Heat activation and inactivation of bacterial spores. Is there an overlap?

Heat activation at a sublethal temperature is widely applied to promote Bacillus species spore germination. This treatment also has potential to be employed in food processing to eliminate undesired bacterial spores by enhancing their germination, and then inactivating the less heat resistant germinated spores at a milder temperature. However, incorrect heat treatment could also generate heat damage in spores, and lead to more heterogeneous spore germination. Here, the heat activation and heat damage profile of Bacillus subtilis spores was determined by testing spore germination and outgrowth at both population and single spore levels. The heat treatments used were 40-80°C, and for 0-300 min. The results were as follows. 1) Heat activation at 40-70°C promoted L-valine and L-asparagine-glucose-fructose-potassium (AGFK) induced germination in a time dependent manner. 2) The optimal heat activation temperatures for AGFK and L-valine germination via the GerB plus GerK or GerA germinant receptors were 65 and 50-65°C, respectively. 3) Heat inactivation of dormant spores appeared at 70°C, and the heat damage of molecules essential for germination and growth began at 70 and 65°C, respectively. 4) Heat treatment at 75°C resulted in both activation of germination and damage to the germination apparatus, and 80°C treatment caused more pronounced heat damage. 5) For the spores that should withstand adverse environmental temperatures in nature, heat activation seems functional for a subsequent optimal germination process, while heat damage affected both germination and outgrowth. Importance Bacterial spores are thermal resistant structures that can thus survive preservation strategies and revive through the process of spore germination. The more heat resistant spores are the more heterogeneous they germinate upon adding germinants. Upon germination spores can cause food spoilage and cause food intoxication. Here we provide new information on both heat activation and inactivation regimes and their effects on the (heterogeneity of) spore germination.

Biological Control of Fusarium culmorum, Fusarium graminearum and Fusarium poae by Antagonistic Yeasts

The genus Fusarium is considered to be one of the most pathogenic, phytotoxic and toxin-producing group of microorganisms in the world. Plants infected by these fungi are characterized by a reduced consumer and commercial value, mainly due to the contamination of crops with mycotoxins. Therefore, effective methods of reducing fungi of the genus Fusarium must be implemented already in the field before harvesting, especially with alternative methods to pesticides such as biocontrol. In this study we identified yeasts that inhibit the growth of the pathogenic fungi Fusarium culmorum, F. graminearum and F. poae. Tested yeasts came from different culture collections, or were obtained from organic and conventional cereals. The greater number of yeast isolates from organic cereals showed antagonistic activity against fungi of the genus Fusarium compared to isolates from the conventional cultivation system. Cryptococcus carnescens (E22) isolated from organic wheat was the only isolate that limited the mycelial growth of all three tested fungi and was the best antagonist against F. poae. Selected yeasts showed various mechanisms of action against fungi, including competition for nutrients and space, production of volatile metabolites, reduction of spore germination, production of siderophores or production of extracellular lytic enzymes: chitinase and β-1,3-glucanase. Of all the investigated mechanisms of yeast antagonism against Fusarium, competition for nutrients and the ability to inhibit spore germination prevailed.

Visualization of SpoVAEa protein dynamics in dormant spores of Bacillus cereus and dynamic changes in their germinosomes and SpoVAEa during germination

Bacillus cereus spores, like most Bacillus spores, can survive for years depending on their specific structure, and germinate when their surroundings become suitable. Spore germination proteins play an important role in the initiation of germination. Because germinated spores lose the extreme resistance of the dormant state, more information related to the function of germination proteins could be useful to develop new strategies to control B. cereus spores. Prior work has shown that: i) the channel protein SpoVAEa exhibits high frequency movement in the outer leaflet of the inner membrane (IM) in dormant spores of B. subtilis; ii) the dynamics of germinosome formation in developing spores of B. cereus indicate that the formation of germinosome foci is slower than foci formation of germinant receptor GerR and scaffold protein GerD. However, the dynamics of movement of SpoVAEa in B. cereus spores, and the complete behavior of the germinosome in germinated spores of B. cereus are still unclear. In this study, we found that the SpoVAEa fluorescent foci in dormant spores of B. cereus redistribute at a lower frequency than in B. subtilis, and likely colocalize with GerD in dormant spores. Our results further indicate that: i) overexpression of GerR(A-C-B)-SGFP2 and SpoVAEa-SGFP2 with GerD-mScarlet-I from a plasmid leads to more heterogeneity and lower efficiency of spore germination in B. cereus; ii), germinosome foci composed of GerR(A-C-B)-SGFP2 and GerD-mScarlet-I were lost prior to the phase transition in germination; and iii) GerD-mScarlet-I foci spread out but continued to exist beyond the phase transition of B. cereus spores.

Chemical Profile, Antimicrobial and Antioxidant Activity Assessment of the Crude Extract and Its Main Flavonoids from Tartary Buckwheat Sprouts

The purpose of this study was to investigate the major flavonoids content and bioactivities of Tartary buckwheat sprouts. The crude methanol extract (ME) of Tartary buckwheat sprouts was abundant in flavonoids, and six major flavonoids, including isoorientin, vitexin, isovitexin, rutin, quercetin, and kaemferol were successfully determined from the sprouts by the high-performance liquid chromatography (HPLC) method. Generally, the flavonoid content of buckwheat sprouts was in the order of rutin > quercetin > isovitexin > vitexin> isoorientin > kaemferol. The highest rutin content of the ME and sprout cultures was 89.81 mg/g and 31.50 mg/g, respectively. Antibacterial activity results indicated the ME displayed notable inhibitory activity against the five tested bacteria, and its minimum inhibitory concentration (MIC) values ranged from 0.8 mg/mL to 3.2 mg/mL. Among the six flavonoids, quercetin was the most active compound, which exhibited strong activity against all tested bacteria except for E. coli and S. epidermidis, with its MIC values ranging from 0.2 mg/mL to 0.4 mg/mL. For the antifungal activity assay, the ME of Tartary buckwheat sprouts and four flavonoids could significantly inhibit the spore germination of two pathogenic fungi, and their inhibitory efficiency was concentration dependent. Quercetin was the most active one, which significantly inhibited the spore germination of F. oxysporum f. sp. vasinfectum and F. oxysporum f. sp. cucumerinum, and its median effective inhibitory concentration (IC50) value was 42.36 and 32.85 µg/mL, respectively. The antioxidant activity results showed that quercetin, kaemferol, and rutin displayed excellent antioxidant activity in the DPPH radical scavenging test, and their IC50 value was calculated as 5.60, 16.23, and 27.95 µg/mL, respectively. This is the first report on the antimicrobial activity of the crude extract of Tartary buckwheat sprouts. These results indicated that the methanol extract of Tartary buckwheat sprouts could be used as a potential antimicrobial or antioxidant agent in the future.

Genetic suppression meets structure prediction: probing a spore germination receptor complex

Despite the thousands of spore germinant receptor operons identified in genomes of Bacilli and Clostridia, understanding how the three essential receptor components act as a signal transduction machine in germination remains limited. The paper by Amon et al in this issue uses the classical genetic approach of suppression to define a region of likely interaction between the GerAA and GerAB proteins: it provides a first glimpse into potential events within the receptor complex.

In Vitro and In Vivo Antifungal Activities of Nine Commercial Essential Oils against Brown Rot in Apples

After harvest, numerous plant pathogenic fungi can infect fresh fruits during transit and storage. Although synthetic fungicides are often used to manage postharvest fruit diseases, their application may lead to problems such as the development of fungicide resistance and residues on fruits. In the present study, the antifungal potential of nine commercial essential oils (EOs) extracted from Eucalyptus radiata ssp. radiata, Mentha pulegium, Rosmarinus officinalis, Origanum compactum, Lavandula angustifolia, Syzygium aromaticum, Thymus vulgaris, Citrus aurantium, and Citrus sinensis were tested against the apple brown rot fungi Monilinia laxa and Monilinia fructigena at different concentrations in vitro (against mycelial growth and spore germination) and in vivo (on detached apple fruit and in semi-commercial postharvest conditions). In addition, fruit quality parameters were evaluated and the composition of the EOs was characterized by Fourier transform infrared (FT-IR) spectroscopy. In vitro results showed significant antifungal activity of all tested EOs on both fungal species. EOs from S. aromaticum were the most effective, whereby inhibition percentages ranged from 64.0 to 94.7% against M. laxa and from 63.9 to 94.4% against M. fructigena for the concentrations 12.5 and 100 µL/mL, respectively, with an EC50 of 6.74 µL/mL for M. laxa and 10.1 µL/mL for M. fructigena. The higher concentrations tested of S. aromaticum, T. vulgaris, C. aurantium, and C. sinensis EOs significantly reduced spore germination, brown rot incidence, and lesion diameter. Evaluation of the treatments during storage for 20 days at 4 °C on apple fruit quality parameters demonstrated the preservation of the fruit quality characteristics studied (weight loss, total soluble solids, titratable acidity, firmness, and maturity index). FT-IR spectra obtained from all tested EO samples presented characteristic peaks and a high diversity of functional groups such as O–H groups, C–H bonds, and C–C stretching. The EOs examined here may have the potential for controlling postharvest fungal diseases of fruit such as brown rot.

Nutrient L-Alanine-Induced Germination of Bacillus Improves Proliferation of Spores and Exerts Probiotic Effects in vitro and in vivo

As alternatives to antibiotics in feed, probiotic Bacillus carries multiple advantages in animal production. Spores undergo strain-related germination in the gastrointestinal tract, but it is still unknown whether the probiotic function of the Bacillus depends on the germination of spores in vivo. In this study, based on 14 potential probiotic Bacillus strains from fermented food and feed, we detected the germination response of these Bacillus spores in relation to different germinating agents. The results showed the germination response was strain-specific and germinant-related, and nutrient germinant L-alanine significantly promoted the growth of strains with germination potential. Two strains of Bacillus subtilis, S-2 and 312, with or without a high spore germination response to L-alanine, were selected to study their morphological and genic differences induced by L-alanine through transmission electron microscopy and comparative transcriptomics analysis. Consequently, after L-alanine treatment, the gray phase was largely increased under microscopy, and the expression of the germination response genes was significantly up-regulated in the B. subtilis S-2 spores compared to the B. subtilis 312 spores (p < 0.05). The protective effect of L-alanine-induced spore germination of the two strains was comparatively investigated both in the IPEC-J2 cell model and a Sprague–Dawley (SD) rat model challenged by enterotoxigenic Escherichia coli K99. The result indicated that L-alanine helped B. subtilis S-2 spores, but not 312 spores, to decrease inflammatory factors (IL-6, IL-8, IL-1 β, TNF-α; p < 0.05) and promote the expression of occludin in IPEC-J2 cells. Besides, supplement with L-alanine-treated B. subtilis S-2 spores significantly improved the growth of the SD rats, alleviated histopathological GIT lesions, and improved the ratio of jejunal villus length to crypt depth in comparison to the B. subtilis S-2 spores alone (p < 0.05). Improved species diversity and abundance of fecal microbiota were only observed in the group with L-alanine-treated S-2 spores (p < 0.05). The study demonstrates L-alanine works well as a probiotic Bacillus adjuvant in improving intestinal health, and it also provides a solution for the practical and accurate regulation of their use as antibiotic alternatives in animal production.

Heat activation and inactivation of bacterial spores. Is there an overlap?

Heat activation at a sublethal temperature is widely applied to promote Bacillus species spore germination. This treatment also has potential to be employed in food processing to eliminate undesired bacterial spores by enhancing their germination, and then inactivating the less heat resistant germinated spores at a milder temperature. However, incorrect heat treatment could also generate heat damage in spores, and lead to more heterogeneous spore germination. Here, the heat activation and heat damage profile of Bacillus subtilis spores was determined by testing spore germination and outgrowth at both population and single spore levels. The heat treatments used were 40-80 degrees Celcius, and for 0-300 min. The results were as follows. 1) Heat activation at 40-70 degrees Celcius promoted L-valine and L-asparagine-glucose-fructose-potassium (AGFK) induced germination in a time dependent manner. 2) The optimal heat activation temperatures for AGFK and L-valine germination via the GerB plus GerK or GerA germinant receptors were 65 and 50-65 degrees Celcius, respectively. 3) Heat inactivation of dormant spores appeared at 70 degrees Celcius, and the heat damage of molecules essential for germination and growth began at 70 and 65 degrees Celcius, respectively. 4) Heat treatment at 75 degrees Celcius resulted in both activation of germination and damage to the germination apparatus, and 80 degrees Celcius treatment caused more pronounced heat damage. 5) For the spores that should withstand adverse environmental temperatures in nature, heat activation seems functional for a subsequent optimal germination process, while heat damage affected both germination and outgrowth.

Evaluation of Fungicides and Bioagents against Fusarium proliferatum under In vitro by Spore Germination Inhibition Technique

Background: Bottle gourd is a cucurbitaceous vegetable of culinary and medicinal importance cultivated in various tropical and sub-tropical regions of world. This crop is exposed to a wide variety of seed and soil mycoflora, out of which Fusarium proliferatum is utmost important as far as seed germination, viability and seedling vigour are concerned. Methods: Study was taken up to evaluate different fungicides and bioagents for their efficacy against the fungus Fusarium proliferatum under in vitro through spore germination inhibition technique. Result: Spore germination inhibition of 86.00%, 85.00% and 81.33% was recorded with hexaconazole (5% SC) @ 0.2% (C3), mancozeb (75% WP) @ 0.3% (C3) and Pseudomonas fluorescens (1% WP) @ 2% (C3), respectively. The inhibition in spore germination by mancozeb (75% WP) and Pseudomonas fluorescens (1% WP) was upto 77.33% and it was 61.78% and 67.33% in treatments involving carbendazim (50% WP) and Trichoderma harzianum (1% WP) that could be exploited to devise integrated approach for disease management.