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Title: Fluorescence spectroscopy for rapid detection and classification of bacterial pathogens

item Sohn, Mi Ryeong
item Himmelsbach, David
item Barton Ii, Franklin
item Cray, Paula

Submitted to: Applied Spectroscopy
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/5/2009
Publication Date: 11/1/2009
Citation: Sohn, M., Himmelsbach, D.S., Barton , F.E., Cray, P.J. 2009. Flourescence spectroscopy for rapid detection and classification of bacterial pathogens. Applied Spectroscopy. 63(11):1251-1255.

Interpretive Summary: With the increasing necessity of hazard analysis and critical control point systems by the food industry, a rapid detection of bacterial pathogens has been required. Several methods have been reported for detecting bacterial pathogens, which include culture method, microbiological methods, antibody-based detection schemes, however the biggest issue is the detection speed and sensitivity. Culture methodology is time-consuming, requiring an enrichment and confirmation of colonies. Microbiological method lacks sensitivity unless bacterial numbers are sufficiently high. Therefore, there is a need for the development of more direct, rapid and sensitivity techniques for detecting bacterial pathogens, particularly in foods. The current study was conducted to investigate the potential of fluorescence spectroscopy to detect bacterial pathogens rapidly without culture enrichment or antibody conjugation. Three different genera of bacteria, which are the most commonly identified commensal (E. coli) and pathogenic (Salmonella and Campylobactor) bacteria in foods, were tested. Synchronous scan technique was employed to determine the optimum excitation and emission wavelengths for each bacterium, and principal component analysis method was used to identify and classify the bacteria, Results showed the fluorescence spectroscopy when coupled with PCA processing has the potential to detect and to rapidly classify bacterial pathogens by their genus as well as concentration. The research result will give an interest to audiences who work in the field of identification of bacteria or microorganisms.

Technical Abstract: This study deals with the rapid detection and classification of three bacteria, Escherichia coli, Salmonella, and Campylobacter, using fluorescence spectroscopy and multivariative analysis. Each bacterial sample was diluted in physiologic saline for analysis. Fluoroscence spectra were collected over a range of 200-900 nm with 0.5 nm intervals on the PerkinElmer Fluorescence Spectrometer. Synchronous scan technique was employeed to find the optimum excitation ('ex) and emission ('em) wavelengths for individual bacteria and the wavelength interval ('') was varied from 10 to 200 nm. The synchronous spectra and two-dimensional plot of the bacterial samples resulted in two maximum 'exs at 225 nm and 280 nm and one maximum 'em at 335-345 nm ('em='ex+''), which corresponds to the 'ex = 225 nm and '' = 110-120 nm and 'ex = 280 nm and '' = 60-65 nm. For all three bacterial genera, the same synchronous scan results were obtained. The emission spectra from the three bacteria groups were very similar, creating difficulty in classification. However, the application of principal component analysis (PCA) to the spectral data resulted in successful classification of the bacteria by their genus as well as concentration. The detection limit was approximately 103-104 cells/ml for each bacterial sample. These results demonstrate that fluorescence spectroscopy, when coupled with PCA processing, has the potential to detect and to rapidly classify bacterial pathogens.