Effects of High Pressure on Bacillus licheniformis Spore Germination and Inactivation

Abstract

Bacillus and Clostridium species form spores, which pose a challenge to the food industry due to their ubiquitous nature and extreme resistance. Pressurization at <300 MPa triggers spore germination by activating germination receptors (GRs), while pressurization at >300 MPa likely triggers germination by opening dipicolinic acid (DPA) channels present in the inner membrane of the spores. In this work, we expose spores of Bacillus licheniformis, a species associated with food spoilage and occasionally with food poisoning, to high pressure (HP) for holding times of up to 2 h. By using mutant spores lacking one or several GRs, we dissect the roles of the GerA, Ynd, and GerK GRs in moderately HP (mHP; 150 MPa)-induced spore germination. We show that Ynd alone is sufficient for efficient mHP-induced spore germination. GerK also triggers germination with mHP, although at a reduced germination rate compared to that of Ynd. GerA stimulates mHP-induced germination but only in the presence of either the intact GerK or Ynd GR. These results suggests that the effectiveness of the individual GRs in mHP-induced germination differs from their effectiveness in nutrient-induced germination, where GerA plays an essential role. In contrast to Bacillus subtilis spores, treatment with very HP (vHP) of 550 MPa at 37°C did not promote effective germination of B. licheniformis spores. However, treatment with vHP in combination with elevated temperatures (60°C) gave a synergistic effect on spore germination and inactivation. Together, these results provide novel insights into how HP affects B. licheniformis spore germination and inactivation and the role of individual GRs in this process.IMPORTANCE Bacterial spores are inherently resistant to food-processing regimes, such as high-temperature short-time pasteurization, and may therefore compromise food durability and safety. The induction of spore germination facilitates subsequent inactivation by gentler processing conditions that maintain the sensory and nutritional qualities of the food. High-pressure (HP) processing is a nonthermal food-processing technology used to eliminate microbes from food. The application of this technology for spore eradication in the food industry requires a better understanding of how HP affects the spores of different bacterial species. The present study provides novel insights into how HP affects Bacillus licheniformis spores, a species associated with food spoilage and occasionally food poisoning. We describe the roles of different germination receptors in HP-induced germination and the effects of two different pressure levels on the germination and inactivation of spores. This study will potentially contribute to the effort to implement HP technology for spore inactivation in the food industry.

Keywords: Bacillus licheniformis; endospores; flow cytometry; germination receptor; high pressure; high-pressure processing; spore germination; spore inactivation.

FIG 1

Plate count data showing the effect of mHP treatment (150 MPa at 37°C) on Bacillus spores. ○, germinated spores; ×, inactivated spores. Each symbol represents an independent spore batch. (A) B. licheniformis strain MW3. (B) B. licheniformis strain NVH-1032. (C) B. subtilis strain PS832.

FIG 2

Contour-density plots of FCM data showing the effect of mHP treatment (150 MPa at 37°C) on the physiological state of B. licheniformis strain MW3 spores. Gating: R1, dormant spores; R2, germinated spores; R3, unknown state 1; R4, unknown state 2; and R5, inactivated spores. Contour-density plots depict the results from analyses performed on the CyFlow ML (∼200,000 events). Instrument-specific gates were constructed as described in “Flow cytometry analysis of mHP-treated B. licheniformis spores,” above.

FIG 3

Plate count data showing the effect of mHP treatment (150 MPa at 37°C) on spores of B. licheniformis ger double- and triple-null mutants. ○, germinated spores; ×, inactivated spores. Each symbol represents an individual spore batch. (A) NVH-1323 (ΔgerAA ΔgerKA-C). (B) NVH-1368 (ΔgerAA ΔyndD). (C) NVH-1376 (ΔgerKA-C ΔyndD). (D) NVH-1370 (ΔgerAA ΔgerKA-C ΔyndD).

FIG 4

Contour-density plots of FCM data showing the effect of mHP treatment (150 MPa at 37°C) on the physiological state of wild-type and ger double- and triple-null mutant spores of the B. licheniformis strain MW3. Gating: R1, dormant spores; R2, germinated spores; R3, unknown state 1; R4, unknown state 2; R5, inactivated spores. Contour-density plots depict the results from analyses of MW3 (wild type), NVH-1323 (ΔgerAA ΔgerKA-C), NVH-1368 (ΔgerAA ΔyndD), and NVH-1370 (ΔgerAA ΔgerKA-C ΔyndD) spores performed on the CyFlow ML (∼200,000 events). NVH-1376 (ΔgerKA-C ΔyndD) analyses were performed on the FACSCalibur (∼10,000 events). Instrument-specific gates constructed as described in “Flow cytometry analysis of mHP-treated B. licheniformis spores,” above.

FIG 5

Plate count data showing the effect of mHP treatment (150 MPa at 37°C) on B. licheniformis ger single-null mutant spores. ○, germinated spores; ×, inactivated spores. Each symbol represents an individual spore batch. (A) NVH-1307 (ΔgerAA), (B) NVH-1324 (ΔgerKA-C), and (C) NVH-1335 (ΔyndD).

FIG 6

Plate count data showing the effect of vHP treatment (550 MPa at 37°C) on Bacillus spores. ○, germinated spores; ×, inactivated spores. Each symbol represents an individual spore batch. (A) B. licheniformis strain MW3. (B) B. licheniformis strain NVH-1032. (C) B. subtilis strain PS832.

FIG 7

Plate count data showing the effect of vHP treatment (550 MPa at 60°C) on B. licheniformis strain MW3 spores. Each symbol represents an individual spore batch. ○, germinated spores; ×, inactivated spores.