Growth Of Vegetative Cells Research Articles

Potential effect of monolaurin and nisin on the growth of three aerobic spore-forming bacteria isolated from Tunisian milk

Antibacterial capacities of monolaurin and nisin on the growth of vegetative cells of three sporeforming bacteria isolated from cow milk collected from Tunisia (Terribacillus aidingensis, Bacillus sporothermodurans and Paenibacillus sp and) were evaluated. Nisin or monolaurin, at different concentrations, can inhibit the growth of three Bacillus. 500 IU/mL and 25 μg/mL corresponded to the minimum inhibitory concentrations of nisin and monolaurin, respectively. The growth of the tested species in the presence of sub-lethal concentrations of monolaurin and nisin was monitored in UHT milk for 7 days at three different temperatures (4°C, 37°C and 25°C). Nisin (250 IU/mL) was able to induce an immediate reduction (after 3 h of incubation at 4°C) of Bacillus species growth (5 log for B. sporothermodurans, 4 log for Paenibacillus sp and 3.2 log for T. aidingensis). Also, monolaurin (25 µg/mL) reduce the growth, at 4°C of B. sporothermodurans by 5 log, Paenibacillus sp by 3 log and T. aidingensis by 4 log, after 3 h of incubation. In the presence of nisin or monolaurin, it can be observed a low reduction in cell growth followed by a rapid regrowth which can attenuate 10 log, at 37°C and 25°C. The obtained data elucidate the potency of nisin and monolaurin, and the application of these antimicrobial agents at low temperatures (4°C) to decontaminate any product from these spoilage bacteria.

Green Tea Catechin Epigallocatechin Gallate Inhibits Vegetative Cell Outgrowth and Expression of Beta-Lactamase Genes in Penicillin-Resistant Bacillus anthracis Strain PCr.

The green tea catechin epigallocatechin gallate (EGCg) has antimicrobial effects on many bacteria. In this study, we investigated the inhibitory effects of EGCg on Bacillus anthracis spores and vegetative cells. The B. anthracis spores were insensitive to EGCg, but the growth of vegetative cells derived from germinated spores was inhibited by EGCg. Moreover, EGCg decreased the minimum inhibitory concentration of penicillin and meropenem for penicillin-resistant B. anthracis. In the penicillin-resistant B. anthracis strain, the transcription levels of the beta-lactamase genes (bla1 and bla2) decreased significantly following the treatment with 50 µg/mL EGCg. These results suggest that the appropriate application of EGCg may effectively control the penicillin-resistant B. anthracis growth and beta-lactamase production.

Antibacterial Activity and Transcriptomic Analysis of Hesperetin against Alicyclobacillus acidoterrestris Vegetative Cells.

The aim of this research was to investigate the antimicrobial characteristics and mechanism of hesperetin against Alicyclobacillus acidoterrestris vegetative cells. The results presented show that hesperetin had effective antimicrobial activity on Alicyclobacillus acidoterrestris vegetative cells, minimum inhibition concentration (MIC) of 0.0625 g/L, and minimum bacterial concentration (MBC) greater than 2 g/L. Moreover, treatment of hesperetin caused significant damage to cell integrity, preventing the growth of Alicyclobacillus acidoterrestris vegetative cells, enhancing the leakage of nucleic acid and proteins, and destroying the vegetative cell morphology. To further investigate the mechanism, transcriptomic analysis was carried out, and 3056 differentially expressed genes (DEGs) were detected. Gene ontology (GO) enrichment analysis revealed that hesperetin inhibits Alicyclobacillus acidoterrestris by affecting the intracellular nitrogen metabolism and amino acid metabolism. The Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis explained that hesperetin was also able to prevent the growth of Alicyclobacillus acidoterrestris by affecting the processes of nutrient transport, energy metabolism, and flagella motility. These results provide new insights into the antimicrobial effects and mechanism of hesperetin against Alicyclobacillus acidoterrestris, which provides a new method for inactive Alicyclobacillus acidoterrestris in the juice industry.

Mechanism of eugenol inhibiting the growth of vegetative cells and spores of Bacillus cereus and its application in rice cakes

Recently, natural active substance has been widely concerned and researched because of its potent effect in the control of foodborne pathogen pollution. In this study, we explored the mechanism of eugenol (EL) against Bacillus cereus (B. cereus) and its spores and the potential of EL in controlling the growth of B. cereus in rice cakes. The results of antibacterial activity showed that EL performed potent antibacterial activity against B. cereus (MIC=0.4 mg/mL, MBC=0.8 mg/mL). EL could increase the permeability of cell membrane, increase the leakage of intracellular protein and disturb the balance of ion exchange within bacteria. Molecular docking and competitive fluorescence experiments revealed that EL interacted with DNA through grooving binding. Under the above multiple effects, the normal metabolic activities within bacteria were severely affected and even leading to death. EL treatment resulted in the decrease of multiple toxin synthesis genes (nheA, nheB, nheC, hblC, hblD, hblA, and cytK) expression levels, and significantly reduced the hemolysis activity of toxins through hydrophobic interactions and hydrogen bonding forces. EL was proved to be effective in inhibiting the growth of B. cereus in rice cakes. The inhibition on spore germination and the improvement of spore thermal sensitivity by EL would also provide a valid method for spore control. This study revealed the antibacterial mechanisms of EL against B. cereus and its spores, and laid a theoretical foundation for expanding its application in the control of foodborne pathogens contamination.

Susceptibility of Vegetative Cells and Endospores of Bacillus cereus to Rhamnolipid Biosurfactants and Their Potential Application in Dairy.

Bacillus cereus is a Gram-positive, endospore-forming bacterium well-known as a food pathogen that causes great losses in the food industry, especially in dairy. In this study, rhamnolipid (RL) biosurfactants were evaluated as a bio-based alternative for controlling the growth of vegetative cells and endospores of B. cereus. RLs were tested against 14 B. cereus strains isolated from different types of foodstuffs. The antimicrobial activity against vegetative cells and endospores revealed minimal inhibitory concentration (MIC) values of 0.098 mg/mL for almost all strains tested and minimal bactericidal concentration (MBC) varying between 0.098 and >25 mg/mL. The presence of RLs inhibited endospore germination by more than 99%, reducing by 5.5 log the outgrowth of strain 0426. Scanning and transmission electron microscopy confirmed that exposure to RL causes damage to the structure of endospores. When skim milk was utilized as a food model, RL inhibited the growth of vegetative cells and endospores of B. cereus, showing MBC of 3.13 mg/mL for the vegetative cells of strain 0426. The surfactant also reduced bacterial growth in milk at refrigerator temperature. The results suggest that RLs are promising candidates for the development of novel strategies to control B. cereus in the food industry.

Engineering probiotics to inhibit Clostridioides difficile infection by dynamic regulation of intestinal metabolism

Clostridioides difficile infection (CDI) results in significant morbidity and mortality in hospitalised patients. The pathogenesis of CDI is intrinsically related to the ability of C. difficile to shuffle between active vegetative cells and dormant endospores through the processes of germination and sporulation. Here, we hypothesise that dysregulation of microbiome-mediated bile salt metabolism contributes to CDI and that its alleviation can limit the pathogenesis of CDI. We engineer a genetic circuit harbouring a genetically encoded sensor, amplifier and actuator in probiotics to restore intestinal bile salt metabolism in response to antibiotic-induced microbiome dysbiosis. We demonstrate that the engineered probiotics limited the germination of endospores and the growth of vegetative cells of C. difficile in vitro and further significantly reduced CDI in model mice, as evidenced by a 100% survival rate and improved clinical outcomes. Our work presents an antimicrobial strategy that harnesses the host-pathogen microenvironment as the intervention target to limit the pathogenesis of infection.

Antibacterial and Sporicidal Activity Evaluation of Theaflavin-3,3'-digallate.

Theaflavin-3,3′-digallate (TFDG), a polyphenol derived from the leaves of Camellia sinensis, is known to have many health benefits. In this study, the antibacterial effect of TFDG against nine bacteria and the sporicidal activities on spore-forming Bacillus spp. have been investigated. Microplate assay, colony-forming unit, BacTiter-GloTM, and Live/Dead Assays showed that 250 µg/mL TFDG was able to inhibit bacterial growth up to 99.97%, while 625 µg/mL TFDG was able to inhibit up to 99.92% of the spores from germinating after a one-hour treatment. Binding analysis revealed the favorable binding affinity of two germination-associated proteins, GPR and Lgt (GerF), to TFDG, ranging from −7.6 to −10.3 kcal/mol. Semi-quantitative RT-PCR showed that TFDG treatment lowered the expression of gpr, ranging from 0.20 to 0.39 compared to the control in both Bacillus spp. The results suggest that TFDG not only inhibits the growth of vegetative cells but also prevents the germination of bacterial spores. This report indicates that TFDG is a promising broad-spectrum antibacterial and anti-spore agent against Gram-positive, Gram-negative, acid-fast bacteria, and endospores. The potential anti-germination mechanism has also been elucidated.

Bovicin HC5 and nisin reduce cell viability and the thermal resistance of Alicyclobacillus acidoterrestris endospores in fruit juices.

Alicyclobacillus acidoterrestris is an important thermoacidophilic spore-forming bacterium in fruit-juice deterioration, and alternative non-thermal methods have been investigated to control fruit juice spoilage. This work aimed to evaluate the capacity of bovicin HC5 and nisin to inhibit the growth of vegetative cells and reduce the thermal resistance of endospores of A. acidoterrestris inoculated (107 CFU mL-1 ) in different fruit juices. The number of viable cells was determined after 12 h incubation at 43 °C in the presence and absence of nisin or bovicin HC5 (10-100 AU mL-1 ). The exposure time (min) required to kill 90% of the initial population (reduction of one log factor) at 90 ºC (D90ºC ) was used to assess the thermal resistance of A. acidoterrestris endospores exposed (80 AU mL-1 ) or non-exposed to the bacteriocins. Additionally, the effect of bovicin and nisin on the morphology and cell structure of A. acidoterrestris was evaluated by atomic force microscopy (AFM). Bovicin HC5 and nisin were bactericidal against A. acidoterrestris inoculated in fruit juices and reduced the D90°C values up to 30-fold. AFM topographical images revealed substantial structural changes in the cellular framework of vegetative cells upon treatment with bovicin HC5 or nisin. These results emphasize the potential application of lantibiotics as additional hurdles in food processing to control thermoacidophilic spoilage bacteria in fruit juices. © 2022 Society of Chemical Industry.

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.

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.

Effect of non-thermal plasma technology on microbial inactivation and total phenolic content of a model liquid food system and black pepper grains

The objectives of this study were to investigate the effects of cold plasma technology on the growth and survival rates of vegetative cells and spores, and total phenolic content of black pepper grains. Plasma treatment was carried out using a non-thermal plasma jet system operating at 20 kHz using atmospheric air at a flow of 11 L/min. A model liquid food system and black pepper grains were both inoculated with Bacillus subtilis vegetative cells and spores. The samples were treated at 15 and 30 kV for treatment times of 3–20 min. The plate count method was used to determine colony-forming units for selected storage times at 4 °C i.e. at 1, 24 and 48 h post treatment. The highest log reduction was observed at 24 h post treatment, i.e. 2.92 log reduction. A 1 log reduction was achieved in the case of black pepper inoculated with spores for all selected storage times. No significant differences in total phenolic content were observed between treated and non-treated black pepper samples (p > 0.05). Optical emission spectroscopy was used to detect reactive species likely to be responsible for cell death. Atomic oxygen, atomic nitrogen, hydroxyl radicals, nitrite oxide and nitrate were detected in light emitted from the plasma. Cell membrane damage caused by non-thermal plasma technology was observed using scanning electron microscopy. This study concludes that cold plasma technology has potential for industry application in food processing to reduce microbial loads in liquid food systems and dried foods with limited impact on food quality.

Antibacterial and Sporicidal Activities of Syzygium polyanthum L. Extract against Bacillus cereus Isolated from Rice

Spore-forming bacteria, Bacillus sp., frequently been associated with the contamination of rice and other starchy products. Spores are more resistant to antimicrobial treatments than its vegetative cells. The extract of Indonesian bay leaf (Syzygium polyanthum L.) was assessed for its antibacterial and sporicidal activities against vegetative cells and spores of B. cereus isolated from rice (25 strains). The results showed that S. polyanthum L. extract was able to inhibit the growth of vegetative cells of all B. cereus isolates with MICs ranged from 0.16 to 0.63 mg/mL and can kill with MBCs ranged from 0.31 to 2.50 mg/mL. The bactericidal endpoint for B. cereus BC-NP.8 in time kill curve was at 1.25 mg/mL (8× MIC) after 4 h of incubation while for B. cereus ATCC 33019 was at 2.50 mg/mL (8× MIC). The sporicidal activity of S. polyanthum L. extract was not affected by different temperatures treatment and alteration of the pHs of extract. Therefore, this indicates that the extract was stable after exposed to pH3, 7 and 10 as well as temperature of 50, 80, and 121°C. Observation under on scanning electron microscope the structure of the B. cereus ATCC 33019 spores was ruptured after being treated with 1% (w/v) S. polyanthum L. extract for 1 h. In conclusion, S. polyanthum L. extract had antibacterial and sporicidal activity against vegetative cells and spores of B. cereus isolated from rice.

The role of life cycle processes on phytoplankton spring bloom composition: a modelling study applied to the Gulf of Finland

Abstract Diatoms are typical representatives of the spring bloom worldwide. In several parts of the Baltic Sea, however, cold-water dinoflagellates such as Biecheleria baltica have become dominant during the past decades. We have investigated the mechanisms behind this trend by using an ecosystem model which includes the life cycles of three main phytoplankton groups (diatoms, dinoflagellates and cyanobacteria). Coupled to a water column model we have applied the model system for the period 1981–2010 to the Gulf of Finland. In agreement with observations, the model results show an increasing trend in the proportion of dinoflagellates in the Gulf of Finland. Temperature and life cycle-related processes explain the relative increase of dinoflagellates and corresponding decrease of diatoms. Warming over the 30 years has enabled a head start of dinoflagellates by reducing the time lag between germination and growth of vegetative cells. Although diatoms have a much higher growth rate, they cannot compete with the high dinoflagellate concentrations that result from the inoculum. Diatoms will only dominate in years when the inoculum concentrations of dinoflagellates or the temperatures are low. Overall, the model results suggest that consideration of life cycle dynamics of competing phytoplankton groups may be crucial to understand trends and shifts in community composition.

A combinatorial strategy of alternative promoter use during differentiation of a heterocystous cyanobacterium.

Heterocystous cyanobacteria such as Nostoc sp. are filamentous photosynthetic organisms that, in response to nitrogen deficiency, undergo a differentiation process transforming certain, semi-regularly spaced cells into heterocysts, devoted to nitrogen fixation. During transition to a nitrogen-fixing regime, growth of most vegetative cells in the filament is temporarily arrested due to nutritional deprivation, but developing heterocysts require intense transcriptional activity. Therefore, the coexistence of arrested vegetative cells and actively developing prospective heterocysts relies on the simultaneous operation of somewhat opposite transcriptional programs. We have identified genes with multiple nitrogen-responsive transcriptional starts appearing in seemingly paradoxical combinations. For instance, sigA, encoding the RNA polymerase housekeeping sigma factor, is transcribed from one major nitrogen stress-repressed promoter and from a second, nitrogen stress-induced promoter. Here, we show that both promoters are expressed with complementary temporal dynamics. Using a gfp reporter we also show that transcription from the inducible promoter takes place exclusively in differentiating heterocysts and is already detected before any morphological or fluorescence signature of differentiation is observed. Tandem promoters with opposite dynamics could operate a compensatory mechanism in which repression of transcription from the major promoter operative in vegetative cells is offset by transcription from a new promoter only in developing heterocyst.

Exponential Growth and Solvents-Production of <i>Clostridium acetobutylicum</i> ATCC 824 on TYA Media Containing Sucrose and Glucose as Different Sole Carbon Sources

Clostridium acetobutylicum ATCC 824 is a solventogenic, obligate anaerobic bacterium that can grow on various types of carbohydrates and are capable of producing spores. In the present study, Clostridium acetobutylicum was successfully grown on TYA medium (tryptone, yeast, acetate medium) containing two different carbon sources, glucose and sucrose coupled with the production of acids (butyric and acetic acid) and solvents, ABE (acetone, butanol and ethanol). An investigation was undertaken to determine the impact of the two types of carbon sources on the solvent production and growth of Clostridium acetobutylicum. HPLC and GC analysis revealed the amount of acids and solvents produced respectively, as well as the amount of unutilized sugars. The amount of combined ABE produced on glucose (0.19g/l A, 0.39g/l B, 0.06g/l E) was higher than on sucrose as carbon source (0.15g/l A, 0.30g/l B, 0.03g/l E). The colony forming units of Clostridium acetobutylicum grown on glucose (4.70 x 105 units/ml) was higher than on sucrose (0.1 x 105) as judged by dilution spread plating on agar. Hence, Glucose was confirmed as the carbon source characterized by the best performance for solvents production and growth of the bacterium. The whole production process on both glucose and sucrose was observed to mainly influence the production of butanol with the concentration of 0.39g/l and 0.30g/l respectively, over the production of other solvents. Higher amount of solvents was produced at lower pH in both cultures with the different carbon sources. Wet-mounts, gram stain and endospore stain were used to determine the motility, type and sporulation of Clostridium acetobutylicum respectively. Acidogenic phase which seems to couple with the growth of vegetative cells, results into production of acetic and butyric acids. Solventogenic phase commences with a drop in pH and is accompanied by the onset of sporulation.

Hopanoids play a role in stress tolerance and nutrient storage in the cyanobacterium Nostoc punctiforme.

Hopanes are abundant in ancient sedimentary rocks at discrete intervals in Earth history, yet interpreting their significance in the geologic record is complicated by our incomplete knowledge of what their progenitors, hopanoids, do in modern cells. To date, few studies have addressed the breadth of diversity of physiological functions of these lipids and whether those functions are conserved across the hopanoid-producing bacterial phyla. Here, we generated mutants in the filamentous cyanobacterium, Nostoc punctiforme, that are unable to make all hopanoids (shc) or 2-methylhopanoids (hpnP). While the absence of hopanoids impedes growth of vegetative cells at high temperature, the shc mutant grows faster at low temperature. This finding is consistent with hopanoids acting as membrane rigidifiers, a function shared by other hopanoid-producing phyla. Apart from impacting fitness under temperature stress, hopanoids are dispensable for vegetative cells under other stress conditions. However, hopanoids are required for stress tolerance in akinetes, a resting survival cell type. While 2-methylated hopanoids do not appear to contribute to any stress phenotype, total hopanoids and to a lesser extent 2-methylhopanoids were found to promote the formation of cyanophycin granules in akinetes. Finally, although hopanoids support symbiotic interactions between Alphaproteobacteria and plants, they do not appear to facilitate symbiosis between N.punctiforme and the hornwort Anthoceros punctatus. Collectively, these findings support interpreting hopanes as general environmental stress biomarkers. If hopanoid-mediated enhancement of nitrogen-rich storage products turns out to be a conserved phenomenon in other organisms, a better understanding of this relationship may help us parse the enrichment of 2-methylhopanes in the rock record during episodes of disrupted nutrient cycling.

Deciphering Adaptation Strategies of the Epidemic Clostridium difficile 027 Strain during Infection through In Vivo Transcriptional Analysis.

Clostridium difficile is responsible for a wide spectrum of infection from asymptomatic carriage to severe, relapsing colitis. Since 2003, C. difficile infections have increased with a higher morbidity and mortality due to the emergence of epidemic and hypervirulent C. difficile strains such as those of the epidemic lineage 027/BI/NAP1. To decipher the hypervirulence and epidemicity of 027 strains, we analyzed gene expression profiles of the R20291 027 strain using a monoxenic mouse model during the first 38h of infection. A total of 741 genes were differentially expressed during the course of infection. They are mainly distributed in functional categories involved in host adaptation. Several genes of PTS and ABC transporters were significantly regulated during the infection, underlying the ability of strain R20291 to adapt its metabolism according to nutrient availability in the digestive tract. In this animal model, despite the early sporulation process, sporulation efficiency seems to indicate that growth of R20291 vegetative cells versus spores were favored during infection. The bacterial mechanisms associated to adaptability and flexibility within the gut environment, in addition to the virulence factor expression and antibiotic resistance, should contribute to the epidemicity and hypervirulence of the C. difficile 027 strains.

Resting cysts, and effects of temperature and salinity on the growth of vegetative cells of the potentially harmful species Alexandrium insuetum Balech (Dinophyceae)

The potentially harmful species Alexandrium insuetum established by the incubation of resting cysts isolated from sediment trap samples collected at Jinhae-Masan Bay, Korea was characterized by morphological and phylogenetic analysis. The effects of temperature and salinity on the growth of A. insuetum were also investigated. The resting cysts are characterized by a spherical shape, a small size (20–25μm) and the presence of either three or four red accumulation bodies. The similarity of morphological features of the resting cysts to those of other species of the minutum group (consisting of Alexandrium minutum and A. tamutum) indicates that the morphological features of resting cysts might improve the accuracy of the grouping of Alexandrium species. A. insuetum germinated from the resting cysts is morphologically consistent with vegetative cells reported from Korean and Japanese coastal areas, and has an partial large subunit (LSU) rDNA sequence identical to that from Japanese strains. The growth of A. insuetum was observed between salinity 20 and 35, with increasing temperature; however at 25°C, A. insuetum could grow even at the salinity of 15. The highest growth rate (0.60d−1) was observed at 25°C and the salinity of 25, which is higher than the previously reported growth rate of A. tamarense, which is responsible for outbreaks of paralytic shellfish poisoining and blooms in Jinhae-Masan Bay. These results suggest that the proliferation of A. insuetum in Jinhae-Masan Bay is likely to be highest during the summer.

Susceptibility of Clostridium perfringens to antimicrobials produced by lactic acid bacteria: Reuterin and nisin

The effectiveness as antimicrobials of lactic acid bacteria produced compounds reuterin and nisin was assessed against vegetative cells and spores of Clostridium perfringens isolates (from ovine milk obtained in farms with diarrheic lambs) and C. perfringens CECT 486 (type A toxin producer). We also tested the inhibitory effect of lysozyme and sodium nitrite on Clostridium. Minimal inhibitory concentrations (MIC) of antimicrobials were determined in modified RCM (mRCM) and milk by using a broth microdilution method, after 7 d at 37 °C under anaerobic conditions. The sensitivity of C. perfringens to the tested antimicrobials was strain and culture medium-dependent. In general, vegetative cells exhibited higher sensitivity than spores. Reuterin (MIC values 2.03–16.25 mM) inhibited the growth of vegetative cells and the outgrowth of spores of all tested C. perfringens, both in mRCM and milk, with higher resistance in milk. Nisin (MIC values 0.78–12.5 μg/ml) was also effective against vegetative cells and spores of tested C. perfringens in both culture media. However, lysozyme (up to 400 μg/ml) did not control the growth of any of the tested Clostridium. Sodium nitrite only inhibited the outgrowth of spores of two C. perfringens isolates at the maximum concentration assayed (300 μg/ml) exclusively in mRCM medium. These results suggest that reuterin and nisin have the potential to control the growth of C. perfringens, and might help to ensure safety at different stages of the food chain. Future studies in food/feed products would be necessary to further corroborate this hypothesis.

Quantitative analysis of the effect of specific tea compounds on germination and outgrowth of Bacillus subtilis spores at single cell resolution

Tea is one of the most widely consumed beverages in the world and known for its antimicrobial activity against many microorganisms. Preliminary studies have shown that tea polyphenols can inhibit the growth of a wide range of Gram-positive bacteria. However, the effect of these compounds on germination and outgrowth of bacterial spores is unclear. Spore-forming bacteria are an aggravating problem for the food industry due to spore formation and their subsequent returning to vegetative state during food storage, thus posing spoilage and food safety challenges. Here we analysed the effect of tea compounds: gallic acid, gallocatechin gallate, Teavigo (>90% epigallocatechin gallate), and theaflavin 3,3′-digallate on spore germination and outgrowth and subsequent growth of vegetative cells of Bacillus subtilis. To quantitatively analyse the effect of these compounds, live cell images were tracked from single phase-bright spores up to microcolony formation and analysed with the automated image analysis tool “SporeTracker”. In general, the tested compounds had a significant effect on most stages of germination and outgrowth. However, germination efficiency (ability of spores to become phase-dark) was not affected. Gallic acid most strongly reduced the ability to grow out. Additionally, all compounds, in particular theaflavin 3,3′-digallate, clearly affected the growth of emerging vegetative cells.