1887

Microbiology Society logo

Volume 155, Issue 8

Detection and identification of specific bacteria in wound biofilms using peptide nucleic acid fluorescent hybridization (PNA FISH) Free

Abstract

Biofilms provide a reservoir of potentially infectious micro-organisms that are resistant to antimicrobial agents, and their importance in the failure of medical devices and chronic inflammatory conditions is increasingly being recognized. Particular research interest exists in the association of biofilms with wound infection and non-healing, i.e. chronic wounds. In this study, fluorescent hybridization (FISH) was used in combination with confocal laser scanning microscopy (CLSM) to detect and characterize the spatial distribution of biofilm-forming bacteria which predominate within human chronic skin wounds (, , sp. and sp.). biofilms were prepared using a constant-depth film fermenter and a reconstituted human epidermis model. biofilms were also studied using biopsy samples from non-infected chronic venous leg ulcers. The specificity of peptide nucleic acid (PNA) probes for the target organisms was confirmed using mixed preparations of planktonic bacteria and multiplex PNA probing. Identification and location of individual bacterial species within multi-species biofilms demonstrated that was predominant. CLSM revealed clustering of individual species within mixed-species biofilms. FISH analysis of archive chronic wound biopsy sections showed bacterial presence and allowed bacterial load to be determined. The application of this standardized procedure makes available an assay for identification of single- or multi-species bacterial populations in tissue biopsies. The technique provides a reliable tool to study bacterial biofilm formation and offers an approach to assess targeted biofilm disruption strategies .

  • Received:
  • Accepted:
  • Revised:
  • Published Online:
Keyword(s): CDFF, constant-depth film fermenter , CLSM, confocal laser scanning microscopy , CVLU, chronic venous leg ulcer , FISH, fluorescent in situ hybridization , PNA, peptide nucleic acid and RHE, reconstituted human epidermis
SGM
Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.028712-0
2009-08-01
2024-04-24
Loading full text...

Full text loading...

/deliver/fulltext/micro/155/8/2603.html?itemId=/content/journal/micro/10.1099/mic.0.028712-0&mimeType=html&fmt=ahah

References

  1. Amann R., Fuchs B. M., Behrens S. 2001; The identification of microorganisms by fluorescence in situ hybridisation. Curr Opin Biotechnol 12:231–236
     [Google Scholar]
  2. Andersen A., Hill K. E., Stephens P., Thomas D. W., Jorgensen B., Krogfelt K. A. 2007; Bacterial profiling using skin grafting, standard culture and molecular bacteriological methods. J Wound Care 16:171–175
     [Google Scholar]
  3. Bjarnsholt T., Kirketerp-Moller K., Jensen P. O., Madsen K. G., Phipps R., Krogfelt K., Hoiby N., Givskov M. 2008; Why chronic wounds will not heal: a novel hypothesis. Wound Repair Regen 16:2–10
     [Google Scholar]
  4. Coull J. J., Hyldig-Nielsen J. J. 2003; US Patent 6664045. PNA probes, probe sets, methods and kits pertaining to the detection of microorganisms.
  5. Davies C. E., Wilson M., Hill K. E., Stephens P., Hill C. M., Harding K. G., Thomas D. W. 2001; Use of molecular techniques to study microbial diversity in the skin: chronic wounds reevaluated. Wound Repair Regen 9:332–340
     [Google Scholar]
  6. Davies C. E., Hill K. E., Wilson M., Stephens P., Hill C. M., Harding K. G., Thomas D. W. 2004; Use of 16S ribosomal DNA PCR and denaturing gradient gel electrophoresis for analysis of the microfloras of healing and nonhealing chronic venous leg ulcers. J Clin Microbiol 42:3549–3557
     [Google Scholar]
  7. Davies C. E., Hill K. E., Newcombe R. G., Stephens P., Wilson M. J., Harding K. G., Thomas D. W. 2007; A prospective study of the microbiology of chronic venous leg ulcers to reevaluate the clinical predictive value of tissue biopsies and swabs. Wound Repair Regen 15:17–22
     [Google Scholar]
  8. Davis S. C., Ricotti C., Cazzaniga A., Welsh E., Eaglstein W. H., Mertz P. M. 2008; Microscopic and physiologic evidence for biofilm-associated wound colonization in vivo. Wound Repair Regen 16:23–29
     [Google Scholar]
  9. Edwards R., Harding K. G. 2004; Bacteria and wound healing. Curr Opin Infect Dis 17:91–96
     [Google Scholar]
  10. Gu F., Lux R., Du-Thumm L., Stokes I., Kreth J., Anderson M. H., Wong D. T., Wolinsky L., Sullivan R., Shi W. 2005; In situ and non-invasive detection of specific bacterial species in oral biofilms using fluorescently labeled monoclonal antibodies. J Microbiol Methods 62:145–160
     [Google Scholar]
  11. Hartmann H., Stender H., Schaefer A., Autenrieth I. B., Kempf V. A. J. 2005; Rapid identification of Staphylococcus aureus in blood cultures by a combination of fluorescence in situ hybridisation using peptide nucleic acid probes and flow cytometry. J Clin Microbiol 43:4855–4857
     [Google Scholar]
  12. Hill K. E., Davies C. E., Wilson M., Stephens P., Harding K. G., Thomas D. W. 2003; Molecular analysis of the microflora in chronic venous leg ulceration. J Med Microbiol 52:365–369
     [Google Scholar]
  13. Howell-Jones R. S., Price P. E., Howard A. J., Thomas D. W. 2006; Antibiotic prescribing for chronic skin wounds in primary care. Wound Repair Regen 14:387–393
     [Google Scholar]
  14. James G. A., Swogger E., Wolcott R., Pulcini E. D., Secor P., Sestrich J., Costerton J. W., Stewart P. S. 2008; Biofilms in chronic wounds. Wound Repair Regen 16:37–44
     [Google Scholar]
  15. Lefmann M., Schweickert B., Buchholz P., Goebel U. B., Ulrichs T., Seiler P., Theegarten D., Moter A. 2006; Evaluation of peptide nucleic acid-fluorescence in situ hybridization for identification of clinically relevant mycobacteria in clinical specimens and tissue sections. J Clin Microbiol 44:3760–3767
     [Google Scholar]
  16. Leid J. G., Willson C. J., Shirtliff M. E., Hassett D. J., Parsek M. R., Jeffers A. K. 2005; The exopolysaccharide alginate protects Pseudomonas aeruginosa biofilm bacteria from IFN- γ-mediated macrophage killing. J Immunol 175:7512–7518
     [Google Scholar]
  17. Lopez C., Pons M. N., Morgenroth E. 2005; Evaluation of microscopic techniques (epifluorescence microscopy, CLSM, TPE-LSM) as a basis for the quantitative image analysis of activated sludge. Water Res 39:456–468
     [Google Scholar]
  18. Marsh P. D. 2003; Are dental diseases examples of ecological catastrophes?. Microbiology 149:279–294
     [Google Scholar]
  19. Mertz P. M. 2003; Cutaneous biofilms: friend or foe?. Wounds 15:129–132
     [Google Scholar]
  20. Moter A., Goebel U. B. 2000; Fluorescence in situ hybridization (FISH) for direct visualization of microorganisms. J Microbiol Methods 41:85–112
     [Google Scholar]
  21. Percival S. L., Bowler P. G. 2004; Biofilms and their potential role in wound healing. Wounds 16:234–240
     [Google Scholar]
  22. Percival S. L., Rogers A. A. 2005; The significance and role of biofilms in chronic wounds. In Biofilms: Persistence and Ubiquity Edited by McBain A., Allison A., Pratten J., Spratt D., Upton M., Verran J. Manchester, UK: The Biofilm Club, University of Manchester;
     [Google Scholar]
  23. Perry-O'Keefe H., Rigby S., Oliveira K., Sorensen D., Stender H., Coull J., Hyldig-Nielsen J. J. 2001a; Identification of indicator microorganisms using a standardized PNA FISH method. J Microbiol Methods 47:281–292
     [Google Scholar]
  24. Perry-O'Keefe H., Stender H., Broomer A., Oliveira K., Coull J., Hyldig-Nielsen J. J. 2001b; Filter-based PNA in situ hybridization for rapid detection, identification and enumeration of specific micro-organisms. J Appl Microbiol 90:180–189
     [Google Scholar]
  25. Pratten J., Wilson M. 1999; Antimicrobial susceptibility and composition of microcosm dental plaques supplemented with sucrose. Antimicrob Agents Chemother 43:1595–1599
     [Google Scholar]
  26. Rhoads D. D., Wolcott R. W., Cutting K. F., Percival S. L. 2007; Evidence of biofilms in wounds and potential ramifications. In Biofilms: Coming of Age pp 131–143 Edited by Gilbert P., Allison D., Brading M., Pratten J., Spratt D., Upton M. Manchester, UK: The Biofilm Club, Manchester University;
     [Google Scholar]
  27. Schaller M., Mailhammer R., Grassl G., Sander C. A., Hube B., Korting H. C. 2002; Infection of human oral epithelia with Candida species induces cytokine expression correlated to the degree of virulence. J Invest Dermatol 118:652–657
     [Google Scholar]
  28. Serralta V. W., Harrison-Balestra C., Cazzaniga A. L., Davis S. C., Mertz P. M. 2001; Lifestyles of bacteria in wounds: presence of biofilms?. Wounds 13:29–34
     [Google Scholar]
  29. Socransky S. S., Haffajee A. D., Cugini M. A., Smith C., Kent R. L. 1998; Microbial complexes in subgingival plaque. J Clin Periodontol 25:134–144
     [Google Scholar]
  30. Stephens P., Wall I. B., Wilson M., Hill K. E., Davies C. E., Hill C. M., Harding K. G., Thomas D. W. 2003; Anaerobic cocci populating the deep tissues of chronic wound impair cellular wound healing responses in vitro. Br J Dermatol 148:456–466
     [Google Scholar]
  31. Sunde P. T., Olsen I., Goebe U. B., Theegarten D., Winter S., Debelian G. J., Tronstad L., Moter A. 2003; Fluorescence in situ hybridization (FISH) for direct visualization of bacteria in periapical lesions of asymptomatic root-filled teeth. Microbiology 149:1095–1102
     [Google Scholar]
  32. Thurnheer T., Gmuer R., Guggenheim B. 2004; Multiplex FISH analysis of a six-species bacterial biofilm. J Microbiol Methods 56:37–47
     [Google Scholar]
  33. Villar C. C., Kashleva H., Mitchell A. P., Dongari-Bagtzoglou A. 2005; Invasive phenotype of Candida albicans affects the host proinflammatory response to infection. Infect Immun 73:4588–4595
     [Google Scholar]
  34. Vroom J. M., De Grauw K. J., Gerritsen H. C., Bradshaw D. J., Marsh P. D., Watson G. K., Birmingham J. J., Allison C. 1999; Depth penetration and detection of pH gradients in biofilms by two-photon excitation microscopy. Appl Environ Microbiol 65:3502–3511
     [Google Scholar]
  35. Wagner M., Horn M., Daims H. 2003; Fluorescence in situ hybridisation for the identification and characterisation of prokaryotes. Curr Opin Microbiol 6:302–309
     [Google Scholar]
  36. Wall I. B., Davies C. E., Hill K. E., Wilson M., Stephens P., Harding K. G., Thomas D. W. 2002; Potential role of anaerobic cocci in impaired human wound healing. Wound Repair Regen 10:346–353
     [Google Scholar]
  37. Wellinghausen N., Bartel M., Essig A., Poppert S. 2007; Rapid identification of clinically relevant Enterococcus species by fluorescence in situ hybridization. J Clin Microbiol 45:3424–3426
     [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.028712-0
Loading
Detection and identification of specific bacteria in wound biofilms using peptide nucleic acid fluorescent in situ hybridization (PNA FISH)
Microbiology 155, 2603 (2009); https://doi.org/10.1099/mic.0.028712-0
/content/journal/micro/10.1099/mic.0.028712-0
/content/journal/micro/10.1099/mic.0.028712-0
Loading

Data & Media loading...

Most cited Most Cited RSS feed

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error