Source: UNIVERSITY OF DELAWARE submitted to
MECHANISMS OF BACILLUS SPORE INACTIVATION BY PRESSURE
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
Reporting Frequency
Annual
Accession No.
0196582
Grant No.
2003-35201-13553
Project No.
DEL00480
Proposal No.
2003-03084
Multistate No.
(N/A)
Program Code
32.0
Project Start Date
Sep 1, 2003
Project End Date
Aug 31, 2007
Grant Year
2003
Project Director
Hoover, D.
Recipient Organization
UNIVERSITY OF DELAWARE
(N/A)
NEWARK,DE 19717
Performing Department
ANIMAL & FOOD SCIENCE
Non Technical Summary
The use of high hydrostatic pressure processing by the food industry is increasing. Consumer appeal for minimally processed foods with fresh-like sensory qualities and nutrient content has grown. Most current products using high pressure processing for their preservation are refrigerated or high-acid foods. The inability of high pressure processing (and other nonthermal processing methods) to control sporeforming bacteria remains and limits broader application of pressure processing while also compromising safety of pressure-treated products. The purpose of this study is to learn more about the manner in which hydrostatic pressure and elevated temperatures affect germination of spores of Bacillus subtilis and Bacillus cereus. Information generated by this work should assist expansion of the application of pressure processing to low-acid foods with minimal compromise to the nonthermal nature of the process. This project should also supply information pertinent to improved understanding of spore physiology as well as contribute towards enhanced control and prediction of germination, outgrowth and inactivation of bacterial spores.
Animal Health Component
75%
Research Effort Categories
Basic
25%
Applied
75%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
5014010110090%
5015010110010%
Goals / Objectives
The overall goal of this project is to study the mechanisms whereby high hydrostatic pressure and mild heat germinate bacterial endospores and subsequently inactivate them in order to better understand and control problematic sporeforming bacteria. The supporting objectives are: 1.) mechanistically determine how hydrostatic pressure (with and without elevated temperatures) triggers spore germination leading to eventual inactivation of the organism, 2.) optimize spore germination induced by pressure, and 3.) evaluate the response of pressure-germinated spores at various stages of germination induced by food.
Project Methods
Experimentally this will be approached by examination of the resistance of spores at various stages of germination as well as measurement of related properties of pressure-germinated spores. We will examine the response of spores of B. subtilis mutants lacking specific germination receptors, having elevated levels of specific germination receptors, lacking dipicolinic acid and/or cortex lytic enzymes, and possessing different core water contents; comparable germination mutants of the pathogen, Bacillus cereus, will be studied concurrently. The responses of spores of B. subtilis and B. cereus to high hydrostatic pressure by post-treatment measurements of concentrations of ATP, small acid-soluble proteins, dipicolinic acid and other small molecules and cortical peptidoglycan as well as the pressure resistance of spores germinated at various pressures, will also be examined. This information will allow us to determine the role these factors play in pressure-induced spore germination.

Progress 09/01/03 to 08/31/07

Outputs
OUTPUTS: Hoover, High pressure processing (HPP)-related outputs: Teaching: FOSC 439/639 Food Microbiology; FOSC 305 Food Science; FOSC 265 Food Science Seminar. Mentoring: Jie Wei (M.S. in Food Science, 2007); Kristen Hirneisen (M.S. partial support). Events: IFT Nonthermal Processing Workshops: 2003 Wageningen, the Netherlands, invited presentation/session moderator; 2004 Sydney, Australia, invited presentation/session moderator/poster presentation; 2005, Philadelphia, attendee; 2006 Cork, Ireland, invited presentation/session moderator. 2004 IFT Annual Meeting & Food Expo, Las Vegas; invited presentation. 2004 ACS National Meeting, Philadelphia; invited presentation. 2004 Interstate Seafood Seminar, Virginia Beach, VA; invited presentation. 2005 Atlantic Fisheries Technology Conference, Norfolk, VA; invited presentation. 2005 Interstate Seafood Seminar, Ocean City, MD.; invited presentation. 2006 Food Safety World Conference, Washington, DC, invited presentation. 2006 IFT Annual Meeting & Food Expo, Orlando, J. Wei presentation. 2007 IFT Annual Meeting & Food Expo, Chicago, invited presentation. Consulting: 10/2007 with a biomedical corporation based in the Chicago area on HPP and spore inactivation. Advisory Board: Naked Juice (Pepsico) Scientific Advisory Board - 2007 HPP of natural juices. Setlow, HPP-related outputs: Mentoring: Donna Cortezzo and Kasia Koziol-Dube (partial support, now medical students); support for work of Barbara Setlow (instructor in biochemistry), V.R. Vepachedu (postdoctoral scientist), and Patricia Pelczar (graduate student). Events: 2004 IFT Annual Meeting & Food Expo; invited symposium presentation. 2005 Natick Army Labs HPP Workshop; invited presentation. 2005 Society for Applied Microbiology Summer Conference; invited presentation. 2006 IFT Annual Meeting & Food Expo; invited symposium presentation. PARTICIPANTS: [Participants are listed under Outputs in report.]

Impacts
The purpose of this study was to learn more about the manner in which HPP and elevated temperatures affect the germination and inactivation of spores of Bacillus subtilis and the foodborne pathogen, Bacillus cereus. It was establishing that different nutrient receptors in spores of B. subtilis varied in initiating germination at 150 MPa. The hierarchy in receptor responsiveness was GerA > GerB > GerK for exposure to either pressure or nutrient mixtures. Increasing the number of nutrient receptors in spores also resulted in increased levels of pressure germination. Spores lacking dipicolinic acid (DPA) did not germinate upon exposure to 150 MPa. The GerA, GerB and GerK nutrient germinant receptors were found to have different requirements for the diacylglycerylation of one receptor subunit for germination with 150 MPa. It was found that wild-type spores of B. subtilis prepared at high temperature germinated more rapidly with 150 MPa than spores prepared at lower temperatures. Spore germination at 150 MPa was unaffected by changes in inner membrane unsaturated fatty acids, lethal treatment with oxidizing agents, or exposure to chemicals inhibiting nutrient germination. The novel sporocide, dimethyldioxirane (DMDO), was found to be an effective inactivation agent of B. subtilis spores. Killed spores germinated at very high pressures (≥500 MPa) and by lysozyme treatment in hypertonic medium, but many of these spores lysed shortly after germination. The spore coat was found to be a major factor in spore resistance to DMDO. DMDO appears to kill spores by damaging the inner membrane of the spore. Tert-butyl hydroperoxide (tBHP) plus the cationic surfactant cetyltrimethyl ammonium bromide (CTAB) and a tetraamido macrocyclic ligand (TAML) was also effective in killing spores of B. subtilis by damaging the inner membrane of the spore. As with spores killed with DMDO, spores killed with the reagent system were also germinated by exposure to pressure (150 or 500 MPa for 15 min) or lysozyme treatment in hypertonic medium, and lysed shortly after germination. Germination of spores of B. subtilis occurred rapidly with exposure to 500 MPa and 50 C. Germination under these conditions did not occur via nutrient-germinant receptors, but by release of DPA through the inner membrane of the spore. Either a membrane protein or the membrane itself is targeted. This work provides new insight into the germination of spores of B. subtilis by exposure to 500 MPa and 50 C. At least partial spore germination by pressure is essential for subsequent spore inactivation, making the understanding of germination mechanisms critical in optimizing effective pressure treatments for the preservation of low-acid foods. Treatment temperature also plays an important role in the pressure germination of spores of Bacillus cereus. Levels of both germination and inactivation increased with increasing temperature. Results suggested that the GerL, GerT and GerN receptors contributed to pressure-induced germination, especially at low pressures (150 MPa) and were also temperature-dependent.

Publications

  • Paul, M., B. Setlow and P. Setlow. 2007. Killing spores of Bacillus subtilis by tert-butylhydroperoxide plus a TAML activator. J. Appl. Microbiol. 102:954-962.
  • Setlow, P. 2008. Germination of spores of Bacillus subtilis by high pressure. In High Pressure Processing of Foods (C. Doona, F. E. Feheery and C.P. Dunne, ed), in press. Blackwell Publishing, London.
  • Setlow, P. 2008. Effects of high pressure on spores. In High-Pressure Microbiology (C. Michiels, A. Aertsen, D. Bartlett and A.A. Yayanos, ed.), in press. ASM Press, Washington, DC.
  • Vepachedu, V.R., K. Hirneisen, D.G. Hoover and P. Setlow. 2007. Studies of the release of small molecules during pressure germination of spores of Bacillus subtilis. Lett. Appl. Microbiol. 45:342-348.
  • Wei, J., P. Setlow and D.G. Hoover. 2006. Effect of high pressure and temperature on inactivation and germination of Bacillus cereus spores. Institute of Food Technologists Annual Meeting & Food Expo, June 24-28, Orlando, FL.
  • Wei, J., P. Setlow and D.G. Hoover. 2008. Germination and inactivation of Bacillus cereus spores by pressure and heat. Manuscript in preparation.
  • Black, E.P., K. Koziol-Dube, D. Guan, D. Cortezzo, D.G. Hoover and P. Setlow. 2005. Studies on the triggering of germination of Bacillus subtilis spores by action of high pressure on nutrient germinant receptors. Appl. Environ. Microbiol. 71:5879-5887.
  • Black, E.P., C.M. Stewart and D.G. Hoover. 2008. High pressure microbiology. In: Handbook of Nonthermal Processing. Farkas, D., and V.M. Balasubramaniam, eds. Blackwell Publishing, London, and the Institute of Food Technologists, Chicago, in press.
  • Black, E.P., P. Setlow, A.D. Hocking, C.M. Stewart, A.L. Kelly and D.G. Hoover. 2007. Response of spores to high pressure processing. Comprehensive Rev. Food Sci. Food Safety 6:103-119.
  • Black, E.P., J. Wei, S. Atluri, D.E. Cortezzo, K. Koziol-Dube, D.G. Hoover and P. Setlow. 2007. Analysis of factors influencing the rate of germination of spore of Bacillus subtilis by very high pressure. J. Appl. Microbiol. 102:65-76.
  • Paul, M., S. Atluri, B. Setlow and P. Setlow. 2006. Mechanisms for killing spores of Bacillus subtilis by dimethyldioxirane. J. Appl. Microbiol. 101:1161-1168.


Progress 01/01/06 to 12/31/06

Outputs
Extended studies have continued on the role of SpoVA proteins in the release of dipicolinic acid (DPA) from spores of Bacillus subtilis upon germination triggered by exposure to 500 MPa at 50 C. This work indicates that SpoVA proteins are involved in DPA release in spore germination under these conditions since increasing the SpoVA protein level in spores increased rates of DPA release and a temperature-sensitive spoVA mutant was also temperature sensitive for DPA release. We are also studying the release of small molecules (Na+, K+, glutamic acid, arginine and AMP) in addition to DPA release during treatment of spores with either 150 or 500 MPa, as well as the role of SpoVA proteins in this release, however, these studies are not yet complete.

Impacts
The study is entering its final phase of work. The expected impact of the overall work will center on its publications presenting information on the various responses of spores of Bacillus subtilis and Bacillus cereus to pressure and elevated temperatures. From this information it is anticipated that greater insight will accrue and allow current and future applications of high pressure processing (HPP) by the food industry to better predict and expect shelf-life and safety successes in low-acid HPP products.

Publications

  • Black, E.P., J. Wei, S. Adluri, D.E. Cortezzo, K. Koziol-Dube, D.G. Hoover and P. Setlow. 2007. Analysis of factors influencing the rate of germination of spores of Bacillus subtilis by very high pressure. J. Appl. Microbiol. 102:65-76.
  • Paul, M., B. Setlow and P. Setlow. 2007. Killing of spores of Bacillus subtilis by tert-butyl hydroperoxide plus a TAML* activator. J. Appl. Microbiol. In press.
  • Black, E.P., P. Setlow, A.D. Hocking, C,M. Stewart, A.L. Kelly, and D.G. Hoover. 2007. Response of spores to high pressure processing. Comprehensive Rev. Food Sci. Food Safety. In preparation.
  • Paul, M., S. Atluri, B. Setlow and P. Setlow. 2006. Mechanisms of killing of spores of Bacillus subtilis by dimethyldioxirane. J. Appl. Microbiol. 101:1161-1168.


Progress 01/01/05 to 12/31/05

Outputs
B. subtilis PS533 is a wild-type strain with plasmid pUB110 (carrying kanamycin resistance). Pressure treatment was typically 500 MPa at 50 C with spores at 1.5-3.0 x 10E8/mL in 50 mM Tris-HCl, pH 7.5. Spore germination was assessed by flow cytometry after Syto 16-staining or by DPA release. 1) Mercuric chloride at 1 mM inhibited germination as measured by flow cytometry, but did not inhibit DPA release. Presumably mercuric chloride inhibited cortex lytic enzymes (CLEs). 2) Dodecylamine at 1 mM completely inhibited germination. 3) Either CLEs, CwlJ or SleB, are sufficient to complete spore germination, but with neither CLE, germination is incomplete; however, germination as measured by DPA release is normal with CLE-deficient spores. 4) Pretreatment of spores with decontaminating agents, dimethyldioxirane or t-butyl hydroperoxide + a TAML activator, gave 97% spore inactivation and greatly slowed nutrient germination of pretreated spores; however, germination by 500 MPa was unaltered although many pressure-germinated spores then lysed. 5) Using a mutant lacking membrane lipoproteins it was shown no membrane lipoprotein is needed for spore germination at 500 MPa. 6) Germination at 500 MPa was optimal at 60 C but reduced at 80 C (CLEs do not work well above 60 C); spore inactivation was slightly greater at 60 than 80 C. With 150-MPa germination, the optimal temperature was 40 C; spore inactivation was greatest at 80 C. 7) Data indicate that 500 MPa DPA is not released in spore activation through the nutrient-germination channel made up by the SpoVA proteins in the inner membrane of the spore. A temperature-sensitive mutation in one SpoVA protein is temperature-sensitive for 150-MPa spore germination (and nutrient germination) but not with 500 MPa. At 150 MPa, increasing the level of SpoVA proteins in the spore 4-fold results in 3-fold increase in germination rate; however, at 500 MPa there is no effect. B. cereus 569 (wild-type strain) and seven derivative germination mutants were screened for pressure response at 150 MPa for 15 min over a temperature range of 20 to 65 C. Extents of inactivation and germination were determined by exposing pressure-treated spores to 70 C for 10 min (the heat shock inactivates germinated spores) with subsequent plate counts. With increase in treatment temperature there was a general progressive increase in spore germination; for example, B. cereus 569 at 150 MPa showed a 1.5-log germination at 20 C and a 4.5-log germination at 65 C (initial level of spores was 10E8/mL). The level of spore inactivation directly correlated with spore germination. Mutant gerI (lacking receptors for inosine and L-alanine) germinated at a significantly lower rate than 569 at all temperatures in the absence of alanine. At low temperatures (20 to 40 C), mutant gerQ (lacking receptor for inosine only) germinated to a greater extent than 569, but rates were similar for both strains at 50 and 65 C. B. cereus 569 and mutant strains with wild-type function to L-alanine demonstrated increased inactivation and germination to treatment in the presence of 100 mM L-alanine.

Impacts
This study examines the responses of wild-type strains and germination mutant strains of the model sporeforming bacterium, Bacillus subtilis, and the foodborne pathogen, Bacillus cereus, to combinations of high hydrostatic pressure and elevated heat. As stated earlier, the anticipated impact deals with further elucidation of germination mechanisms of Bacillus spores. For example, process manipulations of high pressure applications as used in the commercial processing of low-acid foods may be modified as a consequence of information gained in this study.

Publications

  • Paul, M., S. Atluri, B. Setlow and P. Setlow. Mechanisms of killing spores of Bacillus subtilis by dimethyldioxirane. J. Appl. Microbiol. In press, 2006. [Grant cited for partial support of work.]
  • Wei, J., P. Setlow and D.G. Hoover. Effect of high pressure and temperature on inactivation and germination of Bacillus cereus spores. Institute of Food Technologists Annual Meeting & Food Expo, Meeting abstract, June 24-28, 2006, Orlando, FL.


Progress 01/01/04 to 12/31/04

Outputs
1. Spores made at 23C germinated 4-fold faster at 500 MPa/50C than spores made at 44C, but 44C-spores germinated 8-fold faster than 23C-spores upon exposure to 150 MPa/37C. 2. Spores lacking DPA because of spoVF mutation and stabilized by a sleB mutation did not germinate with either 150 MPa/37C or 500 MPa/50C; however, these spores sporulated with DPA germinated as quickly as wild-type spores at either pressure. 3. Spores with elevated or no inner-membrane unsaturated fatty acids germinated at the same rate as wild-type spores at both 150 MPa/37C and 500 MPa/50C. 4. Levels of D-alanine that block L-alanine germination through the GerA nutrient receptor did not inhibit pressure (150 MPa/37C) germination via the GerA receptor. 5. Concentrations of amiloride, ethanol, butanol and octanol that completely inhibit germiation with L-alanine via the GerA receptor did not inhibit germination at 150 MPa/37C via the GerA receptor. 6. Chlorocresol at a concentration that completely inhibits germination with L-alanine via the GerA receptor only inhibited germination at 150 MPa/37C via the GerA receptor by 40%. 7. Mercuric chloride at a concentration that completely inhibits germination by nutrients, also strongly inhibited germination at either pressure. 8. Dodecylamine is a germinant that will also germinate spores lacking all nutrient receptors, strongly inhibited germination at 500 MPa/50C, but less so at 150 MPa/37C. 9. Spores made in high NaCl at either 27 or 37C, germinated slightly faster than spores made at lower NaCl concentrations at 500 MPa/50C (a difference of 2- to 3-fold), but the reverse occurred at 150 MPa/37C. 10. For spores made at 37C, pressure germination at 150 MPa/37C was 5-fold faster with the GerA receptor alone, as compared to the GerB nutrient-germinant receptor alone, and the GerB receptor was 5-fold faster than the GerK receptor alone. 11. Overexpressing the GerA receptor by 25- to 50-fold in spores lacking the GerB and K receptors increased 150 MPa/37C germination 5-fold. 12. Two GerB variants responded more readily to nutrient germinants and also responded 3- to 5-fold better to 150 MPa/37C than wild-type GerB spores. 13. Overexpression (20 to 200-fold) of either the GerB or GerB* receptors in spores that lacked GerA and K receptors increased germination 3-fold at 150 MPa/37C. 14. Inability to add a covalent diacylglycerol to the C-protein component of the GerA receptor which destroys GerA function in nutrient germination, also destroyed GerA function in 150 MPa/37C germination. 15. Inability to add covalent diacylglycerol to the C-protein component of the GerK receptor which has no effect on GerK function in nutrient germination also had no effect on GerK function in germination at 150 MPa/37C. 16. All of the results described above were obtained with Bacillus subtilis spores suspended in 50 mM Tris-HCl (pH 7.5) with 1.5x10e8 spores/mL with spore germination measured by flow cytometry after staining with the nucleic acid stain Syto 16 obtained from Molecular Probes. In addition, almost all of these results were confirmed by measuring the release of dipicolinic acid.

Impacts
Results from this work will hopefully allow more consistent germination and predictions of germination for spores of the genus, Bacillus.

Publications

  • Black, E., K. Koziol-Dube, D. Guan, D. Cortezzo, D.G. Hoover and P. Setlow. 2005. Studies on the triggering of germination of Bacillus subtilis spores by action of high pressure on nutrient germinant receptors. Applied and Environmental Microbiology. In press.


Progress 01/01/03 to 12/31/03

Outputs
In work to date we have conducted a number of experiments to determine the best ways to measure the extent of spore germination induced by pressure and to move spores between the University of Connecticut Health Center (UConn), where the spore crops are produced and analyzed regarding the percentage of spores induced to germinate by high pressure exposure conducted at the University of Delaware (UD). We have been successful in using a fluorescence-activated cell sorter (FACS) to quantitate the percentage of germinated spores in a population of B. subtilis spores. The spores are treated with the nucleic acid stain, Syto9, and discrimination between dormant spores (that stain very poorly) and germinated spores (that stain very brightly) by FACS analysis is simple. We can easily see between 1 to 95% germinated spores in a population of clean spores. We have also shown that freezing spores in dry ice after germination does not affect the results of FACS analysis, and that this freezing does not result in spore germination. We have also been successful in shipping B. subtilis spores from UConn to UD on wet ice, pressure-treating the spores and then return for FACS analysis. Future shipments will be on dry ice. With these preliminary and control experiments completed, we are now beginning the studies designed to elucidate the mechanism(s) whereby high pressure triggers spore germination.

Impacts
Results from this work will hopefully allow more consistent germination and predictions of germination for spores of the genus, Bacillus.

Publications

  • No publications reported this period