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


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.


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



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

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)


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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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