No abstract

Germination experiments with specific germination mutants of Bacillus subtilis, including a newly isolated mutant affected in pressure-induced germination, suggest that a pressure of 100 MPa triggers the germination cascades that are induced by the nutrient germinant alanine (Ala) and by a mixture of asparagine, glucose, fructose, and potassium ions (AGFK), by activating the receptors for alanine and asparagine, GerA and GerB, respectively. As opposed to germination at 100 MPa, germination at 600 MPa apparently shortcuts at least part of the Ala-and AGFK-induced germination pathways. Inhibitors of nutrient-induced germination (HgCl 2 and N␣-P-tosyl-L-arginine methyl ester) also inhibit pressure-induced germination at 600 MPa, suggesting that germination at 600 MPa involves activation of a true physiological germination pathway and is therefore not merely a physico-chemical process in which water is forced into the spore protoplast.

Escherichia coli MG1655 suspensions in 10 mM phosphate buffer (pH 7.0) were subjected to high pressures in the range of 180 to 320 MPa for 15 min. Cell death was evident at 220 MPa and increased exponentially with pressure. Surviving populations were sublethally injured, as demonstrated by their reduced ability to form colonies on violet red bile glucose agar, a selective growth medium containing crystal violet and bile salts. During exposure to high pressure (> 180 MPa), cells were sensitive to lysozyme, nisin, and ethylenediaminetetraacetic acid (EDTA), as was apparent from an increased lethality of pressure in the presence of these agents. Sublethal injury in the surviving population was lower in the presence of nisin and lysozyme, but higher in the presence of EDTA. Combinations of EDTA with nisin or lysozyme present during pressure treatment increased lethality in an additive manner. However, the addition of lysozyme, nisin and/or EDTA to pressurized cell suspensions immediately after pressure treatment did not cause any viable count reduction. Finally, we observed leakage of the periplasmic enzyme β-lactamase from an ampicillin-resistant recombinant E. coli MG1655 under high pressure. These results suggest that high pressure transiently disrupts the permeability of the E. coli outer membrane for water-soluble proteins.

We have studied sublethal injury in Salmonella enterica serovar Typhimurium caused by mild heat and by different emerging nonthermal food preservation treatments, i.e., high-pressure homogenization, high hydrostatic pressure, pulsed white light, and pulsed electric field. Sublethal injury was determined by plating on different selective media, i.e., tryptic soy agar (TSA) plus 3% NaCl, TSA adjusted to pH 5.5, and violet red bile glucose agar. For each inactivation technique, at least five treatments using different doses were applied in order to cover an inactivation range of 0 to 5 log units. For all of the treatments performed with a technique, the logarithm of the viability reductions measured on each of the selective plating media was plotted against the logarithm of the viability reduction on TSA as a nonselective medium, and these points were fined by a straight line. Sublethal injury between different techniques was then compared by the slope and the y intercept of these regression lines. The highest levels of sublethal injury were observed for the heat and high hydrostatic pressure treatments. Sublethal injury after those treatments was observed on all selective plating media. For the heat treatment, but not for the high-pressure treatment, sublethal injury occurred at low doses, which were not yet lethal. The other nonthermal techniques resulted in sublethal injury on only some of the selective plating media, and the levels of injury were much lower. The different manifestations of sublethal injury were attributed to different inactivation mechanisms by each of the techniques, and a mechanistic model is proposed to explain these differences.

We have studied pressure-induced germination of Bacillus subtilis spores at moderate (100 MPa) and high (500 to 600 MPa) pressures. Although we found comparable germination efficiencies under both conditions by using heat sensitivity as a criterion for germination, the sensitivity of pressure-germinated spores to some other agents was found to depend on the pressure used. Spores germinated at 100 MPa were more sensitive to pressure (>200 MPa), UV light, and hydrogen peroxide than were those germinated at 600 MPa. Since small, acid-soluble proteins (SASPs) and dipicolinic acid (DPA) are known to be involved in spore resistance to UV light and hydrogen peroxide, we studied the fate of these compounds during pressure germination. DPA was released upon both low- and high-pressure germination, but SASP degradation, which normally accompanies nutrient-induced germination, occurred upon low-pressure germination but not upon high-pressure germination. These results adequately explain the UV and hydrogen peroxide resistance of spores germinated at 600 MPa. The resistance to pressure inactivation of 600-MPa-germinated spores could also, at least partly, be attributed to α/β-type SASPs, since mutants deficient in α/β-type SASPs were more sensitive to inactivation at 600 MPa. Further, germination at 100 MPa resulted in rapid ATP generation, as is the case in nutrient-induced germination, but no ATP was formed during germination at 600 MPa. These results suggest that spore germination can be initiated by low- and high-pressure treatments but is arrested at an early stage in the latter case. The implications for the use of high pressure as a preservation treatment are discussed.