Stimulation of Germination of Unactivated Bacillus cereus Spores by Ammonia

Preston, Robert A.; Douthit, H. A.

doi:10.1099/00221287-130-5-1041

Cited by 14 publications

(25 citation statements)

References 16 publications

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“…We measured the germinability of the spores without a prior heat activation step, in order to prevent any possible interference by compounds released from the spores themselves (39). Furthermore, spores from YLLG tended to germinate spontaneously after a heat shock for 15 min at 75°C, which reflects their heat sensitivity.…”

Section: Resultsmentioning

confidence: 99%

Influence of Glutamate on Growth, Sporulation, and Spore Properties ofBacillus cereusATCC 14579 in Defined Medium

Vries

Atmadja

Hornstra

et al. 2005

Appl Environ Microbiol

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A chemically defined medium in combination with an airlift fermentor system was used to study the growth and sporulation of Bacillus cereus ATCC 14579. The medium contained six amino acids and lactate as the main carbon sources. The amino acids were depleted during exponential growth, while lactate was metabolized mainly during stationary phase. Two concentrations of glutamate were used: high (20 mM; YLHG) and low (2.5 mM; YLLG). Under both conditions, sporulation was complete and synchronous. Sporulation started and was completed while significant amounts of carbon and nitrogen sources were still present in the medium, indicating that starvation was not the trigger for sporulation. Analysis of amino acids and NH 4 ؉ in the culture supernatant showed that most of the nitrogen assimilated by the bacteria was taken up during sporulation. The consumption of glutamate depended on the initial concentration; in YLLG, all of the glutamate was used early during exponential growth, while in YLHG, almost all of the glutamate was used during sporulation. In YLLG, but not in YLHG, NH 4 ؉ was taken up by the cells during sporulation. The total amount of nitrogen used by the bacteria in YLLG was less than that used by the bacteria in YLHG, although a significant amount of NH 4 ؉ was present in the medium throughout sporulation. Despite these differences, growth and temporal expression of key sigma factors involved in sporulation were parallel, indicating that the genetic time frames of sporulation were similar under both conditions. Nevertheless, in YLHG, dipicolinic acid production started later and the spores were released from the mother cells much later than in YLLG. Notably, spores had a higher heat resistance when obtained after growth in YLHG than when obtained after growth in YLLG, and the spores germinated more rapidly and completely in response to inosine, L-alanine, and a combination of these two germinants.Bacillus cereus is a gram-positive, facultative anaerobic rodshaped bacterium able to form spores. It is a ubiquitous bacterium found in soil and in many raw and processed foods, such as rice, milk and dairy products, spices, and vegetables (8,12,20,44). Many strains of B. cereus are able to produce toxins and cause distinct types of food poisoning (19,31). Concerns over B. cereus contamination have increased over the past few years because of the rapidly expanding market of chilled foods that may be pasteurized but still contain viable spores (8,20,34). Spores from B. cereus can germinate and outgrow during storage, even at low temperatures (8,11,20). To address this increasing problem, major efforts focus on determining the causes of spore resistance and the mechanisms of germination.It has been well established that bacterial spore properties are affected by the conditions during sporulation (1,17,18,33,41). In most studies, spores are routinely produced from fortified agar or rich liquid media, which results in heterogeneous sporulation conditions for the individual cells. This prevents careful analysis of t...

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Section: Resultsmentioning

confidence: 99%

Influence of Glutamate on Growth, Sporulation, and Spore Properties ofBacillus cereusATCC 14579 in Defined Medium

Vries

Atmadja

Hornstra

et al. 2005

Appl Environ Microbiol

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“…Spores were extracted from the wipes into the BBT by sonication in a VWR 250T ultrasonic bath (VWR International, Tempe, AZ) for 15 min at sweeping frequencies between 38.5 and 40.5 kHz and an average power of 180 W. The extraction suspension was then heat treated at 65°C for 60 min to kill any bacterial vegetative cells and fungal spores which may be present in the suspension and to activate the Bacillus spores for rapid germination (9,14). While an estimated 5% of the viable spores were killed by the heat treatment (11), the same relative numbers of viable spores were killed in both the reference and sample suspensions.…”

Section: Methodsmentioning

confidence: 99%

Evaluation of a Wipe Surface Sample Method for Collection of Bacillus Spores from Nonporous Surfaces

Brown

Betty

Brockmann

et al. 2007

Appl Environ Microbiol

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Polyester-rayon blend wipes were evaluated for efficiency of extraction and recovery of powdered Bacillus atrophaeus spores from stainless steel and painted wallboard surfaces. Method limits of detection were also estimated for both surfaces. The observed mean efficiency of polyester-rayon blend wipe recovery from stainless steel was 0.35 with a standard deviation of ؎0.12, and for painted wallboard it was 0.29 with a standard deviation of ؎0. It is critical from a public health perspective that the information obtained is accurate and reproducible. The consequences of an inappropriate public health response founded on information garnered by an ineffective sample collection method or procedure has the potential for undesired social and economic impact. Well-developed and validated procedures for the collection and analysis of biological environmental samples are required to provide the necessary level of confidence in agent characterization information provided.Researchers and investigators are aware that the Centers for Disease Control and Prevention (CDC)-recommended procedures for the collection of B. anthracis spores by swab, wipe, and vacuum filtration collection methods (6) underestimate the number of spores on surfaces, and attempts are being made to address the knowledge gap (4,5,18,19). Additionally, a number of studies have been conducted to determine the efficiency of the swab sample collection method (1,2,3,7,8,10,17,18), but limited studies have been conducted to examine the efficiency of the wipe spore collection method (10, 13). A recent study conducted by Sanderson et al. (19) that compared B. anthracis surface sampling results obtained by swab, wipe, and vacuum filtration methods suggests that the wipe collection method outperforms the swab method, but no independent recovery efficiency was established for the wipe method.The objective of this study was to empirically evaluate the wipe surface sample collection method for recovery efficiency with a polyester-rayon blend wipe material and estimate limits of detection for selected nonporous surfaces seeded with dry deposited Bacillus atrophaeus spores. Additionally, a sonication extraction method was evaluated for effectiveness in removing viable spores from the selected wipe collection material. MATERIALS AND METHODSSpore matrix. The material used as the test agent for this study was a powdered matrix containing B. atrophaeus spores (ATCC 9372; formerly Bacillus subtilis var. niger and subsequently "Bacillus globigii") (12) and silicon dioxide particles obtained from the U.S. Army Dugway Proving Ground Life Science Division. The spore material was prepared by cultivating B. atrophaeus in tryptic soy broth (Difco, Detroit, MI) containing 3 mg/liter MnSO 4 (Fisher Scientific, Pittsburgh, PA). After 80 to 90% sporulation, the spore suspension was centrifuged to obtain a spore suspension containing approximately 20% solids. Dry spore material was then prepared from the unwashed spore suspension with a laboratory spray dryer. The spore material was...

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“…Before each time point, the plate was shaken for 3 s to prevent spore settling. To inhibit Alr, spores were preincubated with 1 mM D-cycloserine (DCS) for 15 min at 30°C before the addition of germinants (46). Several combination of DCS (0.1, 1, and 10 mM) and L-alanine (20,50, and 100 mM) were assessed, with 1 mM DCS and 50 mM L-Ala being the optimal combination.…”

Section: Creation Of B Cereus and B Anthracis Mutantsmentioning

confidence: 99%

A Novel Spore Protein, ExsM, Regulates Formation of the Exosporium in Bacillus cereus and Bacillus anthracis and Affects Spore Size and Shape

2010

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Bacillus cereus spores are assembled with a series of concentric layers that protect them from a wide range of environmental stresses. The outermost layer, or exosporium, is a bag-like structure that interacts with the environment and is composed of more than 20 proteins and glycoproteins. Here, we identified a new spore protein, ExsM, from a ␤-mercaptoethanol extract of B. cereus ATCC 4342 spores. Subcellular localization of an ExsM-green fluorescent protein (GFP) protein revealed a dynamic pattern of fluorescence that follows the site of formation of the exosporium around the forespore. Under scanning electron microscopy, exsM null mutant spores were smaller and rounder than wild-type spores, which had an extended exosporium (spore length for the wt, 2.40 ؎ 0.56 m, versus that for the exsM mutant, 1.66 ؎ 0.38 m [P < 0.001]). Thin-section electron microscopy revealed that exsM mutant spores were encased by a double-layer exosporium, both layers of which were composed of a basal layer and a hair-like nap. Mutant exsM spores were more resistant to lysozyme treatment and germinated with higher efficiency than wild-type spores, and they had a delay in outgrowth. Insertional mutagenesis of exsM in Bacillus anthracis ⌬Sterne resulted in a partial second exosporium and in smaller spores. In all, these findings suggest that ExsM plays a critical role in the formation of the exosporium.

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Stimulation of Germination of Unactivated Bacillus cereus Spores by Ammonia

Cited by 14 publications

References 16 publications

Influence of Glutamate on Growth, Sporulation, and Spore Properties ofBacillus cereusATCC 14579 in Defined Medium

Influence of Glutamate on Growth, Sporulation, and Spore Properties ofBacillus cereusATCC 14579 in Defined Medium

Evaluation of a Wipe Surface Sample Method for Collection of Bacillus Spores from Nonporous Surfaces

A Novel Spore Protein, ExsM, Regulates Formation of the Exosporium in Bacillus cereus and Bacillus anthracis and Affects Spore Size and Shape

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