Triggering Germination Represents a Novel Strategy to Enhance Killing of Clostridium difficile Spores

Nerandzic, Michelle M.; Donskey, Curtis J.

doi:10.1371/journal.pone.0012285

Cited by 53 publications

(40 citation statements)

References 23 publications

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“…While the inhibition or stimulation of spore germination in applied settings is somewhat off the focus of this review, these topics have attracted renewed interest in recent years because of the need for effective decontamination regimens for spores of organisms such as C. difficile and B. anthracis but without the concerns unique to the food industry. Thus, there are some recent reports of promising results in using a germination step prior to spore decontamination for promoting inactivation of B. anthracis and C. difficile spores (80)(81)(82), as well as enzymatic spore coat removal by lytic enzymes such as lysozyme to allow spore killing (83). Several compounds have also been identified that may be effective in inhibiting the germination or outgrowth of spores of organisms such as B. anthracis and C. difficile (84)(85)(86)(87)(88), and perhaps compounds analogous to these could be useful in applied settings.…”

Section: Major Unanswered Questions About Spore Germination By Nutrientsmentioning

confidence: 99%

Germination of Spores of Bacillus Species: What We Know and Do Not Know

2014

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Spores of Bacillus species can remain in their dormant and resistant states for years, but exposure to agents such as specific nutrients can cause spores' return to life within minutes in the process of germination. This process requires a number of sporespecific proteins, most of which are in or associated with the inner spore membrane (IM). These proteins include the (i) germinant receptors (GRs) that respond to nutrient germinants, (ii) GerD protein, which is essential for GR-dependent germination, (iii) SpoVA proteins that form a channel in spores' IM through which the spore core's huge depot of dipicolinic acid is released during germination, and (iv) cortex-lytic enzymes (CLEs) that degrade the large peptidoglycan cortex layer, allowing the spore core to take up much water and swell, thus completing spore germination. While much has been learned about nutrient germination, major questions remain unanswered, including the following. T he germination of the dormant and highly resistant spores formed by members of the Firmicutes phylum, in particular bacilli and clostridia, has long been of significant research interest for four major reasons, as follows: (i) fascinating regulatory systems allow such spores to remain in their dormant, resistant state for years and yet return to active growth in minutes; (ii) while spores of most Firmicutes do not cause disease, spores of some bacilli and clostridia cause food spoilage and food-borne disease, as well as human diseases like gas gangrene, tetanus, botulism, anthrax, and pseudomembranous colitis; (iii) spores of Bacillus anthracis are a major bioterrorism threat; and (iv) spores of Clostridium difficile are an emerging public health threat (1-3). Invariably, it is the germination of spores of these organisms that leads to disease or food spoilage, and yet, when spores germinate and lose their dormancy, they lose their extreme resistance properties and become relatively easy to kill. Germination is thus both an essential part of disease pathogenesis or food spoilage and a major weak spot in these organisms' life cycle. Consequently, there has long been applied interest in spore germination, with researchers seeking to understand this process better in order to either prevent spore germination or accelerate it and then kill the newly sensitive germinated spores. This review will concentrate on the germination of spores of bacilli, primarily because of the large amount of detailed knowledge of the germination of spores of these species compared to that of spores of clostridia. However, some of the differences and similarities between the germination of spores of these related genera will also be presented. Most discussion will focus on the germination of the model sporeformer, Bacillus subtilis, although the mechanisms of germination of B. subtilis spores appear to be similar for spores of other bacilli. The properties of the various proteins that are specifically involved in the germination process will not be discussed in great detail, since these have recently be...

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Section: Major Unanswered Questions About Spore Germination By Nutrientsmentioning

confidence: 99%

Germination of Spores of Bacillus Species: What We Know and Do Not Know

2014

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“…Spore germination is a necessary step in resuming vegetative growth; thus, the probability that the spores germinate in food products needs to be taken into account when assessing the risk of spoilage or outgrowth of pathogenic spore formers. Moreover, both inhibition of spore germination and induction of germination before inactivation treatments (which renders the spore sensitive) are used in the food industry to improve food safety (7)(8)(9)(10)(11)(12). However, none of these strategies is completely effective.…”

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confidence: 99%

Spore Heat Activation Requirements and Germination Responses Correlate with Sequences of Germinant Receptors and with the Presence of a Specific spoVA ^2mob Operon in Foodborne Strains of Bacillus subtilis

Krawczyk

Jong

Omony

et al. 2017

Appl Environ Microbiol

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Spore heat resistance, germination, and outgrowth are problematic bacterial properties compromising food safety and quality. Large interstrain variation in these properties makes prediction and control of spore behavior challenging. Highlevel heat resistance and slow germination of spores of some natural Bacillus subtilis isolates, encountered in foods, have been attributed to the occurrence of the spoVA 2mob operon carried on the Tn1546 transposon. In this study, we further investigate the correlation between the presence of this operon in high-level-heat-resistant spores and their germination efficiencies before and after exposure to various sublethal heat treatments (heat activation, or HA), which are known to significantly improve spore responses to nutrient germinants. We show that high-level-heat-resistant spores harboring spoVA 2mob required higher HA temperatures for efficient germination than spores lacking spoVA 2mob . The optimal spore HA requirements additionally depended on the nutrients used to trigger germination, L-alanine (L-Ala), or a mixture of L-asparagine, D-glucose, D-fructose, and K ϩ (AGFK). The distinct HA requirements of these two spore germination pathways are likely related to differences in properties of specific germinant receptors. Moreover, spores that germinated inefficiently in AGFK contained specific changes in sequences of the GerB and GerK germinant receptors, which are involved in this germination response. In contrast, no relation was found between transcription levels of main germination genes and spore germination phenotypes. The findings presented in this study have great implications for practices in the food industry, where heat treatments are commonly used to inactivate pathogenic and spoilage microbes, including bacterial spore formers.IMPORTANCE This study describes a strong variation in spore germination capacities and requirements for a heat activation treatment, i.e., an exposure to sublethal heat that increases spore responsiveness to nutrient germination triggers, among 17 strains of B. subtilis, including 9 isolates from spoiled food products. Spores of industrial foodborne isolates exhibited, on average, less efficient and slower germination responses and required more severe heat activation than spores from other sources. High heat activation requirements and inefficient, slow germination correlated with elevated resistance of spores to heat and with specific genetic features, indicating a common genetic basis of these three phenotypic traits. Clearly, interstrain variation and numerous factors that shape spore germination behavior challenge standardization of methods to recover highly heat-resistant spores from the environment and have an impact on the efficacy of preservation techniques used by the food industry to control spores.

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“…One strategy is to first germinate spores and then kill the much less resistant germinated spores-the strategy that has been called "germinate to exterminate" for decontaminating spores of Clostridium difficile (21)(22)(23) and spores of Bacillus anthracis. Indeed, spore germination is a crucial mechanistic step in the inactivation of spores by HP processing, which uses conditions of elevated temperature (90 to 121°C) and pressure (Ն600 MPa) to greatly reduce spore loads in certain foodstuffs (24)(25)(26)(27), such as baby food purées (28).…”

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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

Appl Environ Microbiol

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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...

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Triggering Germination Represents a Novel Strategy to Enhance Killing of Clostridium difficile Spores

Cited by 53 publications

References 23 publications

Germination of Spores of Bacillus Species: What We Know and Do Not Know

Germination of Spores of Bacillus Species: What We Know and Do Not Know

Spore Heat Activation Requirements and Germination Responses Correlate with Sequences of Germinant Receptors and with the Presence of a Specific spoVA ^2mob Operon in Foodborne Strains of Bacillus subtilis

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

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Triggering Germination Represents a Novel Strategy to Enhance Killing of Clostridium difficile Spores

Cited by 53 publications

References 23 publications

Germination of Spores of Bacillus Species: What We Know and Do Not Know

Germination of Spores of Bacillus Species: What We Know and Do Not Know

Spore Heat Activation Requirements and Germination Responses Correlate with Sequences of Germinant Receptors and with the Presence of a Specific spoVA 2mob Operon in Foodborne Strains of Bacillus subtilis

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

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Spore Heat Activation Requirements and Germination Responses Correlate with Sequences of Germinant Receptors and with the Presence of a Specific spoVA ^2mob Operon in Foodborne Strains of Bacillus subtilis