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Analytical and Bioanalytical Chemistry

, Volume 411, Issue 27, pp 7315–7325 | Cite as

Multi-technique microscopy investigation on bacterial biofilm matrices: a study on Klebsiella pneumoniae clinical strains

  • Giovanni Birarda
  • Ambra Delneri
  • Cristina Lagatolla
  • Pietro Parisse
  • Paola Cescutti
  • Lisa Vaccari
  • Roberto RizzoEmail author
Research Paper
  • 115 Downloads

Abstract

Biofilms are communities of bacteria living embedded in a highly hydrated matrix composed of polysaccharides, proteins, and extracellular DNA. This life style confers numerous advantages to bacteria including protection against external threats. However, they also contribute to increase bacterial resistance against antimicrobials, an issue particularly relevant in dangerous infections. Due to the complexity of the matrix, few information is present in the literature on details of its architecture including the spatial distribution of the macromolecular components which might give hints on the way the biofilm scaffold is built up by bacteria. In this study, we investigated the possibility to combine well-established microbiological procedures with advanced microscopies to get information on composition and distribution of the macromolecular components of biofilm matrices. To this, confocal microscopy, diffraction-limited infrared (IR) spectral imaging, and atomic force microscopy (AFM) were used to explore biofilm produced by a clinical strain of Klebsiella pneumoniae. IR imaging permitted to have clues on how the biofilm grows and spreads on surfaces, and the local distribution of the components within it. Through the analysis of the pure component spectra, it was possible to assess the chemical and structural composition of the saccaridic matrix, confirming the data obtained by NMR. It was also possible to follow the time course of biofilm from 6 up to 48 h when the biofilm grew into a 3-dimensional multi-layered structure, characteristic of colonies of bacteria linked together by a complex matrix. In addition, nanoFTIR and AFM investigations allowed the estimation of biofilm growth in the vertical direction and the morphological analysis of bacterial colonies at different time points and the evaluation of the chemical composition at the nanoscale.

Keywords

AFM (atomic force microscopy) NanoFTIR Biofilm Multi-variate analysis IR spectroscopy 

Notes

Acknowledgements

We thank Dr. Philip Schäfer from NEASPEC GmbH for the support during these first measurements.

Funding information

This study was funded by the University of Trieste (FRA 13 and FRA15). Images in this paper were generated at the Light Microscopy Imaging Centre (LMIC) of the University of Trieste - Life Sciences Department, funded as detailed at www.units.it/confocal.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflicts of interest.

Supplementary material

216_2019_2111_MOESM1_ESM.pdf (231 kb)
ESM 1 (PDF 230 kb)

References

  1. 1.
    Flemming H-C, Wingender J, Szewzyk U, Steinberg P, Rice SA, Kjelleberg S. Biofilms: an emergent form of bacterial life. Nat Rev Microbiol. 2016;14:563.CrossRefGoogle Scholar
  2. 2.
    Costerton JW, Lewandowski Z, Caldwell DE, Korber DR, Lappin-Scott HM. Microbial biofilms. Annu Rev Microbiol. 1995;49:711.CrossRefGoogle Scholar
  3. 3.
    Branda SS, Vik Å, Friedman L, Kolter R. Biofilms: the matrix revisited. Trends Microbiol. 2005;13:20.CrossRefGoogle Scholar
  4. 4.
    Flemming H-C, Neu T, Wozniak DJ. The EPS matrix: the “house of biofilm cells”. J Bacteriol. 2007;189:7945.CrossRefGoogle Scholar
  5. 5.
    Fux CA, Costerton JW, Stewart PS, Stoodley P. Survival strategies of infectious biofilms. Trends Microbiol. 2005;13:34.CrossRefGoogle Scholar
  6. 6.
    Birarda G, Holman EA, Fu S, Weikel K, Hu P, Blankenberg FG, et al. Synchrotron infrared imaging of advanced glycation endproducts (AGEs) in cardiac tissue from mice fed high glycemic diets. Biomed Spectrosc Imaging. 2013;2:301.PubMedPubMedCentralGoogle Scholar
  7. 7.
    Li B, Zhao Y, Liu C, Chen Z, Zhou D. Molecular pathogenesis of Klebsiella pneumoniae. Future Microbiol. 2014;9:1071.CrossRefGoogle Scholar
  8. 8.
    Munoz-Price LS, Poirel L, Bonomo RA, Schwaber MJ, Daikos GL, Cormican M, et al. Clinical epidemiology of the global expansion of Klebsiella pneumoniae carbapenemases. Lancet Infect Dis. 2013;13:785.CrossRefGoogle Scholar
  9. 9.
    Cescutti P, De Benedetto G, Rizzo R. Structural determination of the polysaccharide isolated from biofilms produced by a clinical strain of Klebsiella pneumoniae. Carbohydr Res. 2016;430:29.CrossRefGoogle Scholar
  10. 10.
    Benincasa M, Lagatolla C, Dolzani L, Milan A, Pacor S, Liut G, et al. Biofilms from Klebsiella pneumoniae: matrix polysaccharide structure and interactions with antimicrobial peptides. Microorganisms. 2016;4:26.CrossRefGoogle Scholar
  11. 11.
    Whitfield C, Richards JC, Perry MB, Clarke BR, MacLean LL. Expression of two structurally distinct D-galactan O antigens in the lipopolysaccharide of Klebsiella pneumoniae serotype O1. J Bacteriol. 1991;173:1420.CrossRefGoogle Scholar
  12. 12.
    Merritt JH, Kadouri DE, O’Toole GA. Growing and analyzing static biofilms. Curr Protoc Microbiol. 2005 Jul;Chapter 1:Unit 1B.1.Google Scholar
  13. 13.
    Heydorn A, Nielsen AT, Hentzer M, Sternberg C, Givskov M, Ersbøll BK, et al. Quantification of biofilm structures by the novel computer program COMSTAT. Microbiology. 2000;146:2395.CrossRefGoogle Scholar
  14. 14.
    Vorregaard M. Comstat2 - a modern 3D image analysis environment for biofilms. Master thesis, Technical University of Denmark (DTU), 2008.Google Scholar
  15. 15.
    Lupi S, Nucara A, Perucchi A, Calvani P, Ortolani M, Quaroni L, et al. Performance of SISSI, the infrared beamline of the ELETTRA storage ring. J Opt Soc Am B. 2007;24:959.CrossRefGoogle Scholar
  16. 16.
    Armitano J, Mejean V, Jourlin-Castelli C. Gram-negative bacteria can also form pellicles. Environ Microbiol Rep. 2014;6:534.CrossRefGoogle Scholar
  17. 17.
    Lewis AT, Jones K, Lewis KE, Jones S, Lewis PD. Detection of Lewis antigen structural change by FTIR spectroscopy. Carbohydr Polym. 2013;92:1294–301.CrossRefGoogle Scholar
  18. 18.
    Socrates G. Infrared and Raman characteristic group frequencies: tables and charts. 3rd ed. Chichester: Wiley; 2004.Google Scholar
  19. 19.
    Probst AJ, Holman HY, DeSantis TZ, Andersen GL, Birarda G, Bechtel HA, et al. Tackling the minority: sulfate-reducing bacteria in an archaea-dominated subsurface biofilm. ISME J. 2013;7:635.CrossRefGoogle Scholar
  20. 20.
    Schlafer S, Meyer RL. Confocal microscopy imaging of the biofilm matrix. J Microbiol Methods. 2017;138:50.CrossRefGoogle Scholar
  21. 21.
    Mandal DK, Bhattacharyya L, Koenig SH, Brown RD, Oscarson S, Brewer CF. Studies of the binding specificity of concanavalin A. Nature of the extended binding site for asparagine-linked carbohydrates. Biochemistry. 1994;33:1157.CrossRefGoogle Scholar
  22. 22.
    Kaewpijit S, Le Moigne J, El-Ghazawi T. Automatic reduction of hyperspectral imagery using wavelet spectral analysis. IEEE Geosci Remote Sensing. 2003;41:863.CrossRefGoogle Scholar
  23. 23.
    Roggo Y, Edmond A, Chalus P, Ulmschneider M. Infrared hyperspectral imaging for qualitative analysis of pharmaceutical solid forms. Anal Chim Acta. 2005;535:79.CrossRefGoogle Scholar
  24. 24.
    Neu TR, Manz B, Volke F, Dynes JJ, Hitchcock AP, Lawrence JR. Advanced imaging techniques for assessment of structure, composition and function in biofilm systems. FEMS Microbiol Ecol. 2010;72(1):1–21.CrossRefGoogle Scholar
  25. 25.
    Neu TR, Swerhone GDW, Lawrence JR. Assessment of lectin-binding analysis for in situ detection of glycoconjugates in biofilm systems. Microbiology. 2001;147:299.CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  • Giovanni Birarda
    • 1
  • Ambra Delneri
    • 2
  • Cristina Lagatolla
    • 2
  • Pietro Parisse
    • 1
  • Paola Cescutti
    • 2
  • Lisa Vaccari
    • 1
  • Roberto Rizzo
    • 2
    Email author
  1. 1.Elettra - Sincrotrone Trieste S.C.p.A.BasovizzaItaly
  2. 2.Department of Life SciencesUniversity of TriesteTriesteItaly

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