This study is devoted to the development of a UV fluorimetry sensor capable of real-time monitoring of the decontamination process of microbiological pathogens by hydrogen peroxide vapour (HPV) treatment. The sensor is operating on the autofluorescence signal of tryptophan. The Bacillus atrophaeus (B. atrophaeus) and Geobacillus stearothermophilus (G. stearothermophilus) spores were exposed to HPV and the resulting dynamic change in tryptophan fluorescence intensity as a function of time was recorded and analysed. It was revealed that the introduced HPV atmosphere caused a 4-time decrease in the fluorescence intensity of the tryptophan emission due to the interaction of HPV with the spores. It was shown that achieving a persistent minimal level of the autofluorescence signal due to the microorganism-bound tryptophan during the defined time period is well correlated with the efficiency of the ongoing decontamination process in the HPV treatment course. Therefore, the progress of the HPV decontamination procedure can be firmly evaluated, using fluorescence data obtained in real time and the validity of the method was demonstrated by comparing the fluorescence data with the reference information obtained by implementing classical microbiology viability tests (incl. time behaviour) for various microorganisms in the HPV atmosphere.
1. Hodgson, M. The role of hydrogen peroxide vapor systems in infection control. Infection Control Today, December 1, 2010.
2. Otter, J. A., Puchowicz, M., Ryan, D., Salkeld, J. A. G., Cooper, T. A., Havill, N. L., et al. Feasibility of routinely using hydrogen peroxide vapor to decontaminate rooms in a busy United States hospital. Infect. Control Hosp. Epidemiol., 2009, 30(6), 574–577.
https://doi.org/10.1086/597544
3. Holmdahl, T., Lanbeck, P., Wullt, M., and Walder, M. H. A head-to-head comparison of hydrogen peroxide vapor and aerosol room decontamination systems. Infect. Control Hosp. Epidemiol., 2011, 32(9), 831–836.
https://doi.org/10.1086/661104
4. APEX Biological Indicators for H2O2 Vapor. MesaLabs Technical Report.
https://biologicalindicators.mesalabs.com/wp-content/uploads/sites/31/2013/11/TR-001-v1.pdf (accessed 2020-10-27).
5. Indicators in H2O2 Bio-Decontamination & Inline, Continuous Measurement.
https://www.vaisala.com/en/blog/2020-05/indicators-h2o2-bio-decontamination-inline-continuous-measurement (accessed 2020-10-27).
6. Sohn, M., Himmelsbach, D. S., Barton, F. E., and Fedorka-Cray, P. J. Fluorescence spectroscopy for rapid detection and classification of bacterial pathogens. Appl. Spectrosc., 2009, 63(11), 1251–1255.
https://doi.org/10.1366/ 000370209789806993
7. Liang, J., Wu, W.-L., Liu, Z.-H., Mei, Y.-J., Cai, R.-X., and Shen P. Study the oxidative injury of yeast cells by NADH autofluorescence. Spectrochim. Acta A Mol. Biomol. Spectrosc., 2007, 67(2), 355–359.
https://doi.org/10.1016/j.saa.2006.07.035
8. Li, R., Goswami, U., King, M., Chen, J., Cesario, T. C., and Rentzepis, P. M. In situ detection of live-to-dead bacteria ratio after inactivation by means of synchronous fluorescence and PCA. Proc. Natl. Acad. Sci. U.S.A., 2018, 115(4), 668–673.
https://doi.org/10.1073/pnas.171651411
9. Yaseen, M. A., Sutin, J., Wu, W., Fu, B., Uhlirova, H., Devor, A., et al. Fluorescence lifetime microscopy of NADH distinguishes alterations in cerebral metabolism in vivo. Biomed. Opt. Express, 2017, 8(5), 2368–2385.
https://doi.org/10.1364/BOE.8.002368
10. Xue, X., Hu, L., and Houdoi, Z. Study on fluorescence spectra of B vitamins. In Proceedings of the 2016 International Conference on Mechanics, Materials and Structural Engineering. Atlantis Press, 2016, 160–165.
https://doi.org/10.2991/icmmse-16.2016.15
11. Vivian, J. T. and Callis, P. R. Mechanisms of tryptophan fluorescence shifts in proteins. Biophys. J., 2001, 80(5), 2093–2109.
https://doi.org/10.1016/S0006-3495(01)76183-8
12. Cavatorta, P., Favilla, R., and Mazzini, A. Fluorescence quenching of tryptophan and related compounds by hydrogen peroxide. Biochim. Biophys. Acta., 1979, 578(2), 541–546.
https://doi.org/10.1016/0005-2795(79) 90185-5
13. Babichenko, S., Gala, J.-L., Bentahir, M., Piette, A.-S., Poryvkina, L., Rebane, O., et al. Non-contact, real-time laser-induced fluorescence detection and monitoring of microbial contaminants on solid surfaces before, during and after decontamination. Biosens. Bioelectron., 2018, 9(2).
https://doi.org/10.4172/2155-6210.1000255
14. Setlow, B. and Setlow, P. Levels of oxidized and reduced pyridine nucleotides in dormant spores and during growth, sporulation, and spore germination of Bacillus megaterium. J Bacteriol., 1977, 129(2), 857–865.
https://doi.org/10.1128/JB.129.2.857-865.1977
15. VCS-100 HPV device description.
https://cleamix.com/products/ (accessed on 2020-10-27).
16. Arreola, J., Keusgen, M., Wagner, T., and Schöning, M. J. Combined calorimetric gas- and spore-based biosensor array for online monitoring and sterility assurance of gaseous hydrogen peroxide in aseptic filling machines. Biosens. Bioelectron., 2019, 143, 111628.
https://doi.org/10.1016/j.bios.2019.111628
17. Wood, J. P., Calfee, M. W., Clayton, M., Griffin‐Gatchalian, N., Touati, A., Ryan, S., et al. A simple decontamination approach using hydrogen peroxide vapour for Bacillus anthracis spore inactivation. J. Appl. Microbiol., 2016, 121(6), 1603–1615.
https://doi.org/10.1111/jam.13284
18. Pan, Y.-L., Hill, S. C., Santarpia, J. L., Brinkley, K., Sickler, T., Coleman, M., et al. Spectrally-resolved fluorescence cross sections of aerosolized biological live agents and simulants using five excitation wavelengths in a BSL-3 laboratory. Opt. Express, 2014, 22(7), 8165–8189.
https://doi.org/10.1364/OE.22.008165
19. H2B fluorometer description.
https://ldi-innovation.com/index.php/h2b/ (accessed 2020-10-27).
20. SFS-Cube device description. https://ldi-innovation.com/index.php/sfs-cube/ (accessed 2020-10-27).
21. Walla, P. J. (ed.). Modern Biophysical Chemistry. Wiley-VCH, Weinheim, Germany, 2014, 45–48.
https://doi.org/10.1002/9783527683505
22. Setlow, B., Loshon, C. A., Genest, P. C., Cowan, A. E., Setlow, C., and Setlow, P. Mechanisms of killing spores of Bacillus subtilis by acid, alkali and ethanol. J. Appl. Microbiol., 2002, 92, 362–375.
https://doi.org/10.1046/j.1365-2672.2002.01540.x
23. Callis, P. R. and Liu, T. Quantitative prediction of fluorescence quantum yields for tryptophan in proteins. J. Phys. Chem. B., 2004, 108(14), 4248–4259.
https://doi.org/10.1021/jp0310551
