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Raman spectroscopic differentiation of planktonic bacteria and biofilms

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Abstract

Both biofilm formations as well as planktonic cells of water bacteria such as diverse species of the Legionella genus as well as Pseudomonas aeruginosa, Klebsiella pneumoniae, and Escherichia coli were examined in detail by Raman microspectroscopy. Production of various molecules involved in biofilm formation of tested species in nutrient-deficient media such as tap water was observed and was particularly evident in the biofilms formed by six Legionella species. Biofilms of selected species of the Legionella genus differ significantly from the planktonic cells of the same organisms in their lipid amount. Also, all Legionella species have formed biofilms that differ significantly from the biofilms of the other tested genera in the amount of lipids they produced. We believe that the significant increase in the synthesis of this molecular species may be associated with the ability of Legionella species to form biofilms. In addition, a combination of Raman microspectroscopy with chemometric approaches can distinguish between both planktonic form and biofilms of diverse bacteria and could be used to identify samples which were unknown to the identification model. Our results provide valuable data for the development of fast and reliable analytic methods based on Raman microspectroscopy, which can be applied to the analysis of tap water-adapted microorganisms without any cultivation step.

Biofilm and planktonic forms of L. pneumophila ssp. pneumophila exhibit different Raman spectra. L. pneumophila ssp. pneumophila in biofilms display a significant increase in the synthesis of lipids compared to the planktonic state

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References

  1. Andrews JS, Rolfe SA, Huang WE, Scholes JD, Banwart SA (2010) Biofilm formation in environmental bacteria is influenced by different macromolecules depending on genus and species. Environ Microbiol 12(9):2496–2507

    Article  CAS  Google Scholar 

  2. Donlan RM (2002) Biofilms: microbial life on surfaces. Emerg Infect Dis 8(9):881–890

    Article  Google Scholar 

  3. Exner M, Kramer A, Lajoie L, Gebel J, Engelhart S, Hartemann P (2005) Prevention and control of health care-associated waterborne infections in health care facilities. Am J Infect Control 33(5):S26–S40

    Article  CAS  Google Scholar 

  4. Potera C (1996) Biofilms invade microbiology. Science 273(5283):1795–1797

    Article  CAS  Google Scholar 

  5. Stewart CR, Muthye V, Cianciotto NP (2012) Legionella pneumophila persists within biofilms formed by Klebsiella pneumoniae, Flavobacterium sp., and Pseudomonas fluorescens under dynamic flow conditions. PLoS One 7(11):e50560

    Article  CAS  Google Scholar 

  6. Brown MR, Allison DG, Gilbert P (1988) Resistance of bacterial biofilms to antibiotics: a growth-rate related effect? J Antimicrob Chemother 22(6):777–780

    Article  CAS  Google Scholar 

  7. Gilbert P, Collier PJ, Brown MR (1990) Influence of growth rate on susceptibility to antimicrobial agents: biofilms, cell cycle, dormancy, and stringent response. Antimicrob Agents Chemother 34(10):1865

    Article  CAS  Google Scholar 

  8. Nahar Q, Fleißner F, Shuster J, Morawitz M, Halfpap C, Stefan M, Langbein U, Southam G, Mittler S (2014) Waveguide evanescent field scattering microscopy: bacterial biofilms and their sterilization response via UV irradiation. J Biophoton 7(7):542–551

    Article  Google Scholar 

  9. Oliver JD (2005) The viable but nonculturable state in bacteria. J Microbiol 43(1):93–100

    Google Scholar 

  10. Oliver JD (2000) The viable but nonculturable state and cellular resuscitation. Microbial biosystems: new frontiers. Atlantic Canada Society for Microbial Ecology, Halifax, pp 723–730

    Google Scholar 

  11. Yee RB, Wadowsky RM (1982) Multiplication of Legionella pneumophila in unsterilized tap water. Appl Environ Microbiol 43(6):1330–1334

    CAS  Google Scholar 

  12. Carson LA, Favero MS, Bond WW, Petersen NJ (1972) Factors affecting comparative resistance of naturally occurring and subcultured Pseudomonas aeruginosa to disinfectants. Appl Microbiol 23(5):863–869

    CAS  Google Scholar 

  13. Pahlow S, Meisel S, Cialla-May D, Weber K, Rösch P, Jürgen P (2015) Isolation and identification of bacteria by means of Raman spectroscopy. Adv Drug Deliv Rev. doi:10.1016/j.addr.2015.04.006

    Google Scholar 

  14. Beier BD, Quivey RG, Berger AJ (2012) Raman microspectroscopy for species identification and mapping within bacterial biofilms. AMB Express 2(1):1–6

    Article  Google Scholar 

  15. Chao Y, Zhang T (2012) Surface-enhanced Raman scattering (SERS) revealing chemical variation during biofilm formation: from initial attachment to mature biofilm. Anal Bioanal Chem 404(5):1465–1475

    Article  CAS  Google Scholar 

  16. Samek O, Al‐Marashi JFM, Telle HH (2010) The potential of Raman spectroscopy for the identification of biofilm formation by Staphylococcus epidermidis. Laser Phys Lett 7(5):378–383

    Article  CAS  Google Scholar 

  17. Kusić D, Kampe B, Rösch P, Popp J (2014) Identification of water pathogens by Raman microspectroscopy. Water Res 48:179–189

    Article  Google Scholar 

  18. Kloß S, Kampe B, Sachse S, Rösch P, Straube E, Pfister W, Kiehntopf M, Popp J (2013) Culture independent Raman spectroscopic identification of urinary tract infection pathogens: a proof of principle study. Anal Chem 85(20):9610–9616

    Article  Google Scholar 

  19. Bocklitz T, Walter A, Hartmann K, Rösch P, Popp J (2011) How to pre-process Raman spectra for reliable and stable models? Anal Chim Acta 704(1):47–56

    Article  CAS  Google Scholar 

  20. Development Core Team R (2011) R: a language and environment for statistical computing. R Foundation for Statistical Computing, Vienna

    Google Scholar 

  21. Morháč M, Kliman J, Matoušek V, Veselský M, Turzo I (1997) Background elimination methods for multidimensional coincidence gamma-ray spectra. Nucl Instrum Methods in Phys Res Sect A 401(1):113–132

    Article  Google Scholar 

  22. Cortes C, Vapnik V (1995) Support-vector networks. Mach Learn 20(3):273–297

    Google Scholar 

  23. Puppels GJ, De Mul FFM, Otto C, Greve J, Robert-Nicoud M, Arndt-Jovin DJ, Jovin TM (1990) Studying single living cells and chromosomes by confocal Raman microspectroscopy. Nature 347:301–303

    Article  CAS  Google Scholar 

  24. Fischer WB, Eysel HH (1992) Polarized Raman spectra and intensities of aromatic amino acids phenylalanine, tyrosine and tryptophan. Spectrochim Acta A: Mol Spectrosc 48(5):725–732

    Article  Google Scholar 

  25. Williams AC, Edwards HGM (1994) Fourier transform Raman spectroscopy of bacterial cell walls. J Raman Spectrosc 25(7-8):673–677

    Article  CAS  Google Scholar 

  26. Oliver JD (2000) Problems in detecting dormant (VBNC) cells, and the role of DNA elements in this response. In: Jansson JK, van Elsas JD, Bailey M (eds) Tracking genetically-engineered microorganisms. Landes Biosciences, Georgetown, pp 1–15

    Google Scholar 

  27. Ciobotă V, Burkhardt E-M, Schumacher W, Rösch P, Küsel K, Popp J (2010) The influence of intracellular storage material on bacterial identification by means of Raman spectroscopy. Anal Bioanal Chem 397(7):2929–2937

    Article  Google Scholar 

  28. James BW, Mauchline WS, Dennis PJ, Keevil CW, Wait R (1999) Poly-3-hydroxybutyrate in Legionella pneumophila, an energy source for survival in low-nutrient environments. Appl Environ Microbiol 65(2):822–827

    CAS  Google Scholar 

  29. Piao Z, Sze CC, Barysheva O, Iida K-i, Yoshida S-i (2006) Temperature-regulated formation of mycelial mat-like biofilms by Legionella pneumophila. Appl Environ Microbiol 72(2):1613–1622

    Article  CAS  Google Scholar 

  30. Steinberger RE, Holden PA (2005) Extracellular DNA in single- and multiple-species unsaturated biofilms. Appl Environ Microbiol 71(9):5404–5410

    Article  CAS  Google Scholar 

  31. Whitchurch CB, Tolker-Nielsen T, Ragas PC, Mattick JS (2002) Extracellular DNA required for bacterial biofilm formation. Science 295(5559):1487

    Article  CAS  Google Scholar 

  32. Karatzoglou A, Smola A, Hornik K (2013) kernlab: kernel-based machine learning lab. R Foundation for Statistical Computing, Vienna

    Google Scholar 

  33. Presti FL, Riffard S, Vandenesch F, Etienne J (1998) Identification of Legionella species by random amplified polymorphic DNA profiles. J Clin Microbiol 36(11):3193–3197

    CAS  Google Scholar 

  34. Moliner C, Ginevra C, Jarraud S, Flaudrops C, Bedotto M, Couderc C, Etienne J, Fournier P-E (2010) Rapid identification of Legionella species by mass spectrometry. J Med Microbiol 59(3):273–284

    Article  CAS  Google Scholar 

  35. Van de Vossenberg J, Tervahauta H, Maquelin K, Blokker-Koopmans CHW, Uytewaal-Aarts M, van der Kooij D, van Wezel AP, van der Gaag B (2013) Identification of bacteria in drinking water with Raman spectroscopy. Anal Methods 5(11):2679–2687

    Article  Google Scholar 

  36. Helm D, Labischinski H, Schallehn G, Naumann D (1991) Classification and identification of bacteria by Fourier-transform infrared spectroscopy. J Gen Microbiol 137(1):69–79

    Article  CAS  Google Scholar 

  37. Vlassov VV, Laktionov PP, Rykova EY (2007) Extracellular nucleic acids. Bioessays 29(7):654–667

    Article  CAS  Google Scholar 

  38. Lang S, Philp JC (1998) Surface-active lipids in rhodococci. Antonie Van Leeuwenhoek 74(1-3):59–70

    Article  CAS  Google Scholar 

  39. Sutherland IW (2001) Biofilm exopolysaccharides: a strong and sticky framework. Microbiology 147(1):3–9

    CAS  Google Scholar 

  40. Sutherland IW (2001) The biofilm matrix—an immobilized but dynamic microbial environment. Trends Microbiol 9(5):222–227

    Article  CAS  Google Scholar 

Download references

Acknowledgments

The authors gratefully acknowledge the assistance of Dr. Oliwia Makarewicz in laser scanning microscopy analysis and Prof. Dr. Eberhard Straube and Svea Sachse for providing us with the bacterial strains and for useful discussions. Funding of the research project RiMaTH (02WRS1276E) from the Federal Ministry of Education and Research, Germany (BMBF), is gratefully acknowledged. Financial support of the European Union via the EU project “HemoSpec” (CN 611682) and of the BMBF via the Integrated Research and Treatment Center “Center for Sepsis Control and Care” (FKZ 01EO1002) is highly acknowledged. This project was realized within the InfectoGnostics Research Campus Jena.

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Authors and Affiliations

  1. Institut für Physikalische Chemie and Abbe Center of Photonics, Friedrich-Schiller-Universität Jena, Helmholtzweg 4, 07743, Jena, Germany

    Dragana Kusić, Bernd Kampe, Petra Rösch & Jürgen Popp

  2. Leibniz Institute of Photonic Technology (IPHT), Albert Einstein Str. 9, 07745, Jena, Germany

    Anuradha Ramoji, Ute Neugebauer & Jürgen Popp

  3. InfectoGnostics Forschungscampus Jena e.V., Zentrum für Angewandte Forschung, Philosophenweg 7, 07743, Jena, Germany

    Jürgen Popp

  4. Center for Sepsis Control and Care (CSCC), Jena University Hospital, KIM II Erlanger Alle 101, 07747, Jena, Germany

    Anuradha Ramoji, Ute Neugebauer & Jürgen Popp

Authors
  1. Dragana Kusić
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  2. Bernd Kampe
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  3. Anuradha Ramoji
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  4. Ute Neugebauer
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  5. Petra Rösch
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  6. Jürgen Popp
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Corresponding author

Correspondence to Petra Rösch.

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Kusić, D., Kampe, B., Ramoji, A. et al. Raman spectroscopic differentiation of planktonic bacteria and biofilms. Anal Bioanal Chem 407, 6803–6813 (2015). https://doi.org/10.1007/s00216-015-8851-7

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  • DOI: https://doi.org/10.1007/s00216-015-8851-7

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