Hostname: page-component-586b7cd67f-rdxmf Total loading time: 0 Render date: 2024-11-23T20:10:28.289Z Has data issue: false hasContentIssue false

Purification procedure sensitizes Bacillus endospores to free radicals from UVA radiation and photocatalysis

Published online by Cambridge University Press:  03 August 2017

Vijay Krishna*
Affiliation:
Department of Materials Science and Engineering, Particle Engineering Research Center, University of Florida, P.O. Box-116135, Gainesville, Florida 32611, USA
Jue Zhao
Affiliation:
Department of Environmental Engineering Sciences, University of Florida, P.O. Box-116135, Gainesville, Florida 32611, USA
Ben Koopman
Affiliation:
Department of Environmental Engineering Sciences, University of Florida, P.O. Box-116135, Gainesville, Florida 32611, USA
Brij Moudgil
Affiliation:
Department of Materials Science and Engineering, Particle Engineering Research Center, University of Florida, P.O. Box-116135, Gainesville, Florida 32611, USA

Abstract

Many researchers are investigating the extreme resilience of bacterial endospores against chemical and physical inactivating agents. The presence of vegetative cells in spore suspensions can result in overly optimistic assessment of inactivating agents; therefore, various spore purification methods have been applied to separate spores from vegetative cells prior to testing. The present study was undertaken to evaluate the effect of two widely used spore purification methodologies on spore integrity and susceptibility to ultraviolet-A (UVA) radiation and free radicals generated from photocatalysts. Bacillus subtilis and Bacillus cereus spores were purified by procedures that involved heat shock alone or chemical washes, lysozyme treatment and heat shock (CLH). The purified spores were exposed to UVA radiation or free radicals generated by photocatalyst and susceptibility were evaluated in terms of survival ratio. The effect of purification procedure on the spore morphology was investigated with electron microscopy. The CLH purification process significantly damages spore coats and increases the susceptibility of Bacillus spores to UVA radiation and photocatalytic inactivation. It is therefore likely that the survival of CLH treated spores in extra-terrestrial environments would be less than that of the same spores purified by a less aggressive procedure.

Type
Research Article
Copyright
Copyright © Cambridge University Press 2017 

Access options

Get access to the full version of this content by using one of the access options below. (Log in options will check for institutional or personal access. Content may require purchase if you do not have access.)

Footnotes

*

Present Address: Biomedical Engineering, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio 44195, USA.

Present address: Public Works Department, City of Salem, Salem, Oregon 97303, USA.

References

Adams, D.M. (1974). Requirement for and sensitivity to lysozyme by Clostridium perfringens spores heated at ultrahigh temperatures. Appl. Microbiol. 27, 797801.Google Scholar
Alderton, G. & Snell, N. (1963). Base exchange and heat resistance in bacterial spores. Biochem. Biophys. Res. Commun. 10, 139143.Google Scholar
ASTM (2001). Standard quantitative carrier test method to evaluate the bactericidal, fungicidal, mycobactericidal and sporicidal potencies of liquid chemical germicides. ASTM Designation: E2111-00.Google Scholar
Atrih, A. & Foster, S.J. (2002). Bacterial endospores the ultimate survivors. Int. Dairy J. 12, 217223.Google Scholar
Bai, W., Krishna, V., Wang, J., Moudgil, B. & Koopman, B. (2012). Enhancement of nano titanium dioxide photocatalysis in transparent coatings by polyhydroxy fullerene. Appl. Catal. B – Environ. 125, 128135.Google Scholar
Bailey-Smith, K., Todd, S.J., Southworth, T.W., Proctor, J. & Moir, A. (2005). The ExsA protein of Bacillus cereus is required for assembly of coat and exosporium onto the spore surface. J. Bacteriol. 187, 38003806.Google Scholar
Block, S.S. (2001). Disinfection, Sterilization, and Preservation. Lippincott Williams & Wilkins, Philadelphia, PA, xxii, 1481 pp.Google Scholar
Boschwitz, H., Milner, Y., Keynan, A., Halvorson, H.O. & Troll, W. (1983). Effect of inhibitors of trypsin-like proteolytic enzymes Bacillus cereus T spore germination. J. Bacteriol. 153, 700708.Google Scholar
Cano, R.J. & Borucki, M.K. (1995). Revival and identification of bacterial spores in 25- to 40-million-year-old Dominican amber. Science 268, 10601064.Google Scholar
CDC (2006). Questions and answers about anthrax. http://www.bt.cdc.gov/agent/anthrax/faq.Google Scholar
Costa, T., Serrano, M., Steil, L., Volker, U., Moran, C.P. Jr. & Henriques, A.O. (2007). The timing of cotE expression affects Bacillus subtilis spore coat morphology but not lysozyme resistance. J. Bacteriol. 189, 24012410.Google Scholar
Demidova, T.N. & Hamblin, M.R. (2005). Photodynamic inactivation of Bacillus spores, mediated by phenothiazinium dyes. Appl. Environ. Microbiol. 71, 69186925.Google Scholar
Driks, A. (1999). Bacillus subtilis spore coat. Microbiol. Mol. Biol. Rev. 63, 120.Google Scholar
Gorman, S.P., Scott, E.M. & Hutchinson, E.P. (1985). Thermal resistance variations due to post-harvest treatments in Bacillus subtilis spores. J. Appl. Bacteriol. 59, 555560.Google Scholar
Henriques, A.O. & Moran, C.P. Jr. (2000). Structure and assembly of the bacterial endospore coat. Methods 20, 95110.Google Scholar
Heritage, J., Evans, E.G.V. & Killington, R.A. (1995). Introductory Microbiology. Cambridge University Press, Cambridge, United Kingdom, 234 pp.Google Scholar
Horneck, G., Rettberg, P., Reitz, G., Wehner, J., Eschweiler, U., Strauch, K., Panitz, C., Starke, V. & Baumstark-Khan, C. (2001). Protection of bacterial spores in space, a contribution to the discussion on Panspermia. Orig. Life Evol. B 31, 527547.Google Scholar
Horneck, G. et al. (2012). Resistance of bacterial endospores to outer space for planetary protection purposes-experiment PROTECT of the EXPOSE-E mission. Astrobiology 12, 445–56.Google Scholar
Jacoby, W.A., Maness, P.C., Wolfrum, E.J., Blake, D.M. & Fennell, J.A. (1998). Mineralization of bacterial cell mass on a photocatalytic surface in air. Environ. Sci Technol. 32, 26502653.Google Scholar
Koch, A. (1981). Growth measurement. In Manual of Methods for General Bacteriology, ed. Gerhardt, P., Murray, R.G.E., Costilow, R., Nester, E., Wood, W., Kreig, N. & Phillips, B. American Society for Microbiology, Washington, DC, pp. 179207.Google Scholar
Krishna, V., Pumprueg, S., Lee, S.H., Zhao, J., Sigmund, W., Koopman, B. & Moudgil, B.M. (2005). Photocatalytic disinfection with titanium dioxide coated multi-wall carbon nanotubes. Process. Saf. Environ. 83, 393397.Google Scholar
Krishna, V.B. (2007). Enhancement of titanium dioxide photocatalysis with polyhydroxy fullerenes. In Materials Science and Engineering, University of Florida, Gainesville, p. 123.Google Scholar
Krishna, V.B., Zhao, J., Pumprueg, S., Koopman, B.L. & Moudgil, B.M. (2016). Improving dispersion of bacterial endospores for enumeration. Kona Powder Part J. 33, 304309.Google Scholar
Mastrapa, R.M.E., Glanzberg, H., Head, J.N., Melosh, H.J. & Nicholson, W.L. (2001). Survival of bacteria exposed to extreme acceleration: Implications for panspermia. Earth Planet. Sci. Lett. 189, 18.Google Scholar
Nagler, K., Setlow, P., Li, Y.Q. & Moeller, R. (2014). High salinity alters the germination behavior of Bacillus subtilis spores with nutrient and nonnutrient germinants. Appl. Environ. Microbiol. 80, 13141321.Google Scholar
Nicholson, W.L. & Galeano, B. (2003). UV resistance of Bacillus anthracis spores revisited: validation of Bacillus subtilis spores as UV surrogates for spores of B. Anthracis Sterne. Appl. Environ. Microbiol. 69, 13271330.Google Scholar
Nicholson, W.L., Munakata, N., Horneck, G., Melosh, H.J. & Setlow, P. (2000). Resistance of Bacillus endospores to extreme terrestrial and extraterrestrial environments. Microbiol. Mol. Biol. Rev. 64, 548–72.Google Scholar
Nicholson, W.L., Schuerger, A.C. & Setlow, P. (2005). The solar UV environment and bacterial spore UV resistance: considerations for Earth-to-Mars transport by natural processes and human spaceflight. Mutat. Res. – Fundam. Mol. M. 571, 249264.Google Scholar
Noell, A.C., Ely, T., Bolser, D.K., Darrach, H., Hodyss, R., Johnson, P.V., Hein, J.D. & Ponce, A. (2015). Spectroscopy and viability of Bacillus subtilis spores after ultraviolet irradiation: implications for the detection of potential bacterial life on Europa. Astrobiology 15, 2031.Google Scholar
Paidhungat, M., Setlow, B., Driks, A. & Setlow, P. (2000). Characterization of spores of Bacillus subtilis which lack dipicolinic acid. J. Bacteriol. 182, 55055512.Google Scholar
Panitz, C., Horneck, G., Rabbow, E., Rettberg, P., Moeller, R., Cadet, J., Douki, T. & Reitz, G. (2015). The SPORES experiment of the EXPOSE-R mission: Bacillus subtilis spores in artificial meteorites. Int. J. Astrobiol. 14, 105114.Google Scholar
Peng, J.S., Tsai, W.C. & Chou, C.C. (2001). Surface characteristics of Bacillus cereus and its adhesion to stainless steel. Int. J. Food Microbiol. 65, 105111.Google Scholar
Pillet, F., Formosa-Dague, C., Baaziz, H., Dague, E. & Rols, M.P. (2016a). Cell wall as a target for bacteria inactivation by pulsed electric fields. Sci. Rep. – UK. 6, 18.Google Scholar
Pillet, F., Marjanovic, I., Rebersek, M., Miklavcic, D., Rols, M.P. & Kotnik, T. (2016b). Inactivation of spores by electric arcs. BMC Microbiol. 16, 15.Google Scholar
Raguse, M., Fiebrandt, M., Stapelmann, K., Madela, K., Laue, M., Lackmann, J.W., Thwaite, J.E., Setlow, P., Awakowicz, P. & Moeller, R. (2016). Improvement of biological indicators by uniformly distributing Bacillus subtilis spores in monolayers to evaluate enhanced spore decontamination technologies. Appl. Environ. Microbiol. 82, 20312038.Google Scholar
Rasmussen, T.M. & Labbe, R.G. (1996). Recoverability of heat-injured Bacillus spores by lysozyme and EDTA or alkaline thioglycollate. World J. Microbiol. Biotechnol. 12, 595599.Google Scholar
Rettberg, P., Rabbow, E., Panitz, C., Reitz, G. & Horneck, G. (2002). Survivability and protection of bacterial spores in space – the BIOPAN experiments. ESA Sp. Publ. 518, 105108.Google Scholar
Riesenman, P.J. & Nicholson, W.L. (2000). Role of the spore coat layers in Bacillus subtilis spore resistance to hydrogen peroxide, artificial UV-C, UV-B, and solar UV radiation. Appl. Environ. Microbiol. 66, 620626.Google Scholar
Setlow, B. & Setlow, P. (1998). Heat killing of Bacillus subtilis spores in water is not due to oxidative damage. Appl. Environ. Microbiol. 64, 41094112.Google Scholar
Sokal, R.R. & Rohlf, F.J. (1997). Biometry: The Principles and Practice of Statistics in Biological Research. Freeman and Company, New York.Google Scholar
Suzuki, Y. & Rode, L.J. (1969). Effect of lysozyme on resting spores of Bacillus megaterium. J. Bacteriol. 98, 238245.Google Scholar
Tauveron, G., Slomianny, C., Henry, C. & Faille, C. (2006). Variability among Bacillus cereus strains in spore surface properties and influence on their ability to contaminate food surface equipment. Int. J. Food Microbiol. 110, 254262.Google Scholar
Wassmann, M. et al. (2012). Survival of spores of the UV-resistant Bacillus subtilis strain MW01 after exposure to Low-Earth orbit and simulated Martian conditions: data from the space experiment ADAPT on EXPOSE-E. Astrobiology 12, 498507.Google Scholar
Xue, Y.M. & Nicholson, W.L. (1996). The two major spore DNA repair pathways, nucleotide excision repair and spore photoproduct lyase, are sufficient for the resistance of Bacillus subtilis spores to artificial UV-C and UV-B but not to solar radiation. Appl. Environ. Microbiol. 62, 22212227.Google Scholar
Zhao, J., Krishna, V., Moudgil, B. & Koopman, B. (2008). Evaluation of endospore purification methods applied to Bacillus cereus. Separ. Purific. Technol. 61, 341347.Google Scholar
Zhao, J., Krishna, V., Hua, B., Moudgil, B. & Koopman, B. (2009). Effect of UVA irradiance on photocatalytic and UVA inactivation of Bacillus cereus spores. J. Photochem. Photobiol. B – Biol. 94, 96100.Google Scholar