Rapid and Reagent-Free Method for Identification of Enterobacteriaceae Using Attenuated Total Reflectance-Fourier Transform Infrared (ATR-FTIR) Spectroscopy

Background: FTIR spectroscopy is a rapid, reagent-free technique for bacterial identification and classification. When implemented in the ATR (as opposed to the commonly employed transmission) mode of spectral acquisition, bacterial colonies from culture plates can be analyzed directly without any sample preparation steps, allowing for identification within minutes after initial culture. In this study we evaluated for the first time the discriminatory power of ATR-FTIR spectroscopy for the rapid classification of Enterobacteriaceae. We demonstrate that this phenotypic fingerprinting technique can complement MALDI-TOF MS by providing additional discriminatory capabilities.

Methods: ATR-FTIR spectra were acquired by transferring isolated colonies from MacConkey agar plates onto the ATR sampling surface of a portable ATR-FTIR spectrometer. ATR-FTIR spectral acquisition time was ~1 min. Four spectra were collected for each isolate from different colonies on the same culture plate, yielding a spectral database containing approximately 1,400 ATR-FTIR spectra of Enterobacteriaceae. Spectral data analysis was performed by hierarchical cluster analysis (HCA) and principal component analysis (PCA) in conjunction with the use of a feature selection algorithm.

Results: Approximately 350 clinical isolates of Enterobacteriaceae, including Enterobacter, Klebsiella, Proteus, Citrobacter, Salmonella, and Shigella spp. as well as pathogenic (STEC, EHEC, EPEC and UPEC) and non-pathogenic E. coli, were employed in this study. Genus-level and species-level classification of all the clinical isolates was achieved by multivariate statistical analysis of the FTIR data with an overall rate of correct classification exceeding 98%, including complete differentiation between E. coli and Shigella spp. Further multivariate statistical analysis of the ATR-FTIR spectra of the E. coli isolates yielded complete differentiation between pathogenic and non-pathogenic strains and discrimination among the four E. coli pathotypes included in this study.

Conclusion: ATR-FTIR spectroscopy can provide a simple and rapid method for species identification of Enterobacteriaceae as well as for discrimination between E. coli and Shigella spp. within minutes after initial culture.