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The effect of chlorine-induced bacterial injury on spectral features using Fourier transform infrared (FT-IR) absorbance spectroscopy was studied using a mixed bacterial culture of (1:1) ca. 500 CFU/mL each Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 15442 in 0.9% saline. Bacterial cells were treated with 0, 0.3, or 1.0 ppm of initial free chlorine (21 degrees C, 1 h of contact time). Chlorine-injured and dead bacterial cells retained the ATR spectral properties of uninjured or live cells in the region of C-O-C stretching vibrations of polysaccharides, indicative of the cell wall peptidoglycan layer and lipopolysaccharide outer leaflet. This confirms the observations of others that extensive bacterial membrane damage is not a key factor in the inactivation of bacteria by chlorine. The bactericidal effect of chlorine caused changes in the spectral features of bacterial ester functional groups of lipids, structural proteins, and nucleic acids, with apparent denaturation reflected between 1800 and 1300 cm (-1) for injured bacterial cells. Three-dimensional principal component analysis (PCA) showed distinct segregation and clustering of chlorine-treated and untreated cells. Cells exposed to chlorine at 0.3 or 1.0 ppm could be distinguished from the untreated control 73 and 80% of the time, respectively, using soft independent modeling of class analogy (SIMCA) analysis. This study suggests that FT-IR spectroscopy may be applicable for detecting the presence of injured and viable but not culturable (VBNC) waterborne pathogens that are underestimated or not discernible using conventional microbial techniques.

This study examined the potential of Fourier transform infrared (FT‐IR) absorbance spectroscopy to detect biochemical changes in bacterial cells that occur during bacterial growth phases in batch culture. Two bacterial strains, Escherichia coli ATCC 25922 and Listeria innocua ATCC 51742 were cultured in brain heart infusion (BHI) broth and incubated at 37C and cells recovered at: the lag phase (3 h), the log phase (7 h), the stationary phase (24 h), and the death phase (10 days) from incubation in BHI (n = 2). Major variations in the biochemical structure of bacterial cells during growth were observed. Over the range of 1800–900 cm−1, loadings 1 (principal component [PC] 1) and 2 (PC2) accounted for 88% of the total variability (76% and 12%, respectively) for E. coli cells, and 80% (72% and 8%, respectively) for L. innocua cells. Changes were attributable to cellular structure of bacterial cells (the variation in nucleic acids, proteins, lipids and polysaccharides) that occur during the growth phases. PRACTICAL APPLICATIONS The focus of this study was to examine Fourier transform infrared (FT‐IR) spectral features of bacterial cells at different growth stages and to determine if variations in composition and distribution of the biochemical components of cells during growth phases can be distinguished using this spectroscopic technique. This could explain the primary functional principle behind detection and discrimination of bacteria by FT‐IR spectroscopy.