Automated species and strain identification of bacteria in complex matrices using FTIR spectroscopy K.A. Puzey 1 , P.J. Gardner 2 , V.K. Petrova 3 , C.W. Donnelly 3 , G.A. Petrucci 4 1 QuantaSpec Inc., 29 B River Rd., Essex Junction, VT 05452 2 Western Carolina University, 333 Belk, Cullowhee NC 28723 3 Department of Nutrition and Food Science, University of Vermont 101 Carrigan Drive, Burlington, VT 05405 4 Department of Chemistry, University of Vermont A218 Cook Physical Sciences Building, Burlington, VT 05405 ABSTRACT Fourier Transform Infrared (FTIR) spectroscopy provides a highly selective and reproducible means for the chemically- based discrimination of intact microbial cells which make the method valuable for large-scale screening of foods. The goals of the present study were to assess the effect of chemical interferents, such as food matrices, different sanitizing compounds and growth media, on the ability of the method to accurately identify and classify L. innocua, L. welshimeri, E. coli, S. cholerasuis, S. subterranea, E. sakazakii, and E. aerogenes. Moreover, the potential of FTIR spectroscopy for discrimination of L. innocua and L. welshimeri of different genotypes and the effect of growth phase on identification accuracy of L. innocua and L. welshimeri were tested. FTIR spectra were collected using two different sample presentation techniques – transmission and attenuated total reflection (ATR), and then analyzed using multivariate discriminant analysis based on the first derivative of the FTIR spectra with the unknown spectra assigned to the species group with the shortest Mahalanobis distance. The results of the study demonstrated 100% correct identification and differentiation of all bacterial strains used in this study in the presence of chemical interferents or food matrices, better than 99% identification rate in presence of media matrices, and 100% correct detection for specific bacteria in mixed flora species. Additionally, FTIR spectroscopy proved to be 100% accurate when differentiating between genotypes of L. innocua and L. welshimeri, with the classification accuracy unaffected by the growth stage. These results suggest that FTIR spectroscopy can be used as a valuable tool for identifying pathogenic bacteria in food and environmental samples. Keywords: FTIR spectroscopy, pathogen detection, Mahalanobis distance 1. INTRODUCTION The lack of a rapid, economical and simple detection and identification method for foodborne pathogens continues to pose an impediment to providing real-time assessments for managing foodborne risks. Conventional cultural procedures for bacterial identification include multiple subcultures and biotype- or serotype- identification steps and require up to a week to obtain confirmed results 1-3 . Moreover, these methods are often misleading and provide only retrospective information about a food product sample 4 . Recently, much effort has been focused on development of new molecular and immunological techniques; however their use for routine bacterial detection and identification is still limited due to high costs and the requirement for trained personnel 3 . In addition, reliability of such nucleic acid amplification methods can be decreased due to the presence of residual matrix components that inhibit enzymatic reactions 5 . Fourier transform infrared (FTIR) spectroscopy offers options for rapid discrimination, classification and identification of intact microbial cells 6 . Agent identification with FTIR spectroscopy involves the observation of vibration properties of chemical bonds when excited by an infrared beam. An infrared spectrum provides information about the biochemical composition of the cell. Each bacterial species produces a unique fingerprint-like FTIR spectra 7-8 . Unlike existing biological technologies that are both labor intensive and expensive due to the high volume of reagents and supplies required for strain identification, FTIR spectroscopy has great potential in the food industry as a rapid and cost-effective pathogen identification method. Please verify that (1) all pages are present, (2) all figures are acceptable, (3) all fonts and special characters are correct, and (4) all text and figures fit within the margin lines shown on this review document. Return to your MySPIE ToDo list and approve or disapprove this submission. 6954-37 V. 3 (p.1 of 9) / Color: No / Format: Letter / Date: 2/28/2008 11:34:40 AM SPIE USE: ____ DB Check, ____ Prod Check, Notes: