Advertisement

Imaging Bacteria and Biofilms on Hardware and Periprosthetic Tissue in Orthopedic Infections

  • Laura Nistico
  • Luanne Hall-Stoodley
  • Paul StoodleyEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1147)

Abstract

Infection is a major complication of total joint arthroplasty (TJA) surgery, and even though it is now as low as 1 % in some hospitals, the increasing number of primary surgeries translates to tens of thousands of revisions due to prosthetic joint infection (PJI). In many cases the only solution is revision surgery in which the hardware is removed. This process is extremely long and painful for patients and is a considerable financial burden for the health-care system. A significant proportion of the difficulties in diagnosis and treatment of PJI are associated with biofilm formation where bacteria attach to the surface of the prosthesis and periprosthetic tissue and build a 3-D biofilm community encased in an extracellular polymeric slime (EPS) matrix. Bacteria in biofilms have a low metabolic rate which is thought to be a major contributor to their recalcitrance to antibiotic treatment. The diagnosis of biofilm infections is difficult due to the fact that bacteria in biofilms are not readily cultured with standard clinical microbiology techniques. To identify and visualize in situ biofilm bacteria in orthopedic samples, we have developed protocols for the collection of samples in the operating room, for molecular fluorescent staining with 16S rRNA fluorescence in situ hybridization (FISH), and for imaging of samples using confocal laser scanning microscopy (CLSM). Direct imaging is the only method which can definitively identify biofilms on implants and complements both culture and culture-independent diagnostic methods.

Key words

Orthopedic samples Hardware Prosthesis Tissues Membranes Biofilm Molecular fluorescent imaging FISH Confocal laser scanning microscopy (CLSM) 

Notes

Acknowledgments

We thank S. Conti, MD, G. Altman MD, D. Altman MD, and N. Sotereanos, MD, Orthopedic Department, Allegheny General Hospital, Pittsburgh, PA, for providing the samples; S. Kathju, MD, PhD, University of Pittsburgh School of Medicine, Pittsburgh, PA and from the Center for Genomic Sciences, Allegheny-Singer Research Institute, Pittsburgh, PA; G.D. Ehrlich, PhD, C.J. Post, MD, PhD, and J.W. Costerton, PhD, for protocol development and provision of resources; and Mary O’Toole for her help in the preparation of the manuscript.

References

  1. 1.
    Ulrich SD, Seyler TM, Bennett D et al (2008) Total hip arthroplasties: what are the reasons for revision? Int Orthop 32:597–604PubMedCentralPubMedCrossRefGoogle Scholar
  2. 2.
    Daigle ME, Weinstein AM, Katz JN et al (2012) The cost-effectiveness of total joint arthroplasty: a systematic review of published literature. Best Pract Res Clin Rheumatol 26:649–658PubMedCrossRefGoogle Scholar
  3. 3.
    Sadoghi P, Liebensteiner M, Agreiter M et al (2013) Revision surgery after total joint arthroplasty: a complication-based analysis using worldwide arthroplasty registers. J Arthroplasty 28:1329–1332PubMedCrossRefGoogle Scholar
  4. 4.
    Gomez E, Cazanave C, Cunningham SA et al (2012) Prosthetic joint infection diagnosis using broad-range PCR of biofilms dislodged from knee and hip arthroplasty surfaces using sonication. J Clin Microbiol 50:3501–3508PubMedCentralPubMedCrossRefGoogle Scholar
  5. 5.
    Rasouli MR, Harandi AA, Adeli B et al (2012) Revision total knee arthroplasty: infection should be ruled out in all cases. J Arthroplasty 27:1239–1243PubMedCrossRefGoogle Scholar
  6. 6.
    Stoodley P, Nistico L, Johnson S et al (2008) Direct demonstration of viable Staphylococcus aureus biofilms in an infected total joint arthroplasty. A case report. J Bone Joint Surg Am 90:1751–1758PubMedCentralPubMedCrossRefGoogle Scholar
  7. 7.
    Stoodley P, Conti SF, DeMeo PJ et al (2011) Characterization of a mixed MRSA/MRSE biofilm in an explanted total ankle arthroplasty. FEMS Immunol Med Microbiol 62:66–74PubMedCrossRefGoogle Scholar
  8. 8.
    Palmer M, Costerton W, Sewecke J et al (2011) Molecular techniques to detect biofilm bacteria in long bone nonunion: a case report. Clin Orthop Relat Res 469:3037–3042PubMedCentralPubMedCrossRefGoogle Scholar
  9. 9.
    Hall-Stoodley L, Stoodley P, Kathju S et al (2012) Towards diagnostic guidelines for biofilm-associated infections. FEMS Immunol Med Microbiol 65:127–145PubMedCrossRefGoogle Scholar
  10. 10.
    Hall-Stoodley L, Costerton JW, Stoodley P (2004) Bacterial biofilms: from the environment to infectious disease. Nat Rev Microbiol 2:95–108PubMedCrossRefGoogle Scholar
  11. 11.
    Post JC, Preston RA, Aul JJ et al (1995) Molecular analysis of bacterial pathogens in otitis media with effusion. JAMA 273:1598–1604PubMedCrossRefGoogle Scholar
  12. 12.
    Amann R, Ludwig V, Schleifer KH (1995) Phylogenetic identification and in situ detection of individual microbial cells without cultivation. Microbiol Rev 59:143–169PubMedCentralPubMedGoogle Scholar
  13. 13.
    Hall-Stoodley L, Hu FZ, Gieseke A et al (2006) Direct detection of bacterial biofilms on the middle-ear mucosa of children with chronic otitis media. JAMA 296:202–211PubMedCentralPubMedCrossRefGoogle Scholar
  14. 14.
    Nistico L, Kreft R, Gieseke A et al (2011) Adenoid reservoir for pathogenic biofilm bacteria. J Clin Microbiol 49:1411–1420PubMedCentralPubMedCrossRefGoogle Scholar
  15. 15.
    Kathju S, Nistico L, Hall-Stoodley L et al (2009) Chronic surgical site infection due to suture-associated polymicrobial biofilm. Surg Infect (Larchmt) 10:457–461CrossRefGoogle Scholar
  16. 16.
    Hall-Stoodley L, Nistico L, Sambanthamoorthy K et al (2008) Characterization of biofilm matrix, degradation by DNase treatment and evidence of capsule downregulation in Streptococcus pneumoniae clinical isolates. BMC Microbiol 8:173PubMedCentralPubMedCrossRefGoogle Scholar
  17. 17.
    Manz W, Amann R, Ludwig W et al (1992) Phylogenetic oligodeoxynucleotide probes for the major subclasses of proteobacteria: problems and solutions. Syst Appl Microbiol 15:593–600CrossRefGoogle Scholar
  18. 18.
    Amann RI, Krumholz L, Stahl DA (1990) Fluorescent-oligonucleotide probing of whole cells for determinative, phylogenetic, and environmental studies in microbiology. J Bacteriol 172:762–770PubMedCentralPubMedGoogle Scholar
  19. 19.
    Pernthaler A, Pernthaler J, Amann R (2002) Fluorescence in situ hybridization and catalyzed reporter deposition for the identification of marine bacteria. Appl Environ Microbiol 68:3094–3101PubMedCentralPubMedCrossRefGoogle Scholar
  20. 20.
    Hugenholtz P, Gene WT, Blackall LL (2001) Design and evaluation of 16S rRNA-targeted oligonucleotide probes for fluorescence in situ hybridization. In: Lieberman BA (ed) Steroid receptors methods: protocols and assays. Humana Press, Totowa, NJ, pp 29–41Google Scholar
  21. 21.
    Thurnheer T, Gmür R, Guggenheim B (2004) Multiplex FISH analysis of a six-species bacterial biofilm. J Microbiol Methods 56:37–47PubMedCrossRefGoogle Scholar
  22. 22.
    Nistico L, Gieseke A, Stoodley P et al (2009) Fluorescence in situ hybridization for the detection of biofilm in the middle ear and upper respiratory tract mucosa. Methods Mol Biol 493:191–215PubMedCrossRefGoogle Scholar
  23. 23.
    Trebesius K, Leitritz L, Adler K et al (2000) Culture independent and rapid identification of bacterial pathogens in necrotising fasciitis and streptococcal toxic shock syndrome by fluorescence in situ hybridisation. Med Microbiol Immunol 188:169–175PubMedCrossRefGoogle Scholar
  24. 24.
    Amann R, Snaidr J, Wagner M et al (1996) In situ visualization of high genetic diversity in a natural microbial community. J Bacteriol 178:3496–3500PubMedCentralPubMedGoogle Scholar
  25. 25.
    Kempf VA, Tresbesius K, Autenrieth IB (2000) Fluorescent in situ hybridization allows rapid identification of microorganisms in blood cultures. J Clin Microbiol 38:830–838PubMedCentralPubMedGoogle Scholar
  26. 26.
    Hodgart M, Trebesius K, Geiger AM et al (2000) Specific and rapid detection by fluorescent in situ hybridization of bacteria in clinical samples obtained from cystic fibrosis patients. J Clin Microbiol 38:818–825Google Scholar
  27. 27.
    Poppert S, Riecker M, Essig A (2010) Rapid identification of Propionibacterium acnes from blood cultures by fluorescence in situ hybridization. Diagn Microbiol Infect Dis 66:214–216PubMedCrossRefGoogle Scholar
  28. 28.
    Wellinghausen N, Bartel M, Essig A et al (2007) Rapid identification of clinically relevant Enterococcus species by fluorescence in situ hybridization. J Clin Microbiol 45:3424–3426PubMedCentralPubMedCrossRefGoogle Scholar
  29. 29.
    Moter A, Leist G, Rudolph R et al (1998) Fluorescence in situ hybridization shows spatial distribution of as yet uncultured treponemes in biopsies from digital dermatitis lesions. Microbiology 144:2459–2467PubMedCrossRefGoogle Scholar
  30. 30.
    Liu WT, Mirzabekov AD, Stalh DA (2001) Optimization of an oligonucleotide microchip for microbial identification studies: a non-equilibrium dissociation approach. Environ Microbiol 3:619PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • Laura Nistico
    • 1
  • Luanne Hall-Stoodley
    • 2
    • 3
  • Paul Stoodley
    • 1
    • 2
    • 3
    Email author
  1. 1.Center for Genomic SciencesAllegheny-Singer Research InstitutePittsburghUSA
  2. 2.National Center for Advanced Tribology, University of SouthamptonSouthamptonUK
  3. 3.Center for Microbial Interface Biology and Department of OrthopeadicsThe Ohio State UniversityColumbusUSA

Personalised recommendations