance (for strain MER-23). The three other non-hGISA MRSA strains of the agr group II (designated 104-1, 22-22, and 19-5) had different PFGE patterns not related to those of European hGISA and GISA strains (Fig. 1). The four methicillin-susceptible hGISA and GISA strains (designated RAB, MER-23, PEY, and PON) had clearly distinct PFGE patterns. Thus, European methicillin-susceptible and methicillin-resistant hGISA and GISA strains are unlikely to have arisen from a single clone; it is more probable that several distinct strains from preexisting MRSA or MSSA strains emerged independently.

Conflicting reports respecting this question have been published. A Korean study of 4,483 consecutive clinical MRSA and MSSA isolates showed that the macrorestricton patterns of glycopeptide-susceptible MRSA isolates were very different from those of GISA strains isolated during the same period (14). In contrast, a retrospective analysis of 457 German MRSA and MSSA isolates showed that the PFGE patterns of most GISA isolates resembled those of common epidemic MRSA strains (1).

In conclusion, almost all European hGISA and GISA strains appear to belong to agr group I or II. The results of macrorestriction analysis, and the fact that these strain groups contain both MSSA and MRSA strains, argue against a unique clonal origin. Moreover, with one exception, there was no obvious genetic relationship between our hGISA and GISA strains and the predominant hospital MRSA strains from the same geographical area.


arrow
ACKNOWLEDGMENTS
 
We are grateful to Keichi Hiramatsu, Fred Tenover, François Denis, Marie-Cécile Ploy, and Marc Struelens for providing us with hGISA and GISA strains. We thank Annie Martra, Martine Rougier, and Chantal Nervi for technical assistance and Françoise Forey for the PFGE analysis. We thank David Young for editing the manuscript.


arrow
FOOTNOTES
 
* Corresponding author. Mailing address: Faculté de Médecine Laennec, National Reference Centre for Staphylococci, IFR62, INSERM E0230, 7 rue Guillaume Paradin, 69372 Lyon cedex 08, France. Phone: 33 4 78 77 86 57. Fax: 33 4 78 77 86 58. E-mail: denesch{at}univ-lyon1.fr. Back


arrow
REFERENCES
 
    1
  1. Bierbaum, G., K. Fuchs, W. Lenz, C. Szekat, and H. G. Sahl. 1999. Presence of Staphylococcus aureus with reduced susceptibility to vancomycin in Germany. Eur. J. Clin. Microbiol. Infect. Dis. 18:691-696.[CrossRef][Medline]
    2. 2
  2. Bobin-Dubreux, S., M. E. Reverdy, C. Nervi, M. Rougier, A. Bolmstrom, F. Vandenesch, and J. Etienne. 2001. Clinical isolate of vancomycin-heterointermediate Staphylococcus aureus susceptible to methicillin and in vitro selection of a vancomycin-resistant derivative. Antimicrob. Agents Chemother. 45:349-352.[Abstract/Free Full Text]
    3. 3
  3. Centers for Disease Control and Prevention. 2000. Staphylococcus aureus with reduced susceptibility to vancomycin—Illinois, 1999. Morb. Mortal. Wkly. Rep. 48:1165-1167.[Medline]
    4. 4
  4. Centers for Disease Control and Prevention. 1997. Staphylococcus aureus with reduced susceptibility to vancomycin—United States, 1997. Morb. Mortal. Wkly. Rep. 46:765-766.[Medline]
    5. 5
  5. Chang, S., D. M. Sievert, J. C. Hageman, M. L. Boulton, F. C. Tenover, F. P. Downes, S. Shah, J. T. Rudrik, G. R. Pupp, W. J. Brown, D. Cardo, and S. K. Fridkin. 2003. Infection with vancomycin-resistant Staphylococcus aureus containing the vanA resistance gene. N. Engl. J. Med. 348:1342-1347.[Free Full Text]
    6. 6
  6. Cui, L., H. Murakami, K. Kuwahara-Arai, H. Hanaki, and K. Hiramatsu. 2000. Contribution of a thickened cell wall and its glutamine nonamidated component to the vancomycin resistance expressed by Staphylococcus aureus Mu50. Antimicrob. Agents Chemother. 44:2276-2285.[Abstract/Free Full Text]
    7. 7
  7. Denis, O., C. Nonhoff, B. Byl, C. Knoop, S. Bobin-Dubreux, and M. J. Struelens. 2002. Emergence of vancomycin-intermediate Staphylococcus aureus in a Belgian hospital: microbiological and clinical features. J. Antimicrob. Chemother. 50:383-391.[Abstract/Free Full Text]
    8. 8
  8. Dunman, P. M., E. Murphy, S. Haney, D. Palacios, G. Tucker-Kellogg, S. Wu, E. L. Brown, R. J. Zagursky, D. Shlaes, and S. J. Projan. 2001. Transcription profiling-based identification of Staphylococcus aureus genes regulated by the agr and/or sarA loci. J. Bacteriol. 183:7341-7353.[Abstract/Free Full Text]
    9. 9
  9. Goering, R. V., and M. A. Winters. 1992. Rapid method for epidemiological evaluation of gram-positive cocci by field inversion gel electrophoresis. J. Clin. Microbiol. 30:577-580.[Abstract/Free Full Text]
    10. 10
  10. Hanaki, H., K. Kuwahara-Arai, S. Boyle-Vavra, R. S. Daum, H. Labischinski, and K. Hiramatsu. 1998. Activated cell-wall synthesis is associated with vancomycin resistance in methicillin-resistant Staphylococcus aureus clinical strains Mu3 and Mu50. J. Antimicrob. Chemother. 42:199-209.[Abstract/Free Full Text]
    11. 11
  11. Hiramatsu, K., N. Aritaka, H. Hanaki, S. Kawasaki, Y. Hosoda, S. Hori, Y. Fukuchi, and I. Kobayashi. 1997. Dissemination in Japanese hospitals of strains of Staphylococcus aureus heterogeneously resistant to vancomycin. Lancet 350:1670-1673.[CrossRef][Medline]
    12. 12
  12. Hiramatsu, K., H. Hanaki, T. Ino, K. Yabuta, T. Oguri, and F. C. Tenover. 1997. Methicillin-resistant Staphylococcus aureus clinical strain with reduced vancomycin susceptibility. J. Antimicrob. Chemother. 40:135-136.[Free Full Text]
    13. 13
  13. Howe, R. A., K. E. Bowker, T. R. Walsh, T. G. Feest, and A. P. MacGowan. 1998. Vancomycin-resistant Staphylococcus aureus. Lancet 351:602.[CrossRef][Medline]
    14. 14
  14. Kim, M. N., S. H. Hwang, Y. J. Pyo, H. M. Mun, and C. H. Pai. 2002. Clonal spread of Staphylococcus aureus heterogeneously resistant to vancomycin in a university hospital in Korea. J. Clin. Microbiol. 40:1376-1380.[Abstract/Free Full Text]
    15. 15
  15. Lina, G., F. Boutite, A. Tristan, M. Bes, J. Etienne, and F. Vandenesch. 2003. Bacterial competition for human nasal cavity colonization: role of staphylococcal agr alleles. Appl. Environ. Microbiol. 69:18-23.[Abstract/Free Full Text]
    16. 16
  16. National Committee for Clinical Laboratory Standards. 2000. Methods for dilution antimicrobial susceptibility testing for bacteria that grow aerobically. Approved standard M7-A5. National Committee for Clinical Laboratory Standards, Wayne, Pa.
    17. 17
  17. Ploy, M. C., C. Grelaud, C. Martin, L. de Lumley, and F. Denis. 1998. First clinical isolate of vancomycin-intermediate Staphylococcus aureus in a French hospital. Lancet 351:1212.[Medline]
    18. 18
  18. Reverdy, M. E., S. Jarraud, S. Bobin-Dubreux, E. Burel, P. Girardo, G. Lina, F. Vandenesch, and J. Etienne. 2001. Incidence of Staphylococcus aureus with reduced susceptibility to glycopeptides in two French hospitals. Clin. Microbiol. Infect. 7:267-272.[CrossRef][Medline]
    19. 19
  19. Sakoulas, G., G. M. Eliopoulos, R. C. Moellering, Jr., C. Wennersten, L. Venkataraman, R. P. Novick, and H. S. Gold. 2002. Accessory gene regulator (agr) locus in geographically diverse Staphylococcus aureus isolates with reduced susceptibility to vancomycin. Antimicrob. Agents Chemother. 46:1492-1502.[Abstract/Free Full Text]
    20. 20
  20. Sieradzki, K., R. B. Roberts, S. W. Haber, and A. Tomasz. 1999. The development of vancomycin resistance in a patient with methicillin-resistant Staphylococcus aureus infection. N. Engl. J. Med. 340:517-523.[Free Full Text]
    21. 21
  21. Smith, T. L., M. L. Pearson, K. R. Wilcox, C. Cruz, M. V. Lancaster, B. Robinson-Dunn, F. C. Tenover, M. J. Zervos, J. D. Band, E. White, and W. R. Jarvis. 1999. Emergence of vancomycin resistance in Staphylococcus aureus. N. Engl. J. Med. 340:493-501.[Abstract/Free Full Text]
    22. 22
  22. Tenover, F. C., J. W. Biddle, and M. V. Lancaster. 2001. Increasing resistance to vancomycin and other glycopeptides in Staphylococcus aureus. Emerg. Infect. Dis. 7:327-332.[Medline]
    23. 22
  23. van Leeuwen, W., W. van Nieuwenhuizen, C. Gijzen, H. Verbrugh, and A. van Belkum. 2000. Population studies of methicillin-resistant and -sensitive Staphylococcus aureus strains reveal a lack of variability in the agrD gene, encoding a staphylococcal autoinducer peptide. J. Bacteriol. 182:5721-5729.[Abstract/Free Full Text]
    24. 23
  24. Wootton, M., R. A. Howe, R. Hillman, T. R. Walsh, P. M. Bennett, and A. P. MacGowan. 2001. A modified population analysis profile (PAP) method to detect hetero-resistance to vancomycin in Staphylococcus aureus in a UK hospital. J. Antimicrob. Chemother. 47:399-403.[Abstract/Free Full Text]

Antimicrobial Agents and Chemotherapy, March 2004, p. 1024-1027, Vol. 48, No. 3
0066-4804/04/$08.00+0     DOI: 10.1128/AAC.48.3.1024-1027.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Howden, B. P., Davies, J. K., Johnson, P. D. R., Stinear, T. P., Grayson, M. L. (2010). Reduced Vancomycin Susceptibility in Staphylococcus aureus, Including Vancomycin-Intermediate and Heterogeneous Vancomycin-Intermediate Strains: Resistance Mechanisms, Laboratory Detection, and Clinical Implications. Clin. Microbiol. Rev. 23: 99-139 [Abstract] [Full Text]  
  • Luczak-Kadlubowska, A., Krzyszton-Russjan, J., Hryniewicz, W. (2006). Characteristics of Staphylococcus aureus Strains Isolated in Poland in 1996 to 2004 That Were Deficient in Species-Specific Proteins. J. Clin. Microbiol. 44: 4018-4024 [Abstract] [Full Text]  
  • Howden, B. P., Johnson, P. D. R., Ward, P. B., Stinear, T. P., Davies, J. K. (2006). Isolates with Low-Level Vancomycin Resistance Associated with Persistent Methicillin-Resistant Staphylococcus aureus Bacteremia. Antimicrob. Agents Chemother. 50: 3039-3047 [Abstract] [Full Text]  
  • Francois, P., Koessler, T., Huyghe, A., Harbarth, S., Bento, M., Lew, D., Etienne, J., Pittet, D., Schrenzel, J. (2006). Rapid Staphylococcus aureus agr Type Determination by a Novel Multiplex Real-Time Quantitative PCR Assay. J. Clin. Microbiol. 44: 1892-1895 [Abstract] [Full Text]  
  • Robinson, D. A., Monk, A. B., Cooper, J. E., Feil, E. J., Enright, M. C. (2005). Evolutionary Genetics of the Accessory Gene Regulator (agr) Locus in Staphylococcus aureus. J. Bacteriol. 187: 8312-8321 [Abstract] [Full Text]  
  • Steenbergen, J. N., Alder, J., Thorne, G. M., Tally, F. P. (2005). Daptomycin: a lipopeptide antibiotic for the treatment of serious Gram-positive infections. J Antimicrob Chemother 55: 283-288 [Abstract] [Full Text]  

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow E-mail this article to a friend
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 Verdier, I.
Right arrow Articles by Vandenesch, F.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Verdier, I.
Right arrow Articles by Vandenesch, F.

Copyright © 2010 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»