Laser Tweezers Raman Spectroscopy (LTRS) to Detect Effects of Chlorine Dioxide on Individual Nosema bombycis Spores

The microsporidium Nosema bombycis (Nb) causes pebrine, a fatal disease in sericulture. Nb is effectively killed by chlorine dioxide (ClO2); however, the precise killing mechanism remains unclear. We used laser tweezers Raman spectroscopy (LTRS) to monitor the action of ClO2 on individual Nb spores in real time. Raman peaks of ClO2 appeared in Nb spores, corresponding to decreased peaks of trehalose that gradually disappeared. A peak (1658 cm(-1)) corresponding to the protein alpha-helix significantly weakened while that (1668 cm(-1)) corresponding to irregular protein structures was enhanced; their intensities were negatively correlated in a certain time range and dependent on ClO2 concentration. The intensities of peaks at 782 cm(-1) (nucleic acids) and 1004 cm(-1) (phenylalanine of protein) did not change evidently even under extremely high ClO2 concentrations. Thus, ClO2 rapidly permeates the Nb spore wall, changing the protein secondary structure to lose biological function and destroy permeability, causing trehalose to leak out. These effects are ClO2 concentration-dependent, but no other obvious changes to biomacromolecules were detected. Single-cell analysis using LTRS is an effective method to monitor the action of chemical sporicides on microbes in real time, providing insight into the heterogeneity of cell stress resistance.