Standardization and internal validation of a bacteria identification method utilizing focal-plane-array Fourier transformed infrared spectroscopy

Food-borne diseases collectively affect significant portions of the world's population. In Canada recent food-borne outbreaks and diseases resulted in significant expenses and resource allocations. Consequently, finding methods that can detect and identify microorganisms in faster, reliable and cost effective ways is a pressing need. Microbial identification methods based on the infrared spectral analysis of microorganisms have been shown to be potentially viable. The recent development of focal plane array Fourier transform infrared (FPA-FTIR) spectroscopy provided the means of acquiring thousands of infrared spectra in the time it takes to record a single spectrum. The increased number of infrared spectra of each organism provides infrared-based microbial differentiation and identification methods with added reliability. Accordingly, we generated a comprehensive standardized and internally validated the FPA-FTIR based bacteria identification method. All infrared spectra were collected from bacterial colonies, lifted from agar and deposited on zinc selenide (ZnSe) slides, on a Agilent Excalibur FTIR spectrometer equipped with a UMA-600 infrared microscope and a 32 x 32 (1024 pixels) mercury-cadmium-telluride focal-plane-array detector and operated under Resolutions Pro 4.0 software (Agilent Technologies, Melbourne, Australia). The effect of growth media formulations, growth time and inactivation techniques on the spectral reproducibility of microorganism was tested. Most of these variables showed some degree of influence on spectral variability, therefore a single combination of these was recommended in the form of a laboratory protocol for consistent microbial preparations. This enabled the construction of a comprehensive spectral database including spectra from 180 different microbial strains. The FPA-FTIR based method was assessed for the discrimination of Campylobacter jejuni and C. coli isolated from poultry. The method was also evaluated as a tool for the identification of Escherichia coli and Listeria monocytogenes strains isolated from deliberately inoculated food matrices. In all cases identification of bacteria based on their FPA-FTIR spectra was highly reliable and comparable to standard methods of bacteria identification; provided that the unknown bacteria samples and reference strains were prepared in a consistent manner, and that appropriate spectral databases were employed.