Skip to main content
×
Home

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

  • Vijay Krishna (a1), Jue Zhao (a2), Ben Koopman (a2) and Brij Moudgil (a1)
Abstract
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.

Copyright
Corresponding author
e-mail: krishnv2@ccf.org
Footnotes
Hide All
*

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.

Footnotes
References
Hide All
Adams D.M. (1974). Requirement for and sensitivity to lysozyme by Clostridium perfringens spores heated at ultrahigh temperatures. Appl. Microbiol. 27, 797801.
Alderton G. & Snell N. (1963). Base exchange and heat resistance in bacterial spores. Biochem. Biophys. Res. Commun. 10, 139143.
ASTM (2001). Standard quantitative carrier test method to evaluate the bactericidal, fungicidal, mycobactericidal and sporicidal potencies of liquid chemical germicides. ASTM Designation: E2111-00.
Atrih A. & Foster S.J. (2002). Bacterial endospores the ultimate survivors. Int. Dairy J. 12, 217223.
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.
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.
Block S.S. (2001). Disinfection, Sterilization, and Preservation. Lippincott Williams & Wilkins, Philadelphia, PA, xxii, 1481 pp.
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.
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.
CDC (2006). Questions and answers about anthrax. http://www.bt.cdc.gov/agent/anthrax/faq.
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.
Demidova T.N. & Hamblin M.R. (2005). Photodynamic inactivation of Bacillus spores, mediated by phenothiazinium dyes. Appl. Environ. Microbiol. 71, 69186925.
Driks A. (1999). Bacillus subtilis spore coat. Microbiol. Mol. Biol. Rev. 63, 120.
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.
Henriques A.O. & Moran C.P. Jr. (2000). Structure and assembly of the bacterial endospore coat. Methods 20, 95110.
Heritage J., Evans E.G.V. & Killington R.A. (1995). Introductory Microbiology. Cambridge University Press, Cambridge, United Kingdom, 234 pp.
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.
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.
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.
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.
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.
Krishna V.B. (2007). Enhancement of titanium dioxide photocatalysis with polyhydroxy fullerenes. In Materials Science and Engineering, University of Florida, Gainesville, p. 123.
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.
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.
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.
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.
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.
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.
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.
Paidhungat M., Setlow B., Driks A. & Setlow P. (2000). Characterization of spores of Bacillus subtilis which lack dipicolinic acid. J. Bacteriol. 182, 55055512.
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.
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.
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.
Pillet F., Marjanovic I., Rebersek M., Miklavcic D., Rols M.P. & Kotnik T. (2016b). Inactivation of spores by electric arcs. BMC Microbiol. 16, 15.
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.
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.
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.
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.
Setlow B. & Setlow P. (1998). Heat killing of Bacillus subtilis spores in water is not due to oxidative damage. Appl. Environ. Microbiol. 64, 41094112.
Sokal R.R. & Rohlf F.J. (1997). Biometry: The Principles and Practice of Statistics in Biological Research. Freeman and Company, New York.
Suzuki Y. & Rode L.J. (1969). Effect of lysozyme on resting spores of Bacillus megaterium . J. Bacteriol. 98, 238245.
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.
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.
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.
Zhao J., Krishna V., Moudgil B. & Koopman B. (2008). Evaluation of endospore purification methods applied to Bacillus cereus . Separ. Purific. Technol. 61, 341347.
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.
Recommend this journal

Email your librarian or administrator to recommend adding this journal to your organisation's collection.

International Journal of Astrobiology
  • ISSN: 1473-5504
  • EISSN: 1475-3006
  • URL: /core/journals/international-journal-of-astrobiology
Please enter your name
Please enter a valid email address
Who would you like to send this to? *
×

Keywords:

Metrics

Full text views

Total number of HTML views: 3
Total number of PDF views: 14 *
Loading metrics...

Abstract views

Total abstract views: 109 *
Loading metrics...

* Views captured on Cambridge Core between 3rd August 2017 - 20th November 2017. This data will be updated every 24 hours.