Analysis of different genetic traits and their association with biofilm formation in Staphylococcus epidermidis isolates from central venous catheter infections

  • D. Petrelli
  • C. Zampaloni
  • S. D’Ercole
  • M. Prenna
  • P. Ballarini
  • S. Ripa
  • L. A. VitaliEmail author


The aim of the present study was to characterize clinical isolates of Staphylococcus epidermidis, one of the bacterial species most often implicated in foreign-body-associated infections, for their ability to form biofilms and for the presence of mecA and IS256 element. Sixty-seven Staphylococcus epidermidis clinical isolates, obtained from implantable medical devices, were investigated. Overall, 70% of the strains were positive for ica operon genes, 85% possessed atlE, and 46% contained aap. In 89% of the population, the Congo red agar test confirmed the correlation between the presence of ica genes and slime expression. Almost all of the strains could be classified as biofilm producers by both the crystal violet assay and microscopy. The bacterial population studied showed a very high frequency of strains positive for mecA as well as for the IS256 element. Although well-structured biofilms have been previously observed only in those strains possessing genes belonging to the ica operon, this study demonstrates that strains lacking specific biofilm-formation determinants can be isolated from catheters and can form a biofilm in vitro. Hence, different and yet-to-be identified factors may work together in the formation and organization of complex staphylococcal microbial communities and sustain infections associated with implanted medical devices.


Crystal Violet IS256 Element Polysaccharide Intercellular Adhesin Oxacillin Resistance atlE Gene 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.



This work was supported in part by grants from the Italian MIUR (FIRB 2001 and PRIN 2003).

We are also grateful to Dr. D. Mack for the generous gift of S. epidermidis strains 1457 and 1457-M11.


  1. 1.
    Donlan RM (2002) Biofilms: microbial life on surfaces. Emerg Infect Dis 8:881–890PubMedGoogle Scholar
  2. 2.
    Parsek MR, Singh PK (2003) Bacterial biofilms: an emerging link to disease pathogenesis. Annu Rev Microbiol 57:677–701PubMedCrossRefGoogle Scholar
  3. 3.
    Rupp ME, Archer GL (1994) Coagulase-negative staphylococci: pathogens associated with medical progress. Clin Infect Dis 19:231–243PubMedGoogle Scholar
  4. 4.
    Vuong C, Otto M (2002) Staphylococcus epidermidis infections. Microbes Infect 4:481–489PubMedCrossRefGoogle Scholar
  5. 5.
    Heilmann C, Hussain M, Peters G, Götz F (1997) Evidence for autolysin-mediated primary attachment of Staphylococcus epidermidis to a polystyrene surface. Mol Microbiol 24:1013–1024PubMedCrossRefGoogle Scholar
  6. 6.
    Costerton JW, Stewart PS, Greenberg EP (1999) Bacterial biofilms: a common cause of persistent infections. Science 284:1318–1322PubMedCrossRefGoogle Scholar
  7. 7.
    Heilmann C, Gerke C, Perdreau-Remington F, Gotz F (1996) Characterization of Tn917 insertion mutants of Staphylococcus epidermidis affected in biofilm formation. Infect Immun 64:277–282PubMedGoogle Scholar
  8. 8.
    Heilmann C, Schweitzer O, Gerke C, Vanittanakom N, Mack D, Gotz F (1996) Molecular basis of intercellular adhesion in the biofilm-forming Staphylococcus epidermidis. Mol Microbiol 20:1083–1091PubMedCrossRefGoogle Scholar
  9. 9.
    Rohde H, Burdelski C, Bartscht K, Hussain M, Buck F, Horstkotte MA, Knobloch JK, Heilmann C, Herrmann M, Mack D (2005) Induction of Staphylococcus epidermidis biofilm formation via proteolytic processing of the accumulation-associated protein by staphylococcal and host proteases. Mol Microbiol 55:1883–1895PubMedCrossRefGoogle Scholar
  10. 10.
    Cramton SE, Gerke C, Schnell NF, Nichols WW, Gotz F (1999) The intercellular adhesion (ica) locus is present in Staphylococcus aureus and is required for biofilm formation. Infect Immun 67:5427–5433PubMedGoogle Scholar
  11. 11.
    Karchmer AW, Gibbons GW (1994) Infections of prosthetic heart valves and vascular grafts. In: Waldvogel FA, Bisno AL (eds) Infections associated with indwelling medical devices, 2nd edn. American Society for Microbiology, Washington DC, pp 213–249Google Scholar
  12. 12.
    Yasuda H, Koga T, Fukuoka T (1999) In vitro and in vivo models of bacterial biofilms. Methods Enzymol 310:577–595PubMedGoogle Scholar
  13. 13.
    Rupp ME, Ulphani JS, Fey PD, Bartscht K, Mack D (1999) Characterization of the importance of polysaccharide intercellular adhesin/hemagglutinin of Staphylococcus epidermidis in the pathogenesis of biomaterial-based infection in a mouse foreign body infection model. Infect Immun 67:2627–2632PubMedGoogle Scholar
  14. 14.
    Cafiso V, Bertuccio T, Santagati M, Campanile F, Amicosante G, Perilli MG, Selan L, Artini M, Nicoletti G, Stefani S (2004) Presence of the ica operon in clinical isolates of Staphylococcus epidermidis and its role in biofilm production. Clin Microbiol Infect 10:1081–1088PubMedCrossRefGoogle Scholar
  15. 15.
    de Silva GD, Kantzanou M, Justice A, Massey RC, Wilkinson AR, Day NP, Peacock SJ (2002) The ica operon and biofilm production in coagulase-negative staphylococci associated with carriage and disease in a neonatal intensive care unit. J Clin Microbiol 40:382–388PubMedCrossRefGoogle Scholar
  16. 16.
    Yao Y, Sturdevant DE, Otto M (2005) Genomewide analysis of gene expression in Staphylococcus epidermidis biofilms: insights into the pathophysiology of Staphylococcus epidermidis biofilms and the role of phenol-soluble modulins in formation of biofilms. J Infect Dis 191:289–298PubMedCrossRefGoogle Scholar
  17. 17.
    Kozitskaya S, Olson ME, Fey PD, Witte W, Ohlsen K, Ziebuhr W (2005) Clonal analysis of Staphylococcus epidermidis isolates carrying or lacking biofilm-mediating genes by multilocus sequence typing. J Clin Microbiol 43:4751–4757PubMedCrossRefGoogle Scholar
  18. 18.
    Galdbart JO, Allignet J, Tung HS, Ryden C, El Solh N (2000) Screening for Staphylococcus epidermidis markers discriminating between skin-flora strains and those responsible for infections of joint prostheses. J Infect Dis 182:351–355PubMedCrossRefGoogle Scholar
  19. 19.
    Gu J, Li H, Li M, Vuong C, Otto M, Wen Y, Gao Q (2005) Bacterial insertion sequence IS256 as a potential molecular marker to discriminate invasive strains from commensal strains of Staphylococcus epidermidis. J Hosp Infect 61:342–348PubMedCrossRefGoogle Scholar
  20. 20.
    Arciola CR, Baldassarri L, Montanaro L (2002) In catheter infections by Staphylococcus epidermidis the intercellular adhesion (ica) locus is a molecular marker of the virulent slime-producing strains. J Biomed Mater Res 59:557–562PubMedCrossRefGoogle Scholar
  21. 21.
    Vandecasteele SJ, Peetermans WE, Merckx R, Rijnders BJ, Van Eldere J (2003) Reliability of the ica, aap and atlE genes in the discrimination between invasive, colonizing and contaminant Staphylococcus epidermidis isolates in the diagnosis of catheter-related infections. Clin Microbiol Infect 9:114–119PubMedCrossRefGoogle Scholar
  22. 22.
    Bradford R, Abdul Manan R, Daley AJ, Pearce C, Ramalingam A, D’Mello D, Mueller Y, Uahwatanasakul W, Qu Y, Grando D, Garland S, Deighton M (2006) Coagulase-negative staphylococci in very-low-birth-weight infants: inability of genetic markers to distinguish invasive strains from blood culture contaminants. Eur J Clin Microbiol Infect Dis 5:283–291CrossRefGoogle Scholar
  23. 23.
    Mermel LA, Farr BM, Sherertz RJ, Raad II, O’Grady N, Harris JS, Crafen DE (2001) Guidelines for the management of intravascular catheter-related infections. Clin Infect Dis 32:1249–1272PubMedCrossRefGoogle Scholar
  24. 24.
    Mack D, Nedelmann M, Krokotsch A, Schwarzkopf A, Heesemann J, Laufs R (1994) Characterization of transposon mutants of biofilm-producing Staphylococcus epidermidis impaired in the accumulative phase of biofilm production: genetic identification of a hexosamine-containing polysaccharide intercellular adhesin. Infect Immun 62:3244–3253PubMedGoogle Scholar
  25. 25.
    Clinical and Laboratory Standards Institute (2005) Performance Standards For Antimicrobial Susceptibility Testing. M100-S15. CLSI. Wayne, PA, USAGoogle Scholar
  26. 26.
    Chung M, de Lencastre H, Matthews P, Tomasz A, Adamsson I, Aires de Sousa M, Camou T, Cocuzza C, Multilaboratory Project Collaborators et al (2000) Molecular typing of methicillin-resistant Staphylococcus aureus by pulsed-field gel electrophoresis: comparison of results obtained in a multilaboratory effort using identical protocols and MRSA strains. Microb Drug Resist 6:189–198PubMedCrossRefGoogle Scholar
  27. 27.
    Arciola CR, Campoccia D, Gamberini S, Cervellati M, Donati E, Montanaro L (2002) Detection of slime production by means of an optimised Congo red agar plate test based on a colorimetric scale in Staphylococcus epidermidis clinical isolates genotyped for ica locus. Biomaterials 23:4233–4239PubMedCrossRefGoogle Scholar
  28. 28.
    O’Toole GA, Pratt LA, Watnick PI, Newman DK, Weaver VB, Kolter R (1999) Genetic approaches to study of biofilms. Methods Enzymol 310:91–109PubMedCrossRefGoogle Scholar
  29. 29.
    Christensen GD, Simpson WA, Younger JJ, Baddour LM, Barrett FF, Melton DM, Beachey EH (1985) Adherence of coagulase-negative staphylococci to plastic tissue culture plates: a quantitative model for the adherence of staphylococci to medical devices. J Clin Microbiol 22:996–1006PubMedGoogle Scholar
  30. 30.
    Arciola CR, Baldassarri L, Montanaro L (2001) Presence of icaA and icaD genes and slime production in a collection of staphylococcal strains from catheter-associated infections. J Clin Microbiol 39:2151–2156PubMedCrossRefGoogle Scholar
  31. 31.
    Carrico JA, Pinto FR, Simas C, Nunes S, Sousa NG, Frazao N, de Lencastre H, Almeida JS (2005) Assessment of band-based similarity coefficients for automatic type and subtype classification of microbial isolates analyzed by pulsed-field gel electrophoresis. J Clin Microbiol 43:5483–5490PubMedCrossRefGoogle Scholar
  32. 32.
    Ziebuhr W, Krimmer V, Rachid S, Lossner I, Gotz F, Hacker J (1999) A novel mechanism of phase variation of virulence in Staphylococcus epidermidis: evidence for control of the polysaccharide intercellular adhesin synthesis by alternating insertion and excision of the insertion sequence element IS256. Mol Microbiol 32:345–356PubMedCrossRefGoogle Scholar
  33. 33.
    Frebourg NB, Lefebvre S, Baert S, Lemeland JF (2000) PCR-based assay for discrimination between invasive and contaminating Staphylococcus epidermidis strains. J Clin Microbiol 38:877–880PubMedGoogle Scholar
  34. 34.
    Vandecasteele SJ, Peetermans WE, Merckx R, Van Eldere J (2003) Expression of biofilm-associated genes in Staphylococcus epidermidis during in vitro and in vivo foreign body infections. J Infect Dis 188:730–737PubMedCrossRefGoogle Scholar
  35. 35.
    Martineau F, Picard FJ, Grenier L, Roy PH, Ouellette M, Bergeron MG (2000) Multiplex PCR assays for the detection of clinically relevant antibiotic resistance genes in staphylococci isolated from patients infected after cardiac surgery. The ESPRIT Trial. J Antimicrob Chemother 46:527–534PubMedCrossRefGoogle Scholar
  36. 36.
    Martineau F, Picard FJ, Lansac N, Menard C, Roy PH, Ouellette M, Bergeron MG (2000) Correlation between the resistance genotype determined by multiplex PCR assays and the antibiotic susceptibility patterns of Staphylococcus aureus and Staphylococcus epidermidis. Antimicrob Agents Chemother 44:231–238PubMedCrossRefGoogle Scholar
  37. 37.
    Berger-Bachi B, Rohrer S (2002) Factors influencing methicillin resistance in staphylococci. Arch Microbiol 178:165–171PubMedCrossRefGoogle Scholar
  38. 38.
    Chambers HF (1997) Methicillin resistance in staphylococci: molecular and biochemical basis and clinical implications. Clin Microbiol Rev 10:781–791PubMedGoogle Scholar
  39. 39.
    Galdbart JO, Allignet J, Tung HS, Ryden C, El Solh N (2000) Screening for Staphylococcus epidermidis markers discriminating between skin-flora strains and those responsible for infections of joint prostheses. J Infect Dis 182:351–355PubMedCrossRefGoogle Scholar
  40. 40.
    Kozitskaya S, Cho SH, Dietrich K, Marre R, Naber K, Ziebuhr W (2004) The bacterial insertion sequence element IS256 occurs preferentially in nosocomial Staphylococcus epidermidis isolates: association with biofilm formation and resistance to aminoglycosides. Infect Immun 72:1210–1215PubMedCrossRefGoogle Scholar
  41. 41.
    Rohde H, Kalitzky M, Kroger N, Scherpe S, Horstkotte MA, Knobloch JK, Zander AR, Mack D (2004) Detection of virulence-associated genes is not useful for discriminating between invasive and commensal Staphylococcus epidermidis strains from a bone marrow transplant unit. J Clin Microbiol 42:5614–5619PubMedCrossRefGoogle Scholar
  42. 42.
    Mack D, Becker P, Chatterjee I, Dobinsky S, Knobloch JK, Peters G, Rohde H, Herrmann M (2004) Mechanisms of biofilm formation in Staphylococcus epidermidis and Staphylococcus aureus: functional molecules, regulatory circuits, and adaptive responses. Int J Med Microbiol 294:203–212PubMedCrossRefGoogle Scholar
  43. 43.
    Conlon KM, Humphreys H, O’Gara JP (2004) Inactivations of rsbU and sarA by IS256 represent novel mechanisms of biofilm phenotypic variation in Staphylococcus epidermidis. J Bacteriol 186:6208–6219PubMedCrossRefGoogle Scholar
  44. 44.
    Kiem S, Oh WS, Peck KR, Lee NY, Lee JY, Song JH, Hwang ES, ES Kim, Cha CY, Choe KW (2004) Phase variation of biofilm formation in Staphylococcus aureus by IS256 insertion and its impact on the capacity adhering to polyurethane surface. J Korean Med Sci 19:779–782PubMedCrossRefGoogle Scholar
  45. 45.
    Gu J, Li H, Li M, Vuong C, Otto M, Wen Y, Gao Q (2005) Bacterial insertion sequence IS256 as a potential molecular marker to discriminate invasive strains from commensal strains of Staphylococcus epidermidis. J Hosp Infect 61:342–348PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2006

Authors and Affiliations

  • D. Petrelli
    • 1
  • C. Zampaloni
    • 1
  • S. D’Ercole
    • 1
  • M. Prenna
    • 1
  • P. Ballarini
    • 1
  • S. Ripa
    • 1
  • L. A. Vitali
    • 1
    Email author
  1. 1.Department of Molecular, Cellular, and Animal BiologyUniversity of CamerinoCamerino (MC)Italy

Personalised recommendations