Multiple modes of inhibition of spore germination and outgrowth by reduced pH and sorbate.

Germination and outgrowth of three strains of Clostridium botulinum in PYEG medium were measured by phase contrast microscopy. Reduction in pH from 7 to 5.5 completely inhibited germination of strain 12885A, reduced the extent of germination of strain 62A and had no effect on the extent of germination of strain 53B. At pH 5.5, 225 mg/l of undissociated sorbic acid had no effect on the germination of strain 53B, while at pH 6.5, 225 mg/l of undissociated sorbic acid completely inhibited germination of strains 62A and 12885A. Outgrowth of germinated spores of strains 62A and 53B was not inhibited at pH 5.5, but the addition of sorbate (225 mg/l undissociated sorbic acid) completely inhibited outgrowth. Sorbate inhibited germination of Cl. botulinum and Bacillus cereus spores triggered to germinate by amino acids. Inhibition occurred after germinant binding, as measured by commitment to germinate.

Inhibition of germinant binding by bacterial spores in acidic environments.

Commitment to germinate occurred in both Clostridium botulinum and Bacillus cereus spores during 0.5 min of exposure to 100 mM L-alanine or L-cysteine, measured by the inability of germination inhibitors (D form of amino acid) to inhibit germination. Spore germination at pH 4.5 was inhibited because the germinant did not bind to the trigger sites. C. botulinum spores exposed to 100 mM L-alanine or L-cysteine at pH 4.5 remained sensitive to D-amino acid inhibition at pH 7, indicating that no germinants had bound to the trigger site at pH 4.5. Inhibition of germinant binding at pH 4.5 was reversible but lagged in commitment to germinate upon transfer to pH 7. Spores sequentially exposed to pH 4.5 buffer and pH 7 buffer with the germinant also demonstrated a lag in commitment to germinate. The pH at which binding was inhibited was not significantly affected by composition of the buffer or by reduced germinant concentrations (10 mM). Nonspecific uptake of L-[3H]alanine by C. botulinum spores was not inhibited at pH 4.5. Inhibition of germinant binding in acidic environments appeared to be due to protonation of a functional group in or near the trigger site. This may represent a general mechanism for inhibition of spore germination in acidic environments.