UV-Induced Spectral and Morphological Changes in Bacterial Spores for Inactivation Assessment

The ability to detect and inactivate spore-forming bacteria is of significance within, for example, industrial, healthcare, and defense sectors. Not only are stringent protocols necessary for the inactivation of spores but robust procedures are also required to detect viable spores after an inactivation assay to evaluate the procedure’s success. UV radiation is a standard procedure to inactivate spores. However, there is limited understanding regarding its impact on spores’ spectral and morphological characteristics. A further insight into these UV-induced changes can significantly improve the design of spore decontamination procedures and verification assays. This work investigates the spectral and morphological changes to Bacillus thuringiensis spores after UV exposure. Using absorbance and fluorescence spectroscopy, we observe an exponential decay in the spectral intensity of amino acids and protein structures, as well as a logistic increase in dimerized DPA with increased UV exposure on bulk spore suspensions. Additionally, using micro-Raman spectroscopy, we observe DPA release and protein degradation with increased UV exposure. More specifically, the protein backbone’s 1600–1700 cm–1 amide I band decays slower than other amino acid-based structures. Last, using electron microscopy and light scattering measurements, we observe shriveling of the spore bodies with increased UV radiation, alongside the leaking of core content and disruption of proteinaceous coat and exosporium layers. Overall, this work utilized spectroscopy and electron microscopy techniques to gain new understanding of UV-induced spore inactivation relating to spore degradation and CaDPA release. The study also identified spectroscopic indicators that can be used to determine spore viability after inactivation. These findings have practical applications in the development of new spore decontamination and inactivation validation methods.

Photoreac on of DPA monitored by 1

H NMR
The photoreaction of DPA was monitored by 1 H NMR spectroscopy at 1, 2 and 6 hours of irradiation. 1 H NMR spectra collected at each time point show the decrease of DPA and the formation of photoproducts over time (Figure S3).The ratio of integrals for 1 H NMR signals of the protons of DPA and the main photoproduct was used to determine the remaining amount of DPA at 1 h and 2 h to be 85 % and 45 % respectively.After 6 h irradiation most 1 H NMR signals are lost, neither DPA nor the photoproduct obtained in the 1 h and 2 h samples can be observed, this is most likely due to formation of insoluble polymers.The main photoproduct observed at 1 h and 2 h irradiation is assumed to be 2,2´-Bipyridine-6,6-dicarboxylic acid formed by decarboxylative photodimerization.This agrees with the observed 1 H NMR signals and a previous spectroscopic study of DPA by Nardi et al. [1] who produced the esterized dimer molecule observed in this work.It should however be noted that the specific photoproduct has not been isolated and characterized due to the low solubility of 2,2´-Bipyridine-6,6-dicarboxylic Acid.LCMS was used to verify the decay of DPA.

Figure
Figure S3: 1H NMR spectra obtained for a solution of 10 µM DPA in deuterium oxide after 1 h, 2 h and 6 h irradiation showing the decay of DPA (triangles) and the formation of the main photoproduct (dots).1H NMR spectra of pre-irradiated DPA is added as reference (0 h).

Figure S4 :
Figure S4: Viability of B. thuringiensis over two minutes of UV-exposure.Total amount of spores in suspension was estimated at 3x10 6 spores.

Figure S9 :
Figure S9: Differences in the crystal structure of 30 mM DPA dried on a glass slide.Untreated DPA forms long fibrous crystals.By contrast, UV-treated DPA no longer forms these fibres, instead forming glassy fragments of different sizes.The images are acquired using a 60× water immersion objective (UPlanSApo, Olympus) in brightfield mode of an inverted microscope (IX71, Olympus).The scale bar is 10 µm.