Skip to main content
SpringerLink
Log in

Characterization and analysis of mycobacteria and Gram-negative bacteria and co-culture mixtures by Raman microspectroscopy, FTIR, and atomic force microscopy

  • Original Paper
  • Published:
Analytical and Bioanalytical Chemistry Aims and scope Submit manuscript

Abstract

The molecular composition of mycobacteria and Gram-negative bacteria cell walls is structurally different. In this work, Raman microspectroscopy was applied to discriminate mycobacteria and Gram-negative bacteria by assessing specific characteristic spectral features. Analysis of Raman spectra indicated that mycobacteria and Gram-negative bacteria exhibit different spectral patterns under our experimental conditions due to their different biochemical components. Fourier transform infrared (FTIR) spectroscopy, as a supplementary vibrational spectroscopy, was also applied to analyze the biochemical composition of the representative bacterial strains. As for co-cultured bacterial mixtures, the distribution of individual cell types was obtained by quantitative analysis of Raman and FTIR spectral images and the spectral contribution from each cell type was distinguished by direct classical least squares analysis. Coupled atomic force microscopy (AFM) and Raman microspectroscopy realized simultaneous measurements of topography and spectral images for the same sampled surface. This work demonstrated the feasibility of utilizing a combined Raman microspectroscopy, FTIR, and AFM techniques to effectively characterize spectroscopic fingerprints from bacterial Gram types and mixtures.

AFM deflection images, Raman spectra, SEM images, and FTIR of Mycobacterium sp. KMS

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8
Fig. 9
Fig. 10

Similar content being viewed by others

References

  1. Hirsch CS, Johnson JL, Ellner JJ (1999) Pulmonary tuberculosis. Curr Opin Pulm Med 5(3):143–150

    Article  CAS  Google Scholar 

  2. Kochi A (1991) The global tuberculosis situation and the new control strategy of the World Health Organization. Tubercle 72(1):1–6

    Article  CAS  Google Scholar 

  3. Griffith DE, Aksamit T, Brown-Elliott BA, Catanzaro A, Daley C, Gordin F, Holland SM, Horsburgh R, Huitt G, Iademarco MF, Iseman M, Olivier K, Ruoss S, von Reyn CF, Wallace RJ, Winthrop K (2007) An official ATS/IDSA statement: diagnosis, treatment, and prevention of nontuberculous mycobacterial diseases. Am J Resp Crit Care 175(4):367–416

    Article  CAS  Google Scholar 

  4. Buijtels PCAM, Willemse-Erix HFM, Petit PLC, Endtz HP, Puppels GJ, Verbrugh HA, van Belkum A, van Soolingen D, Maquelin K (2008) Rapid identification of mycobacteria by Raman spectroscopy. J Clin Microbiol 46(3):961–965

    Article  CAS  Google Scholar 

  5. Nikaido H (2001) Preventing drug access to targets: cell surface permeability barriers and active efflux in bacteria. Semin Cell Dev Biol 12(3):215–223

    Article  CAS  Google Scholar 

  6. Rosenbaum JT, Mcdevitt HO, Guss RB, Egbert PR (1980) Endotoxin-induced uveitis in rats as a model for human disease. Nature 286(5773):611–613

    Article  CAS  Google Scholar 

  7. Carey PR (1999) Raman spectroscopy, the sleeping giant in structural biology, awakes. J Biol Chem 274(38):26625–26628

    Article  CAS  Google Scholar 

  8. Baena JR, Lendl B (2004) Raman spectroscopy in chemical bioanalysis. Curr Opin Chem Biol 8(5):534–539. doi:10.1016/j.cbpa.2004.08.014

    Article  CAS  Google Scholar 

  9. Rosch P, Schmitt M, Kiefer W, Popp J (2003) The identification of microorganisms by micro-Raman spectroscopy. J Mol Struct 661:363–369. doi:10.1016/j.molstruc.2003.06.004

    Article  Google Scholar 

  10. Rosch P, Harz M, Schmitt M, Peschke KD, Ronneberger O, Burkhardt H, Motzkus HW, Lankers M, Hofer S, Thiele H, Popp J (2005) Chemotaxonomic identification of single bacteria by micro-Raman spectroscopy: application to clean-room-relevant biological contaminations. Appl Environ Microbiol 71(3):1626–1637. doi:10.1128/AEM.71.3.1626-1637.2005

    Article  Google Scholar 

  11. De Gelder J, De Gussem K, Vandenabeele P, Vancanneyt M, De Vos P, Moens L (2007) Methods for extracting biochemical information from bacterial Raman spectra: focus on a group of structurally similar biomolecules—fatty acids. Anal Chim Acta 603(2):167–175. doi:10.1016/j.aca.2007.09.049

    Article  Google Scholar 

  12. Petrov GI, Arora R, Yakovlev VV, Wang X, Sokolov AV, Scully MO (2007) Comparison of coherent and spontaneous Raman microspectroscopies for noninvasive detection of single bacterial endospores. Proc Natl Acad Sci U S A 104(19):7776–7779. doi:10.1073/pnas.0702107104

    Article  CAS  Google Scholar 

  13. Neugebauer U, Schmid U, Baumann K, Holzgrabe U, Ziebuhr W, Kozitskaya S, Kiefer W, Schmitt M, Popp J (2006) Characterization of bacterial growth and the influence of antibiotics by means of UV resonance Raman spectroscopy. Biopolymers 82(4):306–311. doi:10.1002/bip.20447

    Article  CAS  Google Scholar 

  14. Harz M, Rosch P, Peschke KD, Ronneberger O, Burkhardt H, Popp J (2005) Micro-Raman spectroscopic identification of bacterial cells of the genus Staphylococcus and dependence on their cultivation conditions. Analyst 130(11):1543–1550. doi:10.1039/b507715j

    Article  CAS  Google Scholar 

  15. Neugebauer U, Schmid U, Baumann K, Ziebuhr W, Kozitskaya S, Deckert V, Schmitt M, Popp J (2007) Towards a detailed understanding of bacterial metabolism—spectroscopic characterization of Staphylococcus epidermidis. ChemPhysChem 8(1):124–137. doi:10.1002/cphc.200600507

    Article  CAS  Google Scholar 

  16. Lu X, Weakley AT, Aston DE, Rasco BA, Wang S, Konkel ME (2012) Examination of nanoparticle inactivation of Campylobacter jejuni biofilms using infrared and Raman spectroscopies. J Appl Microbiol 113(4):952–963. doi:10.1111/j.1365-2672.2012.05373.x

    Article  CAS  Google Scholar 

  17. Suo ZY, Yang XH, Avci R, Kellerman L, Pascual DW, Fries M, Steele A (2007) HEPES-stabilized encapsulation of Salmonella typhimurium. Langmuir 23(3):1365–1374. doi:10.1021/La0621721

    Article  CAS  Google Scholar 

  18. Naumann D, Helm D, Labischinski H (1991) Microbiological characterizations by FT-IR spectroscopy. Nature 351(6321):81–82. doi:10.1038/351081a0

    Article  CAS  Google Scholar 

  19. Maquelin K, Kirschner C, Choo-Smith LP, Ngo-Thi NA, van Vreeswijk T, Stammler M, Endtz HP, Bruining HA, Naumann D, Puppels GJ (2003) Prospective study of the performance of vibrational spectroscopies for rapid identification of bacterial and fungal pathogens recovered from blood cultures. J Clin Microbiol 41(1):324–329. doi:10.1128/Jcm.41.1.324-329.2003

    Article  CAS  Google Scholar 

  20. Holman HYN, Nieman K, Sorensen DL, Miller CD, Martin MC, Borch T, McKinney WR, Sims RC (2002) Catalysis of PAH biodegradation by humic acid shown in synchrotron infrared studies. Environ Sci Technol 36(6):1276–1280. doi:10.1021/Es0157200

    Article  CAS  Google Scholar 

  21. Yadav LDS (2005) Organic spectroscopy. Raman spectroscopy. Kluwer Academic, New Delhi

    Google Scholar 

  22. Rodriguez A, Autio WR, McLandsborough LA (2008) Effects of contact time, pressure, percent relative humidity (%RH), and material type on Listeria biofilm adhesive strength at a cellular level using atomic force microscopy (AFM). Food Biophysics 3(3):305–311. doi:10.1007/s11483-008-9085-4

    Article  Google Scholar 

  23. Yongsunthon R, Lower SK (2006) Force measurements between a bacterium and another surface in situ. Adv Appl Microbiol 58:97–124. doi:10.1016/S0065-2164(06)58003-1

    Google Scholar 

  24. Beckmann MA, Venkataraman S, Doktycz MJ, Nataro JP, Sullivan CJ, Morrell-Falvey JL, Allison DP (2006) Measuring cell surface elasticity on enteroaggregative Escherichia coli wild type and dispersin mutant by AFM. Ultramicroscopy 106(8–9):695–702. doi:10.1016/j.ultramic.2006.02.006

    Article  CAS  Google Scholar 

  25. Miller CD, Hall K, Liang YN, Nieman K, Sorensen D, Issa B, Anderson AJ, Sims RC (2004) Isolation and characterization of polycyclic aromatic hydrocarbon-degrading mycobacterium isolates from soil. Microb Ecol 48(2):230–238. doi:10.1007/s00248-003-1044-5

    Article  CAS  Google Scholar 

  26. Anonymous (1982) Certified host vector systems. Recomb DNA Tech Bull 5(4):206–209

  27. Nelson KE (2002) The complete genome sequence of Pseudomonas putida KT2440 is finally available—foreword. Environ Microbiol 4(12):777–778. doi:10.1046/j.1462-2920.2002.00367.x

    Article  Google Scholar 

  28. Bagdasarian M, Lurz R, Ruckert B, Franklin FCH, Bagdasarian MM, Frey J, Timmis KN (1981) Specific-purpose plasmid cloning vectors. II. Broad host range, high copy number, RSF1010-derived vectors, and a host–vector system for gene cloning in Pseudomonas. Gene 16(1–3):237–247. doi:10.1016/0378-1119(81)90080-9

    Article  CAS  Google Scholar 

  29. Nelson KE, Weinel C, Paulsen IT, Dodson RJ, Hilbert H, dos Santos VAPM, Fouts DE, Gill SR, Pop M, Holmes M, Brinkac L, Beanan M, DeBoy RT, Daugherty S, Kolonay J, Madupu R, Nelson W, White O, Peterson J, Khouri H, Hance I, Lee PC, Holtzapple E, Scanlan D, Tran K, Moazzez A, Utterback T, Rizzo M, Lee K, Kosack D, Moestl D, Wedler H, Lauber J, Stjepandic D, Hoheisel J, Straetz M, Heim S, Kiewitz C, Eisen J, Timmis KN, Dusterhoft A, Tummler B, Fraser CM (2002) Complete genome sequence and comparative analysis of the metabolically versatile Pseudomonas putida KT2440. Environ Microbiol 4(12):799–808. doi:10.1046/j.1462-2920.2002.00366.x

    Article  CAS  Google Scholar 

  30. Pal A, Paul AK (2008) Microbial extracellular polymeric substances: central elements in heavy metal bioremediation. Indian Journal of Microbiology 48(1):49–64

    Article  CAS  Google Scholar 

  31. Tian Y (2008) Behaviour of bacterial extracellular polymeric substances from activated sludge: a review. Int J Environ Pollut 32(1):78–89

    Article  CAS  Google Scholar 

  32. Bhaskar PV, Bhosle NB (2006) Bacterial extracellular polymeric substance (EPS): a carrier of heavy metals in the marine food-chain. Environ Int 32(2):191–198. doi:10.1016/j.envint.2005.08.010

    Article  CAS  Google Scholar 

  33. Kachlany SC, Levery SB, Kim JS, Reuhs BL, Lion LW, Ghiorse WC (2001) Structure and carbohydrate analysis of the exopolysaccharide capsule of Pseudomonas putida G7. Environ Microbiol 3(12):774–784

    Article  CAS  Google Scholar 

  34. McEwen GD, Wu YZ, Zhou AH (2010) Probing nanostructures of bacterial extracellular polymeric substances versus culture time by Raman microspectroscopy and atomic force microscopy. Biopolymers 93(2):171–177. doi:10.1002/Bip.21315

    Article  CAS  Google Scholar 

  35. Beveridge TJ (1981) Ultrastructure, chemistry, and function of the bacterial wall. Int Rev Cytol 72:229–317

    Article  CAS  Google Scholar 

  36. Perry JJ, Staley JT, Lory S (2002) Microbial life. Sinauer Associates Incorporated, Sunderland

    Google Scholar 

  37. Varki A, Cummings R, Esko J, Freeze H, Hart G, Marth J (1999) Essentials of glycobiology. Cold Spring Harbor Laboratory Press, New York

    Google Scholar 

  38. Wu YZ, Zhou AH (2010) Fluctuations in adhesion behavior of dividing/budding Mycobacterium sp. strains JLS, KMS, MCS: an AFM evaluation. Micron 41(7):814–820. doi:10.1016/j.micron.2010.05.008

    Article  Google Scholar 

  39. Zhang L, Henson MJ, Sekulic SS (2005) Multivariate data analysis for Raman imaging of a model pharmaceutical tablet. Anal Chim Acta 545(2):262–278. doi:10.1016/j.aca.2005.04.080

    Article  CAS  Google Scholar 

  40. Maquelin K, Kirschner C, Choo-Smith LP, van den Braak N, Endtz HP, Naumann D, Puppels GJ (2002) Identification of medically relevant microorganisms by vibrational spectroscopy. J Microbiol Methods 51(3):255–271

    Article  CAS  Google Scholar 

  41. Thi NAN, Carolin K, Dieter N (2000) FT-IR microspectrometry: a new tool for characterizing micro-organisms. Proc SPIE 36:36–44

    Google Scholar 

  42. Tripathi A, Jabbour RE, Treado PJ, Neiss JH, Nelson MP, Jensen JL, Snyder AP (2008) Waterborne pathogen detection using Raman spectroscopy. Appl Spectrosc 62(1):1–9. doi:10.1366/000370208783412546

    Article  CAS  Google Scholar 

  43. Berger AJ, Zhu QY (2003) Identification of oral bacteria by Raman microspectroscopy. J Mod Opt 50(15–17):2375–2380. doi:10.1080/0950034032000121055

    CAS  Google Scholar 

  44. le Roux K, Prinsloo LC, Hussein AA, Lall N (2012) A micro-Raman spectroscopic investigation of leukemic U-937 cells treated with Crotalaria agatiflora Schweinf and the isolated compound madurensine. Spectrochim Acta A 95:547–554. doi:10.1016/j.saa.2012.04.048

    Article  Google Scholar 

  45. Parekh SH, Lee YJ, Aamer KA, Cicerone MT (2010) Label-free cellular imaging by broadband coherent anti-Stokes Raman scattering microscopy. Biophys J 99(8):2695–2704. doi:10.1016/j.bpj.2010.08.009

    Article  CAS  Google Scholar 

  46. De Gelder J, De Gussem K, Vandenabeele P, Vancanneyt M, De Vos P, Moens L (2007) Methods for extracting biochemical information from bacterial Raman spectra: focus on a group of structurally similar biomolecules—fatty acids. Anal Chim Acta 603(2):167–175. doi:10.1016/j.aca.2007.09.049

    Article  Google Scholar 

  47. Chan JW, Motton D, Rutledge JC, Keim NL, Huser T (2005) Raman spectroscopic analysis of biochemical changes in individual triglyceride-rich lipoproteins in the pre- and postprandial state. Anal Chem 77(18):5870–5876. doi:10.1021/Ac050692f

    Article  CAS  Google Scholar 

  48. Tortora GJ, Funke BR, Case CL (1998) Microbiology an introduction. Benjamin Cummings, Menlo Park

  49. Goodwin JR, Hafner LM, Fredericks PM (2006) Raman spectroscopic study of the heterogeneity of microcolonies of a pigmented bacterium. J Raman Spectrosc 37(9):932–936. doi:10.1002/Jrs.1523

    Article  CAS  Google Scholar 

  50. Pradhan N, Pradhan SK, Nayak BB, Mukherjee PS, Sukla LB, Mishra BK (2008) Micro-Raman analysis and AFM imaging of Acidithiobacillus ferrooxidans biofilm grown on uranium ore. Res Microbiol 159(7–8):557–561. doi:10.1016/j.resmic.2008.06.006

    Article  CAS  Google Scholar 

  51. Naumann D (2006) Encyclopedia of analytical chemistry infrared spectroscopy in microbiology. Wiley, Chichester

  52. Mauer LJ, Reuhs BL (2008) Mid-infrared sensors for the rapid analysis of select microbial food borne pathogens. In: Voeller JG (ed) Wiley handbook of science and technology for homeland security. Wiley, Chichester

  53. Fountain AW, Pearman WF (2006) Classification of chemical and biological warfare agent simulants by surface-enhanced Raman spectroscopy and multivariate statistical techniques. Appl Spectrosc 60(4):356–365

    Article  Google Scholar 

  54. Naumann D (2001) FT-infrared and FT-Raman spectroscopy in biomedical research. Appl Spectrosc Rev 36(2–3):239–298

    Article  CAS  Google Scholar 

  55. Oust A, Moretro T, Naterstad K, Sockalingum GD, Adt I, Manfait M, Kohler A (2006) Fourier transform infrared and Raman spectroscopy for characterization of Listeria monocytogenes strains. Appl Environ Microbiol 72(1):228–232

    Article  CAS  Google Scholar 

  56. Rasco BA, Lu XN, Al-Qadiri HM, Lin MS (2011) Application of mid-infrared and Raman spectroscopy to the study of bacteria. Food and Bioprocess Technology 4(6):919–935. doi:10.1007/s11947-011-0516-8

    Article  Google Scholar 

  57. Goodacre R, Jarvis RM, Brooker A (2004) Surface-enhanced Raman spectroscopy for bacterial discrimination utilizing a scanning electron microscope with a Raman spectroscopy interface. Anal Chem 76(17):5198–5202. doi:10.1021/ac049663f

    Article  Google Scholar 

  58. Wu YZ, Zhou AH (2009) In situ, real-time tracking of cell wall topography and nanomechanics of antimycobacterial drugs treated Mycobacterium JLS using atomic force microscopy. Chem Commun 45:7021–7023. doi:10.1039/B914605a

    Article  Google Scholar 

  59. Farnia P, Mohammad RM, Merza MA, Tabarsi P, Zhavnerko GK, Ibrahim TA, Kuan HO, Ghanavei J, Ranjbar R, Poleschuyk NN, Titov LP, Owlia P, Kazampour M, Setareh M, Sheikolslami M, Migliori GB, Velayati AA (2010) Growth and cell-division in extensive (XDR) and extremely drug resistant (XXDR) tuberculosis strains: transmission and atomic force observation. Int J Clin Exp Med 3(4):308–314

    Google Scholar 

Download references

Acknowledgments

This work is partially supported by Huntsman Environmental Research Center and Utah Water Research Laboratory, Logan, UT, USA. We also thank Mr. Juan Ciorciari from Thermo Fisher Scientific for helping in the FTIR measurement of bacterial cells. We also thank Dr. Fen-Ann Shen from the Center for Surface Analysis and Applications, USU for the SEM imaging.

Author information

Authors and Affiliations

  1. Department of Biological Engineering, Utah State University, 4105 Old Main Hill, Logan, UT, 84322-4105, USA

    Mingjie Tang, Gerald D. McEwen, Yangzhe Wu, Charles D. Miller & Anhong Zhou

Authors
  1. Mingjie Tang
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Gerald D. McEwen
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Yangzhe Wu
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Charles D. Miller
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Anhong Zhou
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Anhong Zhou.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Tang, M., McEwen, G.D., Wu, Y. et al. Characterization and analysis of mycobacteria and Gram-negative bacteria and co-culture mixtures by Raman microspectroscopy, FTIR, and atomic force microscopy. Anal Bioanal Chem 405, 1577–1591 (2013). https://doi.org/10.1007/s00216-012-6556-8

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00216-012-6556-8

Keywords

Search

Navigation