Assessment of Heat Resistance of Bacterial Spores from Food Product Isolates by Fluorescence Monitoring of Dipicolinic Acid Release

Kort, Remco; O'Brien, Andrea; Stokkum, Ivo H. M. van; Oomes, S.J.C.M.; Crielaard, Wim; Hellingwerf, Klaas J.; Brul, Stanley

doi:10.1128/aem.71.7.3556-3564.2005

Cited by 136 publications

(113 citation statements)

References 46 publications

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“…The results elucidated that the spores were not sensitive to 80 C and 90 C, and survived even up to 100 C, although the survival rate decreased with the increase of the heating time. Furthermore, the spores showed strong resistances to low pH and high bile concentration, which were also reported by many previous studies [4,29,51]. Thus, the isolates got the accession to feed additives.…”

Section: Discussionsupporting

confidence: 80%

Identification and characterization of Bacillus subtilis from grass carp (Ctenopharynodon idellus) for use as probiotic additives in aquatic feed

Guo

Chen

Kong

et al. 2016

Fish & Shellfish Immunology

118

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Section: Discussionsupporting

confidence: 80%

Identification and characterization of Bacillus subtilis from grass carp (Ctenopharynodon idellus) for use as probiotic additives in aquatic feed

Guo

Chen

Kong

et al. 2016

Fish & Shellfish Immunology

118

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“…Although the use of absorbance to monitor germination is convenient, it is not a quantitative measure of CaDPA release and can include cortex hydrolysis at later time points (34). Thus, to provide a quantitative measure of DPA release, we monitored CaDPA release in real-time using an assay based on terbium fluorescence (31,35). C. difficile spores released CaDPA in the presence of taurocholic acid and glycine but not in response to taurocholic acid alone (Fig.…”

Section: Resultsmentioning

confidence: 99%

Spore Cortex Hydrolysis Precedes Dipicolinic Acid Release during Clostridium difficile Spore Germination

2015

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Bacterial spore germination is a process whereby a dormant spore returns to active, vegetative growth, and this process has largely been studied in the model organism Bacillus subtilis. In B. subtilis, the initiation of germinant receptor-mediated spore germination is divided into two genetically separable stages. Stage I is characterized by the release of dipicolinic acid (DPA) from the spore core. Stage II is characterized by cortex degradation, and stage II is activated by the DPA released during stage I. Thus, DPA release precedes cortex hydrolysis during B. subtilis spore germination. Here, we investigated the timing of DPA release and cortex hydrolysis during Clostridium difficile spore germination and found that cortex hydrolysis precedes DPA release. Inactivation of either the bile acid germinant receptor, cspC, or the cortex hydrolase, sleC, prevented both cortex hydrolysis and DPA release. Because both cortex hydrolysis and DPA release during C. difficile spore germination are dependent on the presence of the germinant receptor and the cortex hydrolase, the release of DPA from the core may rely on the osmotic swelling of the core upon cortex hydrolysis. These results have implications for the hypothesized glycine receptor and suggest that the initiation of germinant receptor-mediated C. difficile spore germination proceeds through a novel germination pathway. IMPORTANCEClostridium difficile infects antibiotic-treated hosts and spreads between hosts as a dormant spore. In a host, spores germinate to the vegetative form that produces the toxins necessary for disease. C. difficile spore germination is stimulated by certain bile acids and glycine. We recently identified the bile acid germinant receptor as the germination-specific, protease-like CspC. CspC is likely cortex localized, where it can transmit the bile acid signal to the cortex hydrolase, SleC. Due to the differences in location of CspC compared to the Bacillus subtilis germinant receptors, we hypothesized that there are fundamental differences in the germination processes between the model organism and C. difficile. We found that C. difficile spore germination proceeds through a novel pathway. Clostridium difficile (a Gram-positive, spore-forming, strict anaerobe) has become a significant threat to antibiotic-treated or immunocompromised hosts. Antibiotics are known to disrupt the colonic microbiota, and this perturbation permits C. difficile colonization (1, 2). Due to the strict anaerobic nature of C. difficile cells, spores are generally thought to be the infectious agent (only the spore can survive for extended periods of time in the aerobic environment outside a host) (3, 4). Because the spore form is noninfectious, spores must germinate to actively growing bacteria which initiate infection (5, 6). Thus, germination by C. difficile spores represents one of earliest steps in the pathogenesis of this organism.Endospore germination has been extensively studied in Bacillus sp. and, more recently, in clostridia (7,8). In the spore core,...

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“…The release of dipicolinic acid in the medium during spore germination was monitored by using the terbium fluorescence assay described previously (29).…”

Section: Methodsmentioning

confidence: 99%

Analysis of Temporal Gene Expression duringBacillus subtilisSpore Germination and Outgrowth

et al. 2007

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Bacillus subtilis forms dormant spores upon nutrient depletion. Under favorable environmental conditions, the spore breaks its dormancy and resumes growth in a process called spore germination and outgrowth. To elucidate the physiological processes that occur during the transition of the dormant spore to an actively growing vegetative cell, we studied this process in a time-dependent manner by a combination of microscopy, analysis of extracellular metabolites, and a genome-wide analysis of transcription. The results indicate the presence of abundant levels of late sporulation transcripts in dormant spores. In addition, the results suggest the existence of a complex and well-regulated spore outgrowth program, involving the temporal expression of at least 30% of the B. subtilis genome.A number of bacterial species such as bacilli and clostridia have the ability to form dormant spores. The spore has a specialized and complex structure, enabling the organism to survive for a long time under harsh environmental conditions and in the absence of nutrients. When triggered by specific nutrients, the spore is capable of breaking dormancy (germination) and initiating vegetative growth (34, 52). The Bacillus subtilis spore is composed of a dehydrated central compartment (the spore core) engulfed by two protective outer layers: a thick spore-specific peptidoglycan layer known as the spore cortex and a multilayered protein structure known as the coat (12).The process of endospore formation in Bacillus subtilis has been studied in great detail. Studies have revealed a highly ordered and strictly regulated program ensuring the correct coordination of various aspects of the sporulation process, such as asymmetric cell division, prespore engulfment, spore maturation, and mother cell lysis (22). The sporulation program involves the timed activation of several mother cell and forespore compartment-and sporulation stage-dependent RNA polymerase sigma factors that transcribe specific sets of sporulation genes. Eventually, the sporulation program results in the lysis of the mother cell and the release of a dormant spore (22).The process of spore germination and outgrowth has been studies in less detail. Spore germination is initiated when the spore senses the appropriate trigger molecules, often simple sugars and/or amino acids. The germinant molecules are sensed by germination receptors. This, by an unknown mechanism, leads to an irreversible commitment of a spore to germination. The germinating spore initially releases Zn 2ϩ and H ϩ (65). Simultaneously (and probably as a consequence), the pH of the spore core rises from 6.5 to 7.7. In a second stage, the germinating spore releases the spore core's large supply of dipicolinic acid (pyridine-2,6-dicarboxylic acid), and the spore core is rehydrated. Subsequently, cortex lytic enzymes are activated and the protective spore peptidoglycan cortex is degraded. This enables the germinating spore to hydrate the spore core further and to swell. These germination events coincide with a loss of...

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Assessment of Heat Resistance of Bacterial Spores from Food Product Isolates by Fluorescence Monitoring of Dipicolinic Acid Release

Cited by 136 publications

References 46 publications

Identification and characterization of Bacillus subtilis from grass carp (Ctenopharynodon idellus) for use as probiotic additives in aquatic feed

Identification and characterization of Bacillus subtilis from grass carp (Ctenopharynodon idellus) for use as probiotic additives in aquatic feed

Spore Cortex Hydrolysis Precedes Dipicolinic Acid Release during Clostridium difficile Spore Germination

Analysis of Temporal Gene Expression duringBacillus subtilisSpore Germination and Outgrowth

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