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Applied and Environmental Microbiology, April 2001, p. 1461-1469, Vol. 67, No. 4
0099-2240/01/$04.00+0   DOI: 10.1128/AEM.67.4.1461-1469.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Investigating Microbial (Micro)colony Heterogeneity by Vibrational Spectroscopy

L.-P. Choo-Smith,1 K. Maquelin,1 T. van Vreeswijk,1 H. A. Bruining,1 G. J. Puppels,1,* N. A. Ngo Thi,2 C. Kirschner,2 D. Naumann,2 D. Ami,3 A. M. Villa,3 F. Orsini,3 S. M. Doglia,3 H. Lamfarraj,4 G. D. Sockalingum,4 M. Manfait,4 P. Allouch,5 and H. P. Endtz6

Laboratory for Intensive Care Research and Optical Spectroscopy, Erasmus University Rotterdam, and Department of General Surgery 10M1 and Institute of Medical Microbiology and Infectious Diseases,6 University Hospital Rotterdam "Dijkzigt," 3015 GD Rotterdam, The Netherlands; Robert Koch Institute, Biophysical Structure Analyses, 13353 Berlin, Germany2; UdR INFM Milano-Bicocca, Dipartimento di Biotecnologie e Bioscienze, 20126 Milan, Italy3; and Unité MéDIAN, CNRS FRE 2141, IFR 53, UFR de Pharmacie, Université de Reims Champagne-Ardenne, 51096 Reims Cedex,4 and Service d'Hygiène Hospitalière, Center Hospitalier de Versailles, 78157 Le Chesnay Cedex,5 France

Received 28 September 2000/Accepted 26 January 2001

Fourier transform infrared and Raman microspectroscopy are currently being developed as new methods for the rapid identification of clinically relevant microorganisms. These methods involve measuring spectra from microcolonies which have been cultured for as little as 6 h, followed by the nonsubjective identification of microorganisms through the use of multivariate statistical analyses. To examine the biological heterogeneity of microorganism growth which is reflected in the spectra, measurements were acquired from various positions within (micro)colonies cultured for 6, 12, and 24 h. The studies reveal that there is little spectral variance in 6-h microcolonies. In contrast, the 12- and 24-h cultures exhibited a significant amount of heterogeneity. Hierarchical cluster analysis of the spectra from the various positions and depths reveals the presence of different layers in the colonies. Further analysis indicates that spectra acquired from the surface of the colonies exhibit higher levels of glycogen than do the deeper layers of the colony. Additionally, the spectra from the deeper layers present with higher RNA levels than the surface layers. Therefore, the 6-h colonies with their limited heterogeneity are more suitable for inclusion in a spectral database to be used for classification purposes. These results also demonstrate that vibrational spectroscopic techniques can be useful tools for studying the nature of colony development and biofilm formation.

* Corresponding author. Mailing address: Dept. General Surgery 10M, University Hospital Rotterdam "Dijkzigt," Dr. Molewaterplein 40, 3015 GD Rotterdam, The Netherlands. Phone: 31-10-4635890. Fax: 31-10-4635307. E-mail: PUPPELS{at}HLKD.AZR.NL.

Applied and Environmental Microbiology, April 2001, p. 1461-1469, Vol. 67, No. 4
0099-2240/01/$04.00+0   DOI: 10.1128/AEM.67.4.1461-1469.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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Copyright © 2001 by the American Society for Microbiology. All rights reserved.