Characterization of a halo-acid-tolerant variant of Clostridium botulinum B-aphis

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The germination of spores fromn Clostridium botulinum B-aphis and Ba410 was examined. In a complex medium, heat activation of spores from both strains doubled the germination rates and was required for germination in the presence of 2% NaCl. In a defined medium (CTB [D. B. Rowley and F. Feeherry, J. Bacteriol. 104:1151-1157, 1970]), the parent strain B-aphis germinated at a rate of 0.77% min-' in the absence of NaCl and was not affected by 2% NaCl. A salt-tolerant derivative, strain Ba410, germninated at rates of 0.16% min-' in CTB and 0.04% min-' in CTB containing 2% NaCl. L-Alanine-triggered spores germinated faster than did L-cysteine-triggered spores from both strains. When both amino acids were present, B-aphis germinated rapidly in the absence of NaCl and had biphasic kinetics in the presence of NaCl. Strain Ba410 had biphasic kinetics in the absence of NaCl and germinated slowly with single-phase kinetics in the presence of NaCl. L-Alanineand L-cysteine-triggered germinations were each inhibited by both D-alanine and D-cysteine, indicating a common germinant-binding site for both alanine and cysteine. Attempts to select for variants with amino acid-specific germinant-binding sites were unsuccessful. Differences in the germination kinetics of both strains could not be explained by ultrastructural differences. Transmission electron micrographs revealed striking similarities between the strains. Complete inhibition of botulinal toxigenesis requires NaCl levels of 5% for type E strains (2, 31) and up to 8 and 10o for type A and proteolytic B strains (1, 26) under otherwise optimal conditions. Lower salt concentrations are, however, inhibitory when used with another inhibitor (10, 32). Complex interactions among pH, salt, and nitrite are responsible Relatively little work has been done on the influence of NaCl on clostridial spore germination (2, 4, 26). While examining the interaction of pH and salt on botulinal physiology (22, 24), we isolated and characterized a salt-tolerant variant of Clostridium botulinum B-aphis. This variant, strain Ba410, differs from the parent strain in salt sensitivity, * Corresponding author. t Publication D-10112-1-85 of the New Jersey Agricultural Experiment Station.

Section: Methodssupporting

confidence: 85%

“…protease activity, regulation of cell lysis, and the germination rates of alanineand cysteine-triggered spores (23). The objective of the present study was to compare the spore germination kinetics of C. botulinum B-aphis and Ba410 for different germinants and in the presence or absence of salt.…”

mentioning

confidence: 99%

Germination of spores from Clostridium botulinum B-aphis and Ba410

Montville¹,

Jones²,

Conway³

et al. 1985

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“…At specific times during the incubation, 20-l aliquots were removed from each tube through the rubber stopper by use of a sterile syringe. Germination was determined microscopically as a change in spores from phase bright to phase dark (31). Germination rates were calculated from the change in the percentage of germinated spores with respect to time.…”

Section: Methodsmentioning

confidence: 99%

Characterization of Fatty Acid Composition, Spore Germination, and Thermal Resistance in a Nisin-Resistant Mutant of Clostridium botulinum 169B and in the Wild-Type Strain

Mazzotta

Montville

1999

The membrane fatty acids, thermal resistance, and germination of a nisin-resistant (Nisr) mutant of Clostridium botulinum 169B were compared with those of the wild-type (WT) strain. In the membranes of WT cells, almost 50% of the total fatty acids were unsaturated, but in those of Nisr cells, only 23% of the fatty acids were unsaturated. WT and Nisrspores contained similar amounts (approximately 23%) of unsaturated fatty acids, but the saturated straight-chain/branched-chain ratio was significantly higher in Nisr spores than in WT spores. These fatty acid differences suggest that Nisr cell and spore membranes may be more rigid, a characteristic which would interfere with the pore-forming ability of nisin. Nisr C. botulinum did not produce an extracellular nisin-degrading enzyme, nor were there any differences in the sodium dodecyl sulfate-polyacrylamide gel electrophoresis patterns of coat proteins extracted from WT and Nisr spores, eliminating these as possible reasons for nisin resistance. Nisr spores had thermal resistance parameters similar to those of WT spores. In WT spores, but not in Nisr spores, nisin caused a 40% reduction in thermal resistance and a twofold increase in the germination rate. Because the nisin-induced increase in the germination rate of WT spores occurred only in the presence of a germinant (a molecule that triggers germination), nisin can be classified as a progerminant (a molecule that stimulates germination only in the presence of a germinant).

Synergistic Effects of High Hydrostatic Pressure, Mild Heating, and Amino Acids on Germination and Inactivation of Clostridium sporogenes Spores

Ishimori

Takahashi

Goto

et al. 2012

The synergistic effects of high hydrostatic pressure (HHP), mild heating, and amino acids on the germination of Clostridium sporogenes spores were examined by determining the number of surviving spores that returned to vegetative growth after pasteurization following these treatments. Pressurization at 200 MPa at a temperature higher than 40°C and treatment with some of the 19 L-amino acids at 10 mM or higher synergistically facilitated germination. When one of these factors was omitted, the level of germination was insignificant. Pressures of 100 and 400 MPa were less effective than 200 MPa. The spores were effectively inactivated by between 1.8 and 4.8 logs by pasteurization at 80°C after pressurization at 200 MPa at 45°C for 120 min with one of the amino acids with moderate hydrophobicity, such as Leu, Phe, Cys Met, Ala, Gly, or Ser. However, other amino acids showed poor inactivation effects of less than 0.9 logs. Spores in solutions containing 80 mM of either Leu, Phe, Cys, Met, Ala, Gly, or Ser were successfully inactivated by pasteurization by more than 5.4 logs after pressurization at 200 MPa at 70°C for 15 to 120 min. Ala and Met reduced the spore viability by 2.8 and 1.8 logs, respectively, by pasteurization at a concentration of 1 mM under 200 MPa at 70°C. These results indicate that germination of the spores is facilitated by a combination of high hydrostatic pressure, mild heating, and amino acids.