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  • 1.
    Öberg, Rasmus
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysik. Swedish Defence Research Agency (FOI), Umeå, Sweden.
    Sil, Timir B.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysik.
    Johansson, Alexandra C.
    Swedish Defence Research Agency (FOI), Umeå, Sweden.
    Malyshev, Dmitry
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysik.
    Landström, Lars
    Swedish Defence Research Agency (FOI), Norra Sorunda, Sweden.
    Johansson, Susanne
    Swedish Defence Research Agency (FOI), Umeå, Sweden.
    Andersson, Magnus
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysik. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Andersson, Per Ola
    Swedish Defence Research Agency (FOI), Umeå, Sweden.
    UV-induced spectral and morphological changes in bacterial spores for inactivation assessment2024Ingår i: Journal of Physical Chemistry B, ISSN 1520-6106, E-ISSN 1520-5207, Vol. 128, nr 7, s. 1638-1646Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    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.

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  • 2.
    Öberg, Rasmus
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysik. Swedish Defence Research Agency (FOI), Umeå, Sweden.
    Sil, Timir Baran
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysik.
    Ohlin, C. André
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Andersson, Magnus
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysik.
    Malyshev, Dmitry
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysik.
    Assessing CaDPA levels, metabolic activity, and spore detection through deuterium labeling2024Ingår i: The Analyst, ISSN 0003-2654, E-ISSN 1364-5528, Vol. 149, nr 6, s. 1861-1871Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Many strains among spore-forming bacteria species are associated with food spoilage, foodborne disease, and hospital-acquired infections. Understanding the impact of environmental conditions and decontamination techniques on the metabolic activity, viability, and biomarkers of these spores is crucial for combatting them. To distinguish and track spores and to understand metabolic mechanisms, spores must be labeled. Staining or genetic modification are current methods for this, however, these methods can be time-consuming, and affect the viability and function of spore samples. In this work, we investigate the use of heavy water for permanent isotope labeling of spores and Raman spectroscopy for tracking sporulation/germination mechanisms. We also discuss the potential of this method in observing decontamination. We find that steady-state deuterium levels in the spore are achieved after only ∼48 h of incubation with 30% D2O-infused broth and sporulation, generating Raman peaks at cell silent region of 2200 and 2300 cm−1. These deuterium levels then decrease rapidly upon spore germination in non-deuterated media. We further find that unlike live spores, spores inactivated using various methods do not lose these Raman peaks upon incubation in growth media, suggesting these peaks may be used to indicate the viability of a spore sample. We further observe several Raman peaks exclusive to deuterated DPA, a spore-specific chemical biomarker, at e.g. 988 and 2300 cm−1, which can be used to track underlying changes in spores involving DPA. In conclusion, permanent spore labeling using deuterium offers a robust and non-invasive way of labeling bacterial spores for marking, viability determination, and characterising spore activity.

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