Skip to main content


Log in

Does Dithiothreitol Improve Bacterial Detection from Infected Prostheses? A Pilot Study

  • Basic Research
  • Published:
Clinical Orthopaedics and Related Research®



Sonication and scraping of infected prostheses usually are used to improve diagnosis of prosthetic infections, reducing false negatives. Chemical methods that reduce biofilms also may allow higher levels of detection.


We therefore asked: (1) Do dithiothreitol (DTT) and N-acetylcysteine (NAC) remove bacteria from biofilm formed on prosthetic materials? (2) Is bacterial recovery affected by differing DTT and NAC concentrations and incubation times? (3) Do treatments with DTT and NAC detach the same amounts of bacteria from biofilm on prosthetic materials as sonication and scraping? (4) Are these methods reproducible?


We treated polyethylene and titanium discs covered by biofilm formed by Pseudomonas aeruginosa and Staphylococcus aureus with DTT or NAC solutions at different concentrations for different times. We compared colony counts of S aureus, P aeruginosa, Staphylococcus epidermidis and Escherichia coli after treatment with NAC, DTT, sonication and scraping. We determined colony counts after treatment of biofilm formed by one strain of S aureus and one of P aeruginosa on five discs of each material analyzed on the same day and on five discs analyzed on five consecutive days.


Mean colony counts (LogCFU/mL) obtained after treatment with 1 g/L DTT for 15 minutes (5.3) were similar to those after sonication (4.9) and greater than those obtaining by scraping (3.4) and treatment with 2 g/L NAC for 30 minutes (1.9). DTT and sonication showed good reproducibility.

Clinical Relevance

Our data suggest that treatment of prostheses with DTT may be a reasonable alternative to sonication to improve detection of biofilm-associated bacteria and supplement conventional laboratory culturing techniques for diagnosing periprosthetic infections.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6A–D
Fig. 7A–D
Fig. 8A–D
Fig. 9A–H

Similar content being viewed by others


  1. Achermann Y, Vogt M, Leunig M, Wust J, Trampuz A. Improved diagnosis of periprosthetic joint infection by multiplex PCR of sonication fluid from removed implants. J Clin Microbiol. 2010;48:1208–1214.

    Article  PubMed  Google Scholar 

  2. Aslam S, Trautner BW, Ramanathan V, Darouiche RO. Combination of tigecycline and N-acetylcysteine reduces biofilm-embedded bacteria on vascular catheters. Antimicrob Agents Chemother. 2007;51:1556–1558.

    Article  PubMed  CAS  Google Scholar 

  3. Berbari EF, Marculescu C, Sia I, Lahr BD, Hanssen AD, Steckelberg JM, Gullerud R, Osmon DR. Culture-negative prosthetic joint infection. Clin Infect Dis. 2007;45:1113–1119.

    Article  PubMed  Google Scholar 

  4. Bjerkan G, Witso E, Bergh K. Sonication is superior to scraping for retrieval of bacteria in biofilm on titanium and steel surfaces in vitro. Acta Orthop. 2009;80:245–250.

    Article  PubMed  Google Scholar 

  5. Cataldo MA, Petrosillo N, Cipriani M, Cauda R, Tacconelli E. Prosthetic joint infection: recent developments in diagnosis and management. J Infect. 2010;61:443–448.

    Article  PubMed  Google Scholar 

  6. Choong PF, Dowsey MM, Carr D, Daffy J, Stanley P. Risk factors associated with acute hip prosthetic joint infections and outcome of treatment with a rifampin-based regimen. Acta Orthop. 2007;78:755–765.

    Article  PubMed  Google Scholar 

  7. Christensen GD, Simpson WA, Younger JJ, Baddour LM, Barrett FF, Melton DM, Beachey EH. Adherence of coagulase-negative staphylococci to plastic tissue culture plates: a quantitative model for the adherence of staphylococci to medical device. J Clin Microbiol. 1985;22:996–1006.

    PubMed  CAS  Google Scholar 

  8. Clauss M, Trampuz A, Borens O, Bohner M, Ilchmann T. Biofilm formation on bone grafts and bone graft substitutes: comparison of different materials by a standard in vitro test and microcalorimetry. Acta Biomater. 2010;6:3791–3797.

    Article  PubMed  CAS  Google Scholar 

  9. Costerton JW, Stewart PS, Greenberg EP. Bacterial biofilms: a common cause of persistent infections. Science. 1999;284:1318–1322.

    Article  PubMed  CAS  Google Scholar 

  10. El-Feky MA, El-Rehewy MS, Hassan MA, Abolella HA, Abd El-Baky RM, Gad GF (2009) Effect of ciprofloxacin and N-acetylcysteine on bacterial adherence and biofilm formation on ureteral stent surfaces. Pol J Microbiol. 58: 261–267

    PubMed  CAS  Google Scholar 

  11. Ensing GT, van Horn JR, van der Mei HC, Busscher HJ, Neut D. Copal bone cement is more effective in preventing biofilm formation than Palacos R-G. Clin Orthop Relat Res. 2008;466:1492–1498.

    Article  PubMed  Google Scholar 

  12. Holinka J, Bauer L, Hirschl AM, Graninger W, Windhager R, Presterl E. Sonication cultures of explanted components as an add-on test to routinely conducted microbiological diagnostics improve pathogen detection. J Orthop Res. 2011;29:617–622.

    Article  PubMed  Google Scholar 

  13. Kobayashi H, Oethinger M, Tuohy MJ, Procop GW, Bauer TW. Improved detection of biofilm-formative bacteria by vortexing and sonication: a pilot study. Clin Orthop Relat Res. 2009;467:1360–1364.

    Article  PubMed  Google Scholar 

  14. Kurtz SM, Ong KL, Lau E, Bozic KJ, Berry D, Parvizi J. Prosthetic joint infection risk after TKA in the Medicare population. Clin Orthop Relat Res. 2010;468:52–56.

    Article  PubMed  Google Scholar 

  15. Jämsen E, Huhtala H, Puolakka T, Moilanen T. Risk factors for infection after knee arthroplasty: a register-based analysis of 43,149 cases. J Bone Joint Surg Am. 2009;91:38–47.

    Article  PubMed  Google Scholar 

  16. Naves P, del Prado G, Huelves L, Rodríguez-Cerrato V, Ruiz V, Ponte MC, Soriano F. Effects of human serum albumin, ibuprofen and N-acetyl-L-cysteine against biofilm formation by pathogenic Escherichia coli strains. J Hosp Infect. 2010;76:165–170.

    Article  PubMed  CAS  Google Scholar 

  17. Neut D, van der Mei HC, Bulstra SK, Busscher HJ. The role of small-colony variants in failure to diagnose and treat biofilm infections in orthopedics. Acta Orthop. 2007;78:299–308.

    Article  PubMed  Google Scholar 

  18. Oga M, Arizono T, Sugioka Y. Bacterial adherence to bioinert and bioactive materials studied in vitro. Acta Orthop Scand. 1993;64:273–276.

    Article  PubMed  CAS  Google Scholar 

  19. Olofsson AC, Hermansson M, Elwing H. N-acetyl-L-cysteine affects growth, extracellular polysaccharide production, and bacterial biofilm formation on solid surfaces. Appl Environ Microbiol. 2003;69:4814–4822.

    Article  PubMed  CAS  Google Scholar 

  20. Parvizi J, Zmistowski B, Berbari EF, Bauer TW, Springer BD, Della Valle CJ, Garvin KL, Mont MA, Wongworawat MD, Zalavras CG. New definition for periprosthetic joint infection: from the Workgroup of the Musculoskeletal Infection Society. Clin Orthop Relat Res. 2011;469:2992–2994.

    Article  PubMed  Google Scholar 

  21. Peersman G, Laskin R, Davis J, Peterson M. Infection in total knee replacement: a retrospective review of 6489 total knee replacements. Clin Orthop Relat Res. 2001;392:15–23.

    Article  PubMed  Google Scholar 

  22. Phillips JE, Crane TP, Noy M, Elliott TS, Grimer RJ. The incidence of deep prosthetic infections in a specialist orthopaedic hospital: a 15-year prospective survey. J Bone Joint Surg Br. 2006;88:943–948.

    Article  PubMed  CAS  Google Scholar 

  23. Piper KE, Jacobson MJ, Cofield RH, Sperling JW, Sanchez-Sotelo J, Osmon DR, McDowell A, Patrick S, Steckelberg JM, Mandrekar JN, Fernandez Sampedro M, Patel R. Microbiologic diagnosis of prosthetic shoulder infection by use of implant sonication. J Clin Microbiol. 2009;47:1878–1884.

    Article  PubMed  Google Scholar 

  24. Pulido L, Ghanem E, Joshi A, Purtill JJ, Parvizi J. Periprosthetic joint infection: the incidence, timing, and predisposing factors. Clin Orthop Relat Res. 2008;466:1710–1715.

    Article  PubMed  Google Scholar 

  25. Ramage G, Tunney MM, Patrick S, Gorman SP, Nixon JR. Formation of Propionibacterium acnes biofilms on orthopaedic biomaterials and their susceptibility to antimicrobials. Biomaterials. 2003;24:3221–3227.

    Article  PubMed  CAS  Google Scholar 

  26. Schwandt LQ, Van Weissenbruch R, Stokroos I, Van der Mei HC, Busscher HJ, Albers FW. Prevention of biofilm formation by dairy products and N-acetylcysteine on voice prostheses in an artificial throat. Acta Otolaryngol. 2004;124:726–731.

    Article  PubMed  CAS  Google Scholar 

  27. Sendi P, Frei R, Maurer TB, Trampuz A, Zimmerli W, Graber P. Escherichia coli variants in periprosthetic joint infection: diagnostic challenges with sessile bacteria and sonication. J Clin Microbiol. 2010;48:1720–1725.

    Article  PubMed  Google Scholar 

  28. Trampuz A, Osmon DR, Hanssen AD, Steckelberg JM, Patel R. Molecular and antibiofilm approaches to prosthetic joint infection. Clin Orthop Relat Res. 2003;414:69–88.

    Article  PubMed  Google Scholar 

  29. Trampuz A, Piper KE, Hanssen AD, Osmon DR, Cockerill FR, Steckelberg JM, Patel R. Sonication of explanted prosthetic components in bags for diagnosis of prosthetic joint infection is associated with risk of contamination. J Clin Microbiol. 2006;44:628–631.

    Article  PubMed  Google Scholar 

  30. Trampuz A, Piper KE, Jacobson MJ, Hanssen AD, Unni KK, Osmon DR, Mandrekar JN, Cockerill FR, Steckelberg JM, Greenleaf JF, Patel R. Sonication of removed hip and knee prostheses for diagnosis of infection. N Engl J Med. 2007;357:654–663.

    Article  PubMed  CAS  Google Scholar 

  31. van de Belt H, Neut D, Schenk W, van Horn JR, van Der Mei HC, Busscher HJ. Staphylococcus aureus biofilm formation on different gentamicin-loaded polymethylmethacrylate bone cements. Biomaterials. 2001;22:1607–1611.

    Article  PubMed  Google Scholar 

  32. Wu X, Wang Yu, Tao L. Sulfhydryl compounds reduce Staphylococcus aureus biofilm formation by inhibiting PIA biosynthesis. FEMS Microbiol Lett. 2011;316:44–50.

    Article  PubMed  CAS  Google Scholar 

  33. Zhao T, Liu Y. N-acetylcysteine inhibits biofilms produced by Pseudomonas aeruginosa. BMC Microbiol. 2010;10:140.

    Article  PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations


Corresponding author

Correspondence to Lorenzo Drago PhD.

Additional information

The institution of one or more of the authors (LD, VS, ED) has received, during the study period, funding from Italian Ministry of Health. Each author certifies that he or she, or a member of his or her immediate family, has no commercial associations (eg, consultancies, stock ownership, equity interest, patent/licensing arrangements, etc) that might pose a conflict of interest in connection with the submitted article.

All ICMJE Conflict of Interest Forms for authors and Clinical Orthopaedics and Related Research editors and board members are on file with the publication and can be viewed on request.

This work was performed at Laboratory of Clinical Chemistry and Microbiology, IRCCS Galeazzi Institute, Milan, Italy.

About this article

Cite this article

Drago, L., Romanò, C.L., Mattina, R. et al. Does Dithiothreitol Improve Bacterial Detection from Infected Prostheses? A Pilot Study. Clin Orthop Relat Res 470, 2915–2925 (2012).

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: