High pressure germination of Bacillus subtilis spores with alterations in levels and types of germination proteins

This work analyzes the high-pressure (HP) germination of spores of the food-borne pathogen Clostridium perfringens (with inner membrane [IM] germinant receptors [GRs]) and the opportunistic pathogen Clostridium difficile (with no IM GRs), which has growing implications as an emerging food safety threat. In contrast to those of spores of Bacillus species, mechanisms of HP germination of clostridial spores have not been well studied. HP treatments trigger Bacillus spore germination through spores' IM GRs at ϳ150 MPa or through SpoVA channels for release of spores' dipicolinic acid (DPA) at >400 MPa, and DPA-less spores have lower wet heat resistance than dormant spores. We found that C. difficile spores exhibited no germination events upon 150-MPa treatment and were not heat sensitized. In contrast, 150-MPa-treated unactivated C. perfringens spores released DPA and became heat sensitive, although most spores did not complete germination by fully rehydrating the spore core, but this treatment of heat-activated spores led to almost complete germination and greater heat sensitization. Spores of both clostridial organisms released DPA during 550-MPa treatment, but C. difficile spores did not complete germination and remained heat resistant. Heat-activated 550-MPa-HP-treated C. perfringens spores germinated almost completely and became heat sensitive. However, unactivated 550-MPa-treated C. perfringens spores did not germinate completely and were less heat sensitive than spores that completed germination. Since C. difficile and C. perfringens spores use different mechanisms for sensing germinants, our results may allow refinement of HP methods for their inactivation in foods and other applications and may guide the development of commercially sterile low-acid foods. IMPORTANCESpores of various clostridial organisms cause human disease, sometimes due to food contamination by spores. Because of these spores' resistance to normal decontamination regimens, there is continued interest in ways to kill spores without compromising food quality. High hydrostatic pressure (HP) under appropriate conditions can inactivate bacterial spores. With growing use of HP for food pasteurization, advancement of HP for commercial production of sterile low-acid foods requires understanding of mechanisms of spores' interactions with HP. While much is known about HP germination and inactivation of spores of Bacillus species, how HP germinates and inactivates clostridial spores is less well understood. In this work we have tried to remedy this information deficit by examining germination of spores of Clostridium difficile and Clostridium perfringens by several HP and temperature levels. The results may give insight that could facilitate more efficient methods for spore eradication in food sterilization or pasteurization, biodecontamination, and health care.

Section: Methodsmentioning

confidence: 99%

Effects of High-Pressure Treatment on Spores of Clostridium Species

Doona

Feeherry

Setlow

et al. 2016

“…Spores at an OD 600 of either 1 (B. subtilis spores) or 10 (B. amyloliquefaciens spores) were germinated by treating samples in 1.5 ml of 25 mM K-HEPES buffer (pH 7.4) with an HP of 150 MPa at 37°C or 550 MPa at 50°C for various periods (0 to 5 min), essentially as described previously (42). B. amyloliquefaciens spores were HP treated at a higher spore concentration because these spores did not pellet as tightly as B. subtilis spores upon centrifugation, but rather tended to form thin films of spores on the side of the centrifuge tube that significantly decreased B. amyloliquefaciens spore recovery (see below).…”

Section: Methodsmentioning

confidence: 99%

The Effects of Heat Activation on Bacillus Spore Germination, with Nutrients or under High Pressure, with or without Various Germination Proteins

Luu

Cruz-Mora

Setlow

et al. 2015

bNutrient germination of spores of Bacillus species occurs through germinant receptors (GRs) in spores' inner membrane (IM) in a process stimulated by sublethal heat activation. Bacillus subtilis spores maximum germination rates via different GRs required different 75°C heat activation times: 15 min for L-valine germination via the GerA GR and 4 h for germination with the L-asparagine-glucose-fructose-K ؉ mixture via the GerB and GerK GRs, with GerK requiring the most heat activation. In some cases, optimal heat activation decreased nutrient concentrations for half-maximal germination rates. Germination of spores via various GRs by high pressure (HP) of 150 MPa exhibited heat activation requirements similar to those of nutrient germination, and the loss of the GerD protein, required for optimal GR function, did not eliminate heat activation requirements for maximal germination rates. These results are consistent with heat activation acting primarily on GRs. However, (i) heat activation had no effects on GR or GerD protein conformation, as probed by biotinylation by an external reagent; (ii) spores prepared at low and high temperatures that affect spores' IM properties exhibited large differences in heat activation requirements for nutrient germination; and (iii) spore germination by 550 MPa of HP was also affected by heat activation, but the effects were relatively GR independent. The last results are consistent with heat activation affecting spores' IM and only indirectly affecting GRs. The 150-and 550-MPa HP germinations of Bacillus amyloliquefaciens spores, a potential surrogate for Clostridium botulinum spores in HP treatments of foods, were also stimulated by heat activation. Spores of Bacillus species can remain dormant for long periods in the absence of suitable growth conditions (1, 2). However, if specific nutrients are sensed, spores can rapidly become metabolically active in the process of germination followed by outgrowth. The specific nutrients that trigger spore germination are termed germinants, and these molecules are sensed by germinant receptors (GRs) located in spores' inner membrane (IM). Bacillus subtilis spores have three functional GRs: GerA, which responds to L-alanine or L-valine alone, and GerB and GerK, which together are essential for germination with a mixture of L-asparagine, Dglucose, D-fructose, and K ϩ (termed AGFK), with all four components of the mixture required; neither GerB nor GerK alone triggers germination with any nutrient germinant (1, 3). There is also a variant of the GerB GR, termed GerB*, that responds to L-asparagine alone, although GerB* action can be stimulated by glucose via GerK (3). All GRs in B. subtilis spores appear to be located together in a small cluster in the IM termed the germinosome, and formation of this structure is dependent on the GerD protein, which is also in the IM (2, 4). Since gerD spores do not form a germinosome and exhibit extremely slow GR-dependent germination (4), germinosome formation may be essential for rapid GR-dependent germinatio...

“…Besides germination of spores in response to nutrients, which requires the presence of GRs in the spores (11,12), germination can also be induced by nonnutrient germinants (for instance, Ca-DPA) or by very high hydrostatic pressure (400 to 800 MPa) via mechanisms that are independent of GRs (11,12,49,50). A weak germination response of B. thermoamylovorans spores was observed for nutrient triggers (Fig.…”

Section: Quantification Of Sporesmentioning

confidence: 96%

Bacillus thermoamylovorans Spores with Very-High-Level Heat Resistance Germinate Poorly in Rich Medium despite the Presence of ger Clusters but Efficiently upon Exposure to Calcium-Dipicolinic Acid

Berendsen

Krawczyk

Klaus

et al. 2015

c High-level heat resistance of spores of Bacillus thermoamylovorans poses challenges to the food industry, as industrial sterilization processes may not inactivate such spores, resulting in food spoilage upon germination and outgrowth. In this study, the germination and heat resistance properties of spores of four food-spoiling isolates were determined. Flow cytometry counts of spores were much higher than their counts on rich medium (maximum, 5%). Microscopic analysis revealed inefficient nutrientinduced germination of spores of all four isolates despite the presence of most known germination-related genes, including two operons encoding nutrient germinant receptors (GRs), in their genomes. In contrast, exposure to nonnutrient germinant calcium-dipicolinic acid (Ca-DPA) resulted in efficient (50 to 98%) spore germination. All four strains harbored cwlJ and gerQ genes, which are known to be essential for Ca-DPA-induced germination in Bacillus subtilis. When determining spore survival upon heating, low viable counts can be due to spore inactivation and an inability to germinate. To dissect these two phenomena, the recoveries of spores upon heat treatment were determined on plates with and without preexposure to Ca-DPA. The high-level heat resistance of spores as observed in this study (D 120°C , 1.9 ؎ 0.2 and 1.3 ؎ 0.1 min; z value, 12.2 ؎ 1.8°C) is in line with survival of sterilization processes in the food industry. The recovery of B. thermoamylovorans spores can be improved via nonnutrient germination, thereby avoiding gross underestimation of their levels in food ingredients. Bacillus endospores (spores) are widely present in nature and may contaminate food ingredients and food products. Due to the intrinsic stability of spores, which allows them to withstand environmental insults, sufficient inactivation of spores in commercially sterile food products is a major challenge for the food industry (1-4).Bacillus thermoamylovorans produces spores with high-level heat resistance (3), and the spores are known to survive industrial food sterilization processes. The organism is facultatively anaerobic and has the ability to grow at temperatures between 40°C and 58°C (5, 6). In our experience, strains of B. thermoamylovorans are able to grow at 37°C but not at 30°C. The organism was first described as a nonsporogenous species (5, 7), but in an amended species description the formation of spores was reported (8). The occurrence of B. thermoamylovorans in a gelatin production plant and at dairy farms has been reported (3, 9). The genome sequence of one non-food-related B. thermoamylovorans strain from a biogas plant was published recently (6). Overall, the species has not been well characterized, and little is known about the spore properties that are important for control in foods, including spore resistance to various processing conditions and germination of spores that survive.When spores exit dormancy via germination, food spoilage can occur upon outgrowth. These processes have been well studied in Bacillus subtil...