Optimization of sporulation and purification methods for sporicidal efficacy assessment on Bacillus spores

Li, Liang; Jin, Jinshan; Hu, Haijing; Deveau, Ian F.; Foley, Steven L.; Chen, Huizhong

doi:10.1093/jimb/kuac014

Optimization of sporulation and purification methods for sporicidal efficacy assessment on Bacillus spores

Journal Article · 20 May 2022 · Journal of Industrial Microbiology and Biotechnology

DOI:https://doi.org/10.1093/jimb/kuac014· OSTI ID:1982502

Li, Liang; Jin, Jinshan; Hu, Haijing; Deveau, Ian F.; Foley, Steven L.;

Abstract

Validating the efficacy of sporicidal agents is a critical step in current good manufacturing practices for disinfection requirements. A limitation is that the poor quality of spores can lead to false positive sporicidal results. The aim of this study was to explore optimal sporulation and purification methods in Bacillus spores. Spores of 7 Bacillus strains were produced in 5 different sporulation media. After density centrifugation, spore yields were measured by phase-contrast microscopy and enumeration assays. Effects of purification methods including heat, sonication and lysozyme, and maturation on spore qualities were determined by sodium hypochlorite sporicidal assay. Difco sporulation media was identified as the preferred sporulation medium for 4 out of 7 tested Bacillus strains. Sporulation rates in B. cereus, B. sphaericus, and B. thuringiensis were higher at 30°C than the rates at 37°C at a difference of 5%, 65%, and 20%, respectively. Bacillus licheniformis favored Mn2+-amended 10% Columbia Broth at 37°C for sporulation with 40–72% higher sporulation rates than other media. The maximum sporulation rates of B. cereus and B. thuringiensis were observed on double-strength Schaeffer's-glucose broth. All studied purification methods improved the spore purity with strain variations. However, intense heat (80°C for 20 min) and lysozyme (100 μg/mL) treatment impaired the spore quality of specific Bacillus strains by sensitizing them against sodium hypochlorite. The length of the maturation period had an impact on the spore resistance, and the most optimal maturation periods ranged from 7 to 21 days in Bacillus strains. The results of this study will pave the way for further evaluation of the sporicidal activity of disinfectants.

Research Organization:: Oak Ridge Inst. for Science and Education (ORISE), Oak Ridge, TN (United States)

Sponsoring Organization:: USDOE Office of Science (SC)

DOE Contract Number:: SC0014664

OSTI ID:: 1982502

Journal Information:: Journal of Industrial Microbiology and Biotechnology, Vol. 49, Issue 4; ISSN 1367-5435

Publisher:: Springer

Country of Publication:: United States

Language:: English

References (38)

Activation of the Bacillus subtilis hut operon at the onset of stationary growth phase in nutrient sporulation medium results primarily from the relief of amino acid repression of histidine transport Atkinson, M. R.; Wray, L. V.; Fisher, S. H. Journal of Bacteriology, Vol. 175, Issue 14 https://doi.org/10.1128/jb.175.14.4282-4289.1993	journal	July 1993
Effect of Ultrasonic Waves on the Heat Resistance of Bacillus cereus and Bacillus licheniformis Spores Burgos, J.; Ordóñez, J. A.; Sala, F. Applied Microbiology, Vol. 24, Issue 3 https://doi.org/10.1128/am.24.3.497-498.1972	journal	September 1972
Properties of Aged Spores of Bacillus subtilis Camilleri, Emily; Korza, George; Green, Joshua Journal of Bacteriology, Vol. 201, Issue 14 https://doi.org/10.1128/JB.00231-19	journal	July 2019
Effect of sporulation and recovery medium on the heat resistance and amount of injury of spores from spoilage bacilli Cazemier, A. E.; Wagenaars, S. F. M.; ter Steeg, P. F. Journal of Applied Microbiology, Vol. 90, Issue 5 https://doi.org/10.1046/j.1365-2672.2001.01302.x	journal	May 2001
Protection of Bacillus pumilus Spores by Catalases Checinska, Aleksandra; Burbank, Malcolm; Paszczynski, Andrzej J. Applied and Environmental Microbiology, Vol. 78, Issue 18 https://doi.org/10.1128/AEM.01211-12	journal	September 2012
Investigating synergism during sequential inactivation of Bacillus subtilis spores with several disinfectants Cho, Min; Kim, Jae-Hong; Yoon, Jeyong Water Research, Vol. 40, Issue 15 https://doi.org/10.1016/j.watres.2006.05.042	journal	August 2006
Analysis of damage due to moist heat treatment of spores ofBacillus subtilis Coleman, W. H.; Setlow, P. Journal of Applied Microbiology, Vol. 106, Issue 5 https://doi.org/10.1111/j.1365-2672.2008.04127.x	journal	May 2009
The Timing of cotE Expression Affects Bacillus subtilis Spore Coat Morphology but Not Lysozyme Resistance Costa, Teresa; Serrano, Mónica; Steil, Leif Journal of Bacteriology, Vol. 189, Issue 6 https://doi.org/10.1128/JB.01353-06	journal	March 2007
Multifaceted toxin profile, an approach toward a better understanding of probioticBacillus cereus Cui, Yifang; Märtlbauer, Erwin; Dietrich, Richard Critical Reviews in Toxicology, Vol. 49, Issue 4 https://doi.org/10.1080/10408444.2019.1609410	journal	April 2019
Bacillus subtilis Spore Coat Driks, Adam Microbiology and Molecular Biology Reviews, Vol. 63, Issue 1 https://doi.org/10.1128/MMBR.63.1.1-20.1999	journal	March 1999
Development of a sporicidal test method for Clostridium difficile Fraise, A. P.; Wilkinson, M. A. C.; Bradley, C. R. Journal of Hospital Infection, Vol. 89, Issue 1 https://doi.org/10.1016/j.jhin.2014.09.014	journal	January 2015
Sensitization of Bacterial Spores to Lysozyme and to Hydrogen Peroxide with Agents which Rupture Disulphide Bonds Gould, G. W.; Hitchins, A. D. Journal of General Microbiology, Vol. 33, Issue 3 https://doi.org/10.1099/00221287-33-3-413	journal	December 1963
Potential Risks of Pharmacy Compounding Gudeman, Jennifer; Jozwiakowski, Michael; Chollet, John Drugs in R&D, Vol. 13, Issue 1 https://doi.org/10.1007/s40268-013-0005-9	journal	March 2013
Inactivation of Bacillus anthracis spores in murine primary macrophages Hu, Haijing; Sa, Qila; Koehler, Theresa M. Cellular Microbiology, Vol. 8, Issue 10 https://doi.org/10.1111/j.1462-5822.2006.00738.x	journal	October 2006
The Bacillus cereus Food Infection as Multifactorial Process Jessberger, Nadja; Dietrich, Richard; Granum, Per Einar Toxins, Vol. 12, Issue 11 https://doi.org/10.3390/toxins12110701	journal	November 2020
Proteomic Analysis of the Spore Coats of Bacillus subtilis and Bacillus anthracis Lai, Erh-Min; Phadke, Nikhil D.; Kachman, Maureen T. Journal of Bacteriology, Vol. 185, Issue 4 https://doi.org/10.1128/JB.185.4.1443-1454.2003	journal	February 2003
The Stability of Messenger Ribonucleic Acid during Sporulation in Bacillus subtilis Leighton, T. J.; Doi, Roy H. Journal of Biological Chemistry, Vol. 246, Issue 10 https://doi.org/10.1016/S0021-9258(18)62213-6	journal	May 1971
Regulatory framework of pharmaceutical compounding and actual developments of legislation in Europe Minghetti, Paola; Pantano, Doriana; Gennari, Chiara Grazia Milena Health Policy, Vol. 117, Issue 3 https://doi.org/10.1016/j.healthpol.2014.07.010	journal	September 2014
A Procedure for High-Yield Spore Production by Bacillus s ubtilis Monteiro, Sandra M.; Clemente, João J.; Henriques, Adriano O. Biotechnology Progress, Vol. 21, Issue 4 https://doi.org/10.1021/bp050062z	journal	January 2005
Resistance of Bacillus Endospores to Extreme Terrestrial and Extraterrestrial Environments Nicholson, W. L.; Munakata, N.; Horneck, G. Microbiology and Molecular Biology Reviews, Vol. 64, Issue 3 https://doi.org/10.1128/MMBR.64.3.548-572.2000	journal	September 2000
Bacillus cereus keratitis associated with contact lens wear1 1The authors have no financial interest in any material used in this study. Pinna, Antonio; Sechi, Leonardo A.; Zanetti, Stefania Ophthalmology, Vol. 108, Issue 10 https://doi.org/10.1016/S0161-6420(01)00723-0	journal	October 2001
Method for obtaining free bacterial spores of Bacillus subtilis var. niger Powers, E. M. Applied Microbiology, Vol. 16, Issue 1 https://doi.org/10.1128/am.16.1.180-181.1968	journal	January 1968
Inactivation ofBacillus subtilisspores by combining ultrasonic waves under pressure and mild heat treatment Raso, J.; Palop, A.; Pagán, R. Journal of Applied Microbiology, Vol. 85, Issue 5 https://doi.org/10.1046/j.1365-2672.1998.00593.x	journal	November 1998
Evaluation of Anaerobic Growth of Bacillus licheniformis and Bacillus subtilis in Tomato Juice Rodriguez, J. H.; Cousin, M. A.; Nelson, P. E. Journal of Food Protection, Vol. 55, Issue 9 https://doi.org/10.4315/0362-028X-55.9.672	journal	September 1992
Ineffectiveness of Hospital Disinfectants Against Bacteria: A Collaborative Study Rutala, William A.; Cole, Eugene C. Infection Control, Vol. 8, Issue 12 https://doi.org/10.1017/S0195941700067564	journal	December 1987
Maturation of Released Spores Is Necessary for Acquisition of Full Spore Heat Resistance during Bacillus subtilis Sporulation Sanchez-Salas, Jose-Luis; Setlow, Barbara; Zhang, Pengfei Applied and Environmental Microbiology, Vol. 77, Issue 19 https://doi.org/10.1128/AEM.05031-11	journal	October 2011
A Review of Cleanroom Microflora: Types, Trends, and Patterns Sandle, T. PDA Journal of Pharmaceutical Science and Technology, Vol. 65, Issue 4 https://doi.org/10.5731/pdajpst.2011.00765	journal	July 2011
RNA Dynamics in Aging Bacterial Spores Segev, Einat; Smith, Yoav; Ben-Yehuda, Sigal Cell, Vol. 148, Issue 1-2 https://doi.org/10.1016/j.cell.2011.11.059	journal	January 2012
Life cycle and spore resistance of spore-forming Bacillus atrophaeus Sella, Sandra R. B. R.; Vandenberghe, Luciana P. S.; Soccol, Carlos Ricardo Microbiological Research, Vol. 169, Issue 12 https://doi.org/10.1016/j.micres.2014.05.001	journal	December 2014
Spores of Bacillus subtilis: their resistance to and killing by radiation, heat and chemicals Setlow, P. Journal of Applied Microbiology, Vol. 101, Issue 3 https://doi.org/10.1111/j.1365-2672.2005.02736.x	journal	September 2006
Evaluation of the sporicidal activity of different chemical disinfectants used in hospitals against Clostridium difficile Speight, S.; Moy, A.; Macken, S. Journal of Hospital Infection, Vol. 79, Issue 1 https://doi.org/10.1016/j.jhin.2011.05.016	journal	September 2011
Density Gradient Centrifugation for the Separation of Sporulating Forms of Bacteria Tamir, Hadassah; Gilvarg, Charles Journal of Biological Chemistry, Vol. 241, Issue 5 https://doi.org/10.1016/S0021-9258(18)96806-7	journal	March 1966
Current physical and SDS extraction methods do not efficiently remove exosporium proteins from Bacillus anthracis spores Thompson, Brian M.; Binkley, Jana M.; Stewart, George C. Journal of Microbiological Methods, Vol. 85, Issue 2 https://doi.org/10.1016/j.mimet.2011.02.009	journal	May 2011
Evaluating the Sporicidal Activity of Disinfectants against Clostridium difficile and Bacillus amyloliquefaciens Spores by Using the Improved Methods Based on ASTM E2197-11 Uwamahoro, Marie Christine; Massicotte, Richard; Hurtubise, Yves Frontiers in Public Health, Vol. 6 https://doi.org/10.3389/fpubh.2018.00018	journal	February 2018
Pharmaceutical Compounding: a History, Regulatory Overview, and Systematic Review of Compounding Errors Watson, C. James; Whitledge, James D.; Siani, Alicia M. Journal of Medical Toxicology, Vol. 17, Issue 2 https://doi.org/10.1007/s13181-020-00814-3	journal	November 2020
Manganese Requirement for Sporulation and Other Secondary Biosynthetic Processes of Bacillus Weinberg, Eugene D. Applied Microbiology, Vol. 12, Issue 5 https://doi.org/10.1128/am.12.5.436-441.1964	journal	September 1964
Impact of standard test protocols on sporicidal efficacy Wesgate, R.; Rauwel, G.; Criquelion, J. Journal of Hospital Infection, Vol. 93, Issue 3 https://doi.org/10.1016/j.jhin.2016.03.018	journal	July 2016
Mechanisms of killing of Bacillus subtilis spores by hypochlorite and chlorine dioxide Young, S. B.; Setlow, P. Journal of Applied Microbiology, Vol. 95, Issue 1 https://doi.org/10.1046/j.1365-2672.2003.01960.x	journal	July 2003

Similar Records

Identification of Sporulated and Vegetative Bacteria using Statistical Analysis of Fourier Transform Mid-Infrared Transmission Data

Journal Article · 2004 · Applied Spectroscopy, 58(2):203-211 · OSTI ID:15007110

Foster, Nancy S; Thompson, Sandra E; Valentine, Nancy B; +2 more

Development of ultraviolet resistance in sporulating Bacillus cereus T.

Journal Article · 1973 · J. Bacteriol., v. 116, no. 2, pp. 823-831 · OSTI ID:4421800

Germaine, G R; Coggiola, E; Murrell, W G

Plasmid-associated sensitivity of Bacillus thuringiensis to UV light

Journal Article · 1990 · Applied and Environmental Microbiology; (USA) · OSTI ID:6236335

Benoit, T G; Wilson, G R; Bull, D L; +1 more

Related Subjects

Biotechnology & Applied Microbiology

Optimization of sporulation and purification methods for sporicidal efficacy assessment on Bacillus spores

Citation Formats

References (38)

Similar Records

Related Subjects