Spore Outgrowth Research Articles

The Indigenous Volatile Inhibitor 2-Methyl-2-butene Impacts Biofilm Formation and Interspecies Interaction of the Pathogenic Mucorale Rhizopus arrhizus.

2-Methyl-2-butene has recently been reported to be a quorum-based volatile self-inhibitor of spore germination and growth in pathogenic Mucorale Rhizopus arrhizus. The present study aimed to elucidate if this compound can influence R. arrhizus biofilm formation and interspecies interaction. The compound was found to significantly decrease R. arrhizus biofilm formation (p < 0.001), with nearly 25% and 50% lesser biomass in the biofilms cultured with exposure to 4 and 32µg/ml of 2-methyl-2-butene, respectively. The growth of pre-formed biofilms was also impacted, albeit to a lesser extent. Additionally, 2-methyl-2-butene was found to self-limit R. arrhizus growth during interspecies interaction with Staphylococcus aureus and was detected at a substantially greater concentration in the headspace of co-cultures (2338.75µg/ml) compared with monocultures (69.52µg/ml). Some of the C5 derivatives of this compound (3-methyl-1-butanol, 2-methyl-2-butanol, and 3-methyl-1-butyne) were also observed to partially mimic its action, such as inhibition of spore germination, but did not impact R. arrhizus biofilm formation. Finally, the treated R. arrhizus displayed changes in fungal morphology suggestive of cytoskeletal alterations, such as filopodia formation, blebs, increased longitudinal folds and/or corrugations, and finger-like and sheet-like surface protrusions, depending upon the concentration of the compound(s) and the planktonic or biofilm growth mode.

SIGNALING PATHWAYS OF CONIDIAL GERMINATION AND GROWTH OF BOTRYTIS CINEREA: HOST DETECTION, PATHOGENESIS ON VITIS VINIFERA AND PREFERENCE FOR WINE GRAPES

Botrytis cinerea is a phytopathogenic filamentous fungus that infects and causes severe damages to numerous crops. However, the European grapevine ( Vitis vinifera ) remains the major host for the pathogen. Botrytis infects mainly ripe grapes (usually over 10 degrees Baume) unless primary infections by other fungi, or infestations by viticultural insect pests, or mechanical damages have occurred. The disease is commonly known in viticulture as “Botrytis bunch rot” or “grey mold” and can spread rapidly causing devastating losses on the field and post-harvest. Regardless of its generalist action, B. cinerea must develop strategies to recognise and invade its host. The fungus responds to physical and chemical stimuli, emanated from the environment to activate spore germination and growth. Once a signal from the external environment meets a target molecule of the cell, the signaling process begins. The target molecule is a protein that acts as a receptor. The physical and chemical signals that stimulate conidial germination depend on factors such as surface hardness, surface hydrophobicity, carbon sources and nutrient-rich substrates. Among sugars, fructose is the best growth inducer of B. cinerea. Heterotrimeric guanine nucleotide binding proteins (G-proteins) are involved in the regulation of a range of functions including germination, growth and host detection. G-proteins consist of three subunits, α, β and γ. Two of these subunits attach to the cell membrane with small lipid tails. At rest, unit α is bound to Guanosine Diphosphate (GDP) and the G-protein is inactive. When an extracellular ligand binds to the receptor, the latter interacts with the G-protein and activates it, forcing the α subunit to excrete the bound GDP and replace it with Guanosine Triphosphate (GTP). In addition to signals that trigger spore germination, pathogenesis is also influenced by several biotic and abiotic factors. In general terms, interactions between pathogen, its potential host and the environment affect level, speed and nature of the infection and must be substantially considered in integrated control strategies.

The Selenophosphate Synthetase Gene, selD, Is Important for Clostridioides difficile Physiology.

The endospore-forming pathogen Clostridioides difficile is the leading cause of antibiotic-associated diarrhea and is a significant burden on the community and health care. C. difficile, like all forms of life, incorporates selenium into proteins through a selenocysteine synthesis pathway. The known selenoproteins in C. difficile are involved in a metabolic process that uses amino acids as the sole carbon and nitrogen source (Stickland metabolism). The Stickland metabolic pathway requires the use of two selenium-containing reductases. In this study, we built upon our initial characterization of the CRISPR-Cas9-generated selD mutant by creating a CRISPR-Cas9-mediated restoration of the selD gene at the native locus. Here, we use these CRISPR-generated strains to analyze the importance of selenium-containing proteins on C. difficile physiology. SelD is the first enzyme in the pathway for selenoprotein synthesis, and we found that multiple aspects of C. difficile physiology were affected (e.g., growth, sporulation, and outgrowth of a vegetative cell post-spore germination). Using transcriptome sequencing (RNA-seq), we identified multiple candidate genes which likely aid the cell in overcoming the global loss of selenoproteins to grow in medium which is favorable for using Stickland metabolism. Our results suggest that the absence of selenophosphate (i.e., selenoprotein synthesis) leads to alterations to C. difficile physiology so that NAD+ can be regenerated by other pathways. IMPORTANCE C. difficile is a Gram-positive, anaerobic gut pathogen which infects thousands of individuals each year. In order to stop the C. difficile life cycle, other nonantibiotic treatment options are in urgent need of development. Toward this goal, we find that a metabolic process used by only a small fraction of the microbiota is important for C. difficile physiology: Stickland metabolism. Here, we use our CRISPR-Cas9 system to "knock in" a copy of the selD gene into the deletion strain to restore selD at its native locus. Our findings support the hypothesis that selenium-containing proteins are important for several aspects of C. difficile physiology, from vegetative growth to spore formation and outgrowth postgermination.

Viable and not viable spore concentrations in National Gallery of Umbria (Italy)

The conservation actions towards artworks holding a common patrimony for the community are of primary importance, but also those related to their "container" as museums, libraries or archives are to consider. Fungal spores and bacteria carried by air flows to the artwork surface can colonize it causing biodeterioration through physical and/or chemical alterations of the materials with the irreversible loss of their value. The quality control of the indoor air surrounding the historic building is essential, as well as for the protection and conservation of the artwork, also for the protection of the health of operators and visitors. The aim of this study was to monitor airborne fungal particles, through volumetric spore traps, for improving the knowledge about the conservation and protection of artworks in the museum environment analysing the principal relationships between indoor environmental conditions and potentially biodeteriogen fungal spore growth. The evidence of no significant relationships between spore concentrations and environmental conditions recorded inside the different expositive rooms testified the regular and correct maintenance of the air conditioning system inside the considered building (National Gallery of Umbria, central Italy). Moreover, in a specific museum room a significant spore concentration decreasing trend was recorded mainly due to a structural modification in the same building.

Ultraviolet radiation stress response of haploid and diploid spores of Mazzaella laminarioides: Do bio-optical traits matter?

Early stages of macroalgae are known to be more vulnerable to changes in the abiotic environment than their adult phases. However, in algal groups attaining intricate life-cycles, the ecophysiological responses of haploid and diploid propagules have not been sufficiently studied. In the present study, stress responses of haploid tetraspores and diploid carpospores of sub-Antarctic red alga Mazzaella laminarioides exposed to ultraviolet (UV) radiation were studied. We tested the hypothesis that UV stress responses of two types of spores attaining similar size are determined by light use characteristics by comparing the photobiological and bio-optical traits. Fluorescence-based photosynthetic efficiency was measured during a 5- h exposure and after a 5- h recovery. Germination and growth of spores exposed to UV treatments were also assessed. Diploid carpospores exhibited higher chlorophyll a content and higher absorption in the UV band than tetraspores. Although the maximal quantum yield was higher in carpospores than tetraspores, no differences in light demands were detected. Exposure to photosynthetically active radiation (PAR) caused inhibition of photosynthetic activity only in carpospores (43%), while exposure to a combination of PAR + UV reduced the maximal quantum yield significantly in tetraspores (36%) and carpospores (60%). In both types of spores, a recovery from UV stress was observed, allowing their germination and further development. The different responses to radiation found in haploid and diploid propagules highlight the ecophysiological versatility of different life-cycle phases to cope with light stress factors ensuring finally successful recruitment and survival.

Visualization of fidaxomicin association with the exosporium layer of Clostridioides difficile spores

BackgroundFidaxomicin has novel pharmacologic effects on C. difficile spore formation including outgrowth inhibition and persistent spore attachment. However, the mechanism of fidaxomicin attachment on spores has not undergone rigorous microscopic studies. Materials & methodsFidaxomicin attachment to C. difficile spores of three distinct ribotypes and C. difficile mutant spores with inactivation of exosporium or spore-coat protein-coding genes were visualized using confocal microscopy with a fidaxomicin-bodipy compound (green fluorescence). The pharmacologic effect of the fidaxomicin-bodipy compound was determined. Confocal microscopy experiments included direct effect on C. difficile wild-type and mutant spores, effect of exosporium removal, and direct attachment to a comparator spore forming organism, Bacillus subtilis. ResultsThe fidaxomicin-bodipy compound MIC was 1 mg/L compared to 0.06 mg/L for unlabeled fidaxomicin, a 16-fold increase. Using confocal microscopy, the intracellular localization of fidaxomicin into vegetative C. difficile cells was observed consistent with its RNA polymerase mechanism of action and inhibited spore outgrowth. The fidaxomicin-bodipy compound was visualized outside of the core of C. difficile spores with no co-localization with the membrane staining dye FM4-64. Exosporium removal reduced fidaxomicin-bodipy association with C. difficile spores. Reduced fidaxomicin-bodipy was observed in C. difficile mutant spores for the spore surface proteins CdeC and CotE. ConclusionThis study visualized a direct attachment of fidaxomicin to C. difficile spores that was diminished with mutants of specific exosporium and spore coat proteins. These data provide advanced insight regarding the anti-spore properties of fidaxomicin.

Combining Celery Oleoresin, Limonene and Rhamnolipid as New Strategy to Control Endospore-Forming Bacillus cereus.

Foodborne diseases (FBD) are a great problem worldwide, leading millions of people to seek medical help and to significant economic losses for industry. Among the agents implicated in FDB is Bacillus cereus, a Gram-positive, toxigenic and endospore-forming bacterium. In this study, rhamnolipid (RL) biosurfactant, celery oleoresin (OR) and limonene (LN) were evaluated as bio-based alternatives for controlling the growth of vegetative cells and endospores of B. cereus. To address their antimicrobial activity, the compounds were tested separately and in combination. Results demonstrate that, when combined with RL, both OR and LN have lower minimal inhibitory concentration (MIC) values and increased endospore inhibition potential. A percentage of endospore inhibition from 73% to 98%, corresponding to a 2.8–3.6 log reduction in spore outgrowth, was observed. RL inhibited B. cereus growth and endospore germination and potentially enhanced the antimicrobial efficacy of the natural hydrophobic compounds tested.

Lecanicillium lecanii (Zimmermann) Zare & Gams, as an efficient biocontrol agent of tea thrips, Scirtothrips bispinosus Bagnall (Thysanoptera: Thripidae)

BackgroundContinuous and non-judicial application of synthetic insecticides to control the tea thrips, Scirtothrips bispinosus (Bagnall), one of the major tea pests in South India has led to certain undesirable issues in the ecosystem besides the presence of the pesticide residues in manufactured tea. Biological control agents are of immense importance in tea cultivation. The present study was designed to isolate Lecanicillium lecanii (Zimmermann) Zare & Gama from the field-collected cadavers of the insects/mites infected by fungi of tea growing areas of Anamallais (Tamil Nadu, South India), and to evaluate their field bio-efficacy against the tea thrips.ResultsLecanicillium lecanii isolated from the tea ecosystem had been formulated into a wettable powder (WP) formulation and evaluated against tea thrips under both laboratory and field conditions. Among the several media evaluated, the PDAY (Potato Dextrose Agar + 1% Yeast powder) was found to be the best suitable medium for the growth and germination of spores. Optimum conditions for the growth of L. lecanii were found in PDAY medium at the pH 6-7, temperature 25-30°C and 90-95% RH. Exposure to UV light for more than 30 min significantly inhibited the growth of the fungus. Lecanicillium lecanii at (1 × 107 spore/ha) was found significantly effective against thrips. Fungal development index (FDI) of L. lecanii + jaggery significantly differed than other treatments. Lecanicillium lecanii at 1500g (1×107 conidia/ml) mixed in 400 l of water was effective against the tea thrips. Addition of equal amount of jaggery with L. lecanii wettable powder in the tank mixture could increase the efficacy of the mycopesticide against tea thrips.ConclusionThe powder formulation of L. lecanii was found safer to natural enemies present in the tea ecosystem. After fulfilling the requirements for its registration and label claim on tea, this strain of L. lecanii could be commercialized for the benefit of the tea industry for the management of tea thrips in an eco-friendly manner.

Analysis of tRNA Cys processing under salt stress in Bacillus subtilis spore outgrowth using RNA sequencing data

Background: In spore-forming bacteria, the molecular mechanisms of accumulation of transfer RNA (tRNA) during sporulation must be a priority as tRNAs play an essential role in protein synthesis during spore germination and outgrowth. However, tRNA processing has not been extensively studied in these conditions, and knowledge of these mechanisms is important to understand long-term stress survival. Methods:To gain further insight into tRNA processing during spore germination and outgrowth, the expression of the single copy tRNA Cys gene was analyzed in the presence and absence of 1.2 M NaCl in Bacillus subtilis using RNA-Seq data obtained from the Gene Expression Omnibus (GEO) database. The CLC Genomics work bench 12.0.2 (CLC Bio, Aarhus, Denmark, https://www.qiagenbioinformatics.com/) was used to analyze reads from the tRNA Cys gene. Results:The results show that spores store different populations of tRNA Cys-related molecules. One such population, representing 60% of total tRNA Cys, was composed of tRNA Cys fragments. Half of these fragments (3´-tRF) possessed CC, CCA or incorrect additions at the 3´end. tRNA Cys with correct CCA addition at the 3´end represented 23% of total tRNA Cys, while with CC addition represented 9% of the total and with incorrect addition represented 7%. While an accumulation of tRNA Cys precursors was induced by upregulation of the rrnD operon under the control of σ A -dependent promoters under both conditions investigated, salt stress produced only a modest effect on tRNA Cys expression and the accumulation of tRNA Cys related species. Conclusions:The results demonstrate that tRNA Cys molecules resident in spores undergo dynamic processing to produce functional molecules that may play an essential role during protein synthesis.

Antimicrobial effects of a bioactive glycolipid on spore-forming spoilage bacteria in milk

The growth of psychrotolerant aerobic spore-forming bacteria during refrigerated storage often results in the spoilage of fluid milk, leading to off-flavors and curdling. Because of their low toxicity, biodegradability, selectivity, and antimicrobial activity over a range of conditions, glycolipids are a novel and promising intervention to control undesirable microbes. The objective of this study was to determine the efficacy of a commercial glycolipid product to inhibit spore germination, spore outgrowth, and the growth of vegetative cells of Paenibacillus odorifer, Bacillus weihenstephanensis, and Viridibacillus arenosi, which are the predominant spore-forming spoilage bacteria in milk. For spore germination and outgrowth assays, varying concentrations (25-400 mg/L) of the glycolipid product were added to commercial UHT whole and skim milk inoculated with ∼4 log10 spores/mL of each bacteria and incubated at 30°C for 5 d. Inhibition of spore germination in inoculated UHT whole milk was only observed for V. arenosi, and only when glycolipid was added at 400 mg/L. However, concentrations of 400 and 200 mg/L markedly inhibited the outgrowth of vegetative cells from spores of P. odorifer and B. weihenstephanensis, respectively. No inhibition of spore germination or outgrowth was observed in inoculated UHT skim milk for any strain at the concentrations tested (25 and 50 mg/L). The effect of glycolipid addition on vegetative cell growth in UHT whole and skim milk when inoculated with ∼4 log10 cfu/mL of each bacteria was also determined over 21 d of storage at 7°C. Glycolipid addition at 50 mg/L was bactericidal against P. odorifer and B. weihenstephanensis in inoculated UHT skim milk through 21 d of storage, whereas 100 mg/L was needed for similar control of V. arenosi. Concentrations of 100 and 200 mg/L inhibited the growth of vegetative cells of B. weihenstephanensis and P. odorifer, respectively, in inoculated UHT whole milk, whereas 200 mg/L was also bactericidal to B. weihenstephanensis. Additional studies are necessary to identify effective concentrations for the inhibition of Viridibacillus spp. growth in whole milk beyond 7 d. Findings from this study demonstrate that natural glycolipids have the potential to inhibit the growth of dairy-spoilage bacteria and extend the shelf life of milk.

SER-287, AN INVESTIGATIONAL MICROBIOME THERAPEUTIC FOR PATIENTS WITH ACTIVE MILD-TO-MODERATE ULCERATIVE COLITIS, PRODUCES REMISSION ASSOCIATED METABOLITES, REDUCES INTESTINAL EPITHELIAL IL-8 SECRETION AND PROTECTS EPITHELIAL BARRIER FUNCTION IN VITRO

In a Phase 1b study of mild-to-moderate ulcerative colitis (UC) (Henn et al. Gastro 2020), oral daily dosing of SER-287, a purified consortium of Firmicutes spores administered after vancomycin pre-conditioning, led to significantly higher rates of clinical remission compared to placebo (40% versus 0%, respectively; p=0.024). Engraftment, defined as SER-287 spore germination and growth of metabolically-active bacteria, was associated with remission and shifts in disease relevant microbe-associated metabolites. To better understand the mechanism underlying these favorable clinical observations, we evaluated metabolite production by SER-287 and their impact on specific inflammatory pathways and barrier epithelial integrity in vitro.

SER-287, AN INVESTIGATIONAL MICROBIOME THERAPEUTIC FOR PATIENTS WITH ACTIVE MILD-TO-MODERATE ULCERATIVE COLITIS, PRODUCES REMISSION ASSOCIATED METABOLITES, REDUCES INTESTINAL EPITHELIAL IL-8 SECRETION AND PROTECTS EPITHELIAL BARRIER FUNCTION IN VITRO

Abstract Background In a Phase 1b study of mild-to-moderate ulcerative colitis (UC) (Henn et al. Gastro 2020), oral daily dosing of SER-287, a purified consortium of Firmicutes spores administered after vancomycin pre-conditioning, led to significantly higher rates of clinical remission compared to placebo (40% versus 0%, respectively; p=0.024). Engraftment, defined as SER-287 spore germination and growth of metabolically-active bacteria, was associated with remission and shifts in disease relevant microbe-associated metabolites. To better understand the mechanism underlying these favorable clinical observations, we evaluated metabolite production by SER-287 and their impact on specific inflammatory pathways and barrier epithelial integrity in vitro. Methods Three SER-287 lots and a negative control pro-inflammatory consortium including three strains isolated from UC subjects (Escherichia coli, Bifidobacterium dentium, and Streptococcus parasanguinis) were grown in vitro in a complex medium designed to mimic the nutrient composition found in the human colon. Culture supernatants were assessed for the presence of anti-inflammatory and remission associated metabolites by GC-MS or LC-MS. Anti- and pro-inflammatory activities were determined by measuring IL-8 secretion, an inflammatory cytokine whose expression is elevated in UC patients, by HT29 epithelial cells after incubation with bacterial supernatants in the presence or absence of TNF-α. Epithelial integrity was assayed by measuring FITC-dextran leakage in a trans-well culture of differentiated primary human colonic epithelial cells challenged with IFN-γ in the presence or absence of culture supernatants. Results When cultured in vitro, all SER-287 lots tested produced the anti-inflammatory short-chain fatty acids butyrate and propionate and other metabolites associated with remission in the Phase 1b trial, including tryptophan metabolites, bile acids and medium-chain fatty acids. SER-287 culture supernatants significantly reduced IL-8 secretion in TNF-α stimulated HT29 cells while the pro-inflammatory consortium increased IL-8 secretion compared to TNF-α alone (Figure 1A). SER-287 lots induced minimal IL-8 secretion in the absence of TNF-α while the pro-inflammatory consortium strongly induced IL-8 (Figure 1B). SER-287 supernatants significantly decreased IFN-γ-mediated barrier disruption, while the pro-inflammatory consortium was not protective (Figure 2). Conclusions Production of remission-associated metabolites, reduction of epithelial inflammation, and protection from epithelial barrier damage are shown to be pharmacological properties of SER-287 which may underlie the mechanism of action of this microbiome therapeutic for mild-to-moderate UC.

N-vanillyl-octanamide represses growth of fungal phytopathogens in vitro and confers postharvest protection in tomato and avocado fruits against fungal-induced decay.

Plant diseases caused by pathogenic fungi result in considerable losses in agriculture. The use of fungicides is an important alternative to combat these pathogens, but may affect both the environment and human health. Plants produce many bioactive compounds to defend themselves from biotic challenges and an increasing number of secondary metabolites have been identified, which may be used to control fungal infections. Here, the bioactivity of a synthetic capsaicinoid, N-vanillyl-octanamide, also termed ABX-I, in the growth of five phytopathogenic fungi was assessed in vitro. The compound inhibited growth of Colletotrichum gloeosporioides, Botrytis cinerea, Colletotrichum acutatum, Fusarium sp., and Rhizoctonia solani AG2, while the magnitude of this effect differed from capsaicin. To investigate if ABX-I could effectively protect crops against phytopathogens, fungal challenges were performed in tomato leaves and fruits, as well as avocado fruits co-infiltrated with Botrytis cinerea or Colletotrichum gloeosporioides, respectively. In both tomato leaves and fruits and avocado fruits, ABX-I decreased the fungal damage not only in vegetative but also in edible tissues, and diminished decay symptoms compared with untreated fruits, which were highly sensitive to the pathogens. Furthermore, ABX-I spray application to tomato or avocado plants did not compromise growth and development, whereas it repressed spore germination and growth of C. gloeosporioides, which suggests its potential as an affordable and promising resource to control fungal diseases in the agronomic sector.

Kinetic Modeling and Meta-Analysis of the Bacillus subtilis SigB Regulon during Spore Germination and Outgrowth.

The exponential increase in the number of conducted studies combined with the development of sequencing methods have led to an enormous accumulation of partially processed experimental data in the past two decades. Here, we present an approach using literature-mined data complemented with gene expression kinetic modeling and promoter sequence analysis. This approach allowed us to identify the regulon of Bacillus subtilis sigma factor SigB of RNA polymerase (RNAP) specifically expressed during germination and outgrowth. SigB is critical for the cell’s response to general stress but is also expressed during spore germination and outgrowth, and this specific regulon is not known. This approach allowed us to (i) define a subset of the known SigB regulon controlled by SigB specifically during spore germination and outgrowth, (ii) identify the influence of the promoter sequence binding motif organization on the expression of the SigB-regulated genes, and (iii) suggest additional sigma factors co-controlling other SigB-dependent genes. Experiments then validated promoter sequence characteristics necessary for direct RNAP–SigB binding. In summary, this work documents the potential of computational approaches to unravel new information even for a well-studied system; moreover, the study specifically identifies the subset of the SigB regulon, which is activated during germination and outgrowth.

Temporal Changes of the Epiphytic Bacteria Community From the Marine Macroalga Ulva lactuca (Santa Marta, Colombian-Caribbean).

Microbial communities live on macroalgal surfaces. The identity and abundance of the bacteria making these epiphytic communities depend on the macroalgal host and the environmental conditions. Macroalgae rely on epiphytic bacteria for basic functions (spore settlement, morphogenesis, growth, and protection against pathogens). However, these marine bacterial-macroalgal associations are still poorly understood for macroalgae inhabiting the Colombian Caribbean. This study aimed at characterizing the epiphytic bacterial community from macroalgae of the species Ulva lactuca growing in La Punta de la Loma (Santa Marta, Colombia). We conducted a 16S rRNA gene sequencing-based study of these microbial communities sampled twice a year between 2014 and 2016. Within these communities, the Proteobacteria, Bacterioidetes, Cyanobacteria, Deinococcus-Thermus and Actinobacteria were the most abundant phyla. At low taxonomic levels, we found high variability among epiphytic bacteria from U. lactuca and bacterial communities associated with macroalgae from Germany and Australia. We observed differences in the bacterial community composition across years driven by abundance shifts of Rhodobacteraceae Hyphomonadaceae, and Flavobacteriaceae, probably caused by an increase of seawater temperature. Our results support the need for functional studies of the microbiota associated with U. lactuca, a common macroalga in the Colombian Caribbean Sea.

Spore Heat Resistance and Growth Ability at Refrigeration Temperatures of Bacillus spp. and Paenibacillus spp.

In this study, spore heat resistance and growth ability at refrigeration temperatures of Bacillus spp. and Paenibacillus spp. were determined. The spore D90°C of 67.6% (23 of 34 strains) of Bacillus and 73.9% (17 of 23 strains) of Paenibacillus was less than 15 min. The growth abilities of both genera were equivalent at 10°C. However, 71.1% (32 of 45 strains) of Paenibacillus and only 6.3% (3 of 48 strains) of Bacillus cereus group could grow at 4°C. Eight B. cereus strains formed spores with higher heat resistance compared to the other Bacillus strains assessed; however, they did not grow at tempreratures below 10°C. Conversely, four Paenibacillus strains formed spores with heat resistance equivalent to that of the eight B. cereus strains and grew at 6°C or lower. In particular, Paenibacillus sp. JCM13343 formed the highest heat-resistant spores (D90°C = 136.1 min) and grew well at 4°C. These results indicate that Paenibacillus can grow in processed foods during refrigerated storage and has the potential to cause spoilage as well as Bacillus. Therefore, Paenibacillus should be considered as one of the targets for microbiological control in refrigerated processed foods.

Effect of vacuum cooling followed by ozone repressurization on Clostridium perfringens germination and outgrowth in cooked pork meat under temperature-abuse conditions

The effect of combining vacuum cooling with an ozone-based inhibition process (InhVac) on Clostridium perfringens spore germination and outgrowth in cooked pork meat after exponential chilling (from 54.4 to 7.2 °C in 12, 15, 18, or 21 h) and isothermal storage (20, 25, 30, 36, or 45 °C) was evaluated. Ice cooling (IC) and vacuum cooling (VC) were used to compare the effects with InhVac. The samples were inoculated with a three-strain mixture of C. perfringens spores to obtain concentration of ca. 3 log10 CFU/g. C. perfringens growth in samples treated by InhVac were 0.1, 0.37 and 0.9 log10 CFU/g after 15, 18 and 21 h of cooling from 54.4 to 7.2 °C respectively, significantly lower (P<0.05) than those in samples subjected to IC (1.01, 2.10 and 2.8 log10 CFU/g) and VC (0.56, 1.01 and 2.13 log10 CFU/g). Compared to VC and IC, InhVac treatment increased the lag phase (λ), decreased the growth rates (μmax), and extended the sample shelf-life (the time until a 1 log10 CFU/g increase in C. perfringes from the initial concentration value) at all storage temperatures. InhVac-treated samples not only had a longer shelf-life than those treated by VC, but also exhibited almost two times longer shelf-life compared to those subjected to IC regardless of storage temperatures. Additionally, statistical indexes showed that a primary modified Gompertz model and a secondary Square Root model could fit the data well. Industrial relevanceIn this study, an innovative inhibition approach (InhVac) was found to show a better antimicrobial effect on C. perfringens germination and outgrowth in cooked pork meat compared to ice cooling and vacuum cooling under temperature-abuse conditions. A primary modified Gompertz model and a secondary Square Root model could be used to predict the C. perfringens growth in samples subjected to InhVac treatment.

Characterization of Heterogeneity and Dynamics of Lysis of Single Bacillus subtilis Cells upon Prophage Induction During Spore Germination, Outgrowth, and Vegetative Growth Using Raman Tweezers and Live-Cell Phase-Contrast Microscopy.

A prophage comprises a bacteriophage genome that has integrated into a host bacterium's DNA, which generally permits the cell to grow and divide normally. However, the prophage can be induced by various stresses, or induction can occur spontaneously. After prophage induction, viral replication and production of endolysins begin until the cell lyses and phage particles are released. However, the heterogeneity of prophage induction and lysis of individual cells in a population and the dynamics of a cell undergoing lysis by prophage induction have not been fully characterized. Here, we used Raman tweezers and live-cell phase-contrast microscopy to characterize the Raman spectral and cell length changes that occur during the lysis of individual Bacillus subtilis cells from spores that carry PBSX prophage during spores' germination, outgrowth, and then vegetative growth. Major findings of this work are as follows: (i) After addition of xylose to trigger prophage induction, the intensities of Raman spectral bands associated with nucleic acids of single cells in induced cultures gradually fell to zero, in contrast to the much smaller changes in protein band intensities and no changes in nucleic acid bands in uninduced cultures; (ii) the nucleic acid band intensities from an individual induced cell exhibited a rapid decrease, following a long lag period; (iii) after the addition of nutrient-rich medium with xylose, single spores underwent a long period (228 ± 41.4 min) for germination, outgrowth, and vegetative growth, followed by a short period of cell burst in 1.5 ± 0.8 min at a cell length of 8.2 ± 5.5 μm; (iv) the latent time (Tlatent) between the addition of xylose and the start of cell burst was heterogeneous in cell populations; however, the period (ΔTburst) from the latent time to the completion of cell lysis was quite small; (v) in a poor medium with l-alanine alone, addition of xylose caused prophage induction following spore germination but with longer Tlatent and ΔTburst times and without cell elongation; (vi) spontaneous prophage induction and lysis of individual cells from spores in a minimal nutrient medium were observed without xylose addition, and cell length prior to cell lysis was ∼4.1 μm, but spontaneous prophage induction was not observed in a rich medium; (vii) in a rich medium, addition of xylose at a time well after spore germination and outgrowth significantly shortened the average Tlatent time. The results of this study provide new insights into the heterogeneity and dynamics of lysis of individual B. subtilis cells derived from spores upon prophage induction.

Corticosteroids alter alveolar macrophage control of Lichtheimia corymbifera spores in an ex vivo mouse model.

The aim of this study was to describe the impact of corticosteroids on alveolar macrophage (AM) capacities to control Mucorales growth in a new murine ex vivo model. Corticosteroids enhanced fungal growth of L. corymbifera through AM phagocytosis inhibition and burst oxidative decrease.

Micro propagation of Adiantum Capillus plant through a culture of plant spores

This experiment was conducted in Biotechnology research center BTRC for the purpose of studying the possibility of propagate Adiantum Capillus plant by using spores through plant tissues culture technology, which is one of the endangered plants in Libya. MS media was used in this study supplemented with some growth regulators (cytokines) of benzyl adenine BA and Kinten K at different concentrations (0.0, 0.5, 1.0, 2.0) mg / l. The results indicate that the growth of spores represented by the increase in the length and size of the vegetative masses in the MS nutritional medium equipped with BA growth regulator at a concentration of 2.0 mg / l is significantly higher than other treatments. The obtained plants were adapted by using growth medium of the Betmos and sand mixture in a ratio of 1: 2 in the growth room. The branches and roots formed when the plant moved to the greenhouse in the same medium of development