Vegetative Cell Growth Research Articles

Influence of propiconazole and metconazole formulations on Bacillus subtilis vegetative cell growth and disease control of fruit crops.

Biological control agent (BCA) Bacillus subtilis formulated as Theia® is registered for control of fungal and bacterial diseases of fruit crops. Combinations of Theia® and strategic concentrations of two demethylation inhibitor (DMI) fungicides were investigated to explore potential synergisms. Bacteria were cultured in nutrient broth and combined with technical grades and two formulations of propiconazole (EC and WP) and metconazole (EC and WDG) at 0, 10, 50, 100, and 150 µg/ml active ingredient. After co-cultivation, optical density (OD600) and colony forming units (CFU/ml) were evaluated. In contrast to EC formulations, the WP or WDG formulations at 10 or 50 µg/ml of both DMIs did not affect vegetative cell growth. The mixture of Theia® and each formulated DMI at 50 µg/ml active ingredient resulted in a significant reduction of Monilinia fructicola lesion development on apple, Colletotrichum siamense lesion development on cherry, and Botrytis cinerea lesion development on cherry. The combination of Theia® with EC formulations showed weaker disease reduction due to antagonism. Only Theia® + non-EC formulated propiconazole and metconazole significantly reduced brown rot disease incidence of apple compared to the respective solo treatments and anthracnose disease incidence of cherry compared to the untreated control. Our results indicated that at least some DMI fungicides possess bactericidal effects depending on the formulation and concentration. The combination of Theia® with a lower than label rate concentration (50 µg/ml) of the DMI fungicides propiconazole and metconazole showed potential for synergistic effects especially when non-EC formulations were used.

The roles of yeast formins and their regulators Bud6 and Bil2 in the pheromone response.

In response to pheromone Saccharomyces cerevisiae extend a mating projection. This process depends on the formation of polarized actin cables which direct secretion to the mating tip and translocate the nucleus for karyogamy. Here, we demonstrate that proper mating projection formation requires the formin Bni1, as well as the actin nucleation promoting activities of Bud6, but not the formin Bnr1. Further, Bni1 is required for pheromone gradient tracking. Our work also reveals unexpected new functions for Bil2 in the pheromone response. Previously we identified Bil2 as a direct inhibitor of Bnr1 during vegetative cell growth. Here, we show that Bil2 has Bnr1-independent functions in spatially focusing Bni1-GFP at mating projection tips, and in vitro Bil2 and its binding partner Bud6 organize Bni1 into clusters that nucleate actin assembly. bil2∆ cells also display entangled Bni1-generated actin cable arrays and defects in secretory vesicle transport and nuclear positioning. At low pheromone concentrations, bil2∆ cells are delayed in establishing a polarity axis, and at high concentrations they prematurely form a second and a third mating projection. Together, these results suggest that Bil2 promotes the proper formation and timing of mating projections by organizing Bni1 and maintaining a persistent axis of polarized growth.

Mechanistic insights into the adaptive evolvability of spore heat resistance in Bacillus cereus sensu lato

Wet heat treatment is a commonly applied method in the food and medical industries for the inactivation of microorganisms, and bacterial spores in particular. While many studies have delved into the mechanisms underlying wet heat killing and spore resistance, little attention has so far been dedicated to the capacity of spore-forming bacteria to tune their resistance through adaptive evolution. Nevertheless, a recent study from our group revealed that a psychrotrophic strain of the Bacillus cereus sensu lato group (i.e. Bacillus weihenstephanensis LMG 18989) could readily and reproducibly evolve to acquire enhanced spore wet heat resistance without compromising its vegetative cell growth ability at low temperatures. In the current study, we demonstrate that another B. cereus strain (i.e. the mesophilic B. cereus sensu stricto ATCC 14579) can acquire significantly increased spore wet heat resistance as well, and we subjected both the previously and currently obtained mutants to whole genome sequencing. This revealed that five out of six mutants were affected in genes encoding regulators of the spore coat and exosporium pathway (i.e. spoIVFB, sigK and gerE), with three of them being affected in gerE. A synthetically constructed ATCC 14579 ΔgerE mutant likewise yielded spores with increased wet heat resistance, and incurred a compromised spore coat and exosporium. Further investigation revealed significantly increased spore DPA levels and core dehydration as the likely causes for the observed enhanced spore wet heat resistance. Interestingly, deletion of gerE in Bacillus subtilis 168 did not impose increased spore wet heat resistance, underscoring potentially different adaptive evolutionary paths in B. cereus and B. subtilis.

Survival and Development Strategies of Cyanobacteria through Akinete Formation and Germination in the Life Cycle

Eutrophic freshwater ecosystems are vulnerable to toxin-producing cyanobacteria growth or harmful algal blooms. Cyanobacteria belonging to the Nostocales order form akinetes that are similar to the seeds of vascular plants, which are resting cells surrounded by a thick membrane. They overwinter in sediment and germinate when conditions become favorable, eventually developing into vegetative cells and causing blooms. This review covers the cyanobacterial akinete of the Nostocales order and summarizes the environmental triggers and cellular responses involved in akinete germination and formation based on data from the literature. It also emphasizes the intimate and dynamic relationship that exists between the germination and formation of akinete in the annual life cycle of cyanobacteria. After comparing many published data, it is found that the tolerance ranges for factors affecting both akinete germination and formation do not differ significantly and are broadly consistent with the tolerance ranges for vegetative cell growth. However, the optimal range varies with different species and strains of cyanobacteria. The life cycle of cyanobacteria, as a result of akinete germination and formation, has a seasonal periodicity and spatial connectivity between the water column and the sediment. However, during the summer growing season, intimate coupling between akinete formation and germination can occur in the water column, and this can contribute to high population densities being maintained in the water column. During this time, shallow sediment could also provide suitable conditions for akinete germination, thereby contributing to the establishment of water column populations. The information summarized in this review is expected to help improve our shared understanding of the life cycle of the Nostocales cyanobacteria while also providing insights into the monitoring and management of harmful algal blooms.

Prevalence, Virulence Potential, and Growth in Cheese of Bacillus cereus Strains Isolated from Fresh and Short-Ripened Cheeses Sold on the Italian Market.

This study investigated B. cereus presence in 122 samples belonging to 34 typologies of fresh or short-ripened cheeses made from cow, sheep, goat, or buffalo pasteurized milk, and sold on the Italian market. B. cereus was isolated at a prevalence of 9.8%, with a marked variability among cheese categories, and at low counts (always below 2.26 Log CFU/g). Twelve isolates were identified by MALDI-TOF analysis and typified by RAPD PCR as belonging to different B. cereus strains. All the strains were tested for the production of hemolysin BL, phosphatidylcholine-specific phospholipase C, proteases, and biofilm formation, and for the presence of chromosomal toxin-encoding genes (sph, plcA, cytK, entFM, bcet, nheA, nheB, nheC). Overall, 92% of strains harbored bcet, 75% the three genes nheA, nheB, and nheC, as well as plcA and sph, 67% entFM, and 33% cytK. All strains showed biofilm-forming ability. A chemical-physical characterization of the cheeses was also performed to show their suitability as substrates for B. cereus growth, showing high heterogeneity in terms of pH, aw, salt content, and concentration of organic acids. Finally, the ability to support spore germination and vegetative cell growth of a selected cheese was investigated in spores-inoculated samples maintained at 10 °C and 15 °C, showing the inhibitory effect of low storage temperatures.

Deficiency of nitrogen but not phosphorus triggers the life cycle transition of the dinoflagellate Scrippsiella acuminata from vegetative growth to resting cyst formation

Nitrogen (N) and phosphorus (P) are essential elements for algal growth. When N and P are deficient, dinoflagellates will take a series of measures to achieve population continuation including formation of resting cysts, an important ecological strategy of dinoflagellates that plays a key role in the initiation and termination of harmful algal blooms (HABs). How the deficiency of N and P affects algal growth and cyst formation has been investigated in some dinoflagellate species, but how it affects the life cycle transition in dinoflagellates has been poorly understood. In this study, we further explored the effect of N and P deficiency on the algal growth and resting cyst production in the cosmopolitan HABs-causing species Scrippsiella acuminata via refining the N and P concentration gradients. Further, we tracked the expression patterns of one CyclinB and one CDK1 genes of S. acuminata at different growth stages under three deficiency concentrations (1/1000 dilutions of N, P, and both N and P). The results suggest that N deficiency always triggered the cyst formation but P deficiency mainly inhibited the vegetative growth instead of inducing cyst formation. We also observed the highest cyst production when S. acuminata was cultured in the f/2-Si medium that was a one-thousandth dilution of N and P (N∼ 0.882 μM; P∼ 0.0362 μM). Our results for the expressions of CyclinB and CDK1 were well consistent with the results of algal growth and cyst formation at different deficiencies of N and P in terms of that higher expressions of these two genes were corresponding to higher rates of vegetative cell growth, while their expressions in resting cysts maintained to be moderate but significantly lower than that in fast-growing vegetative cells. Although we are still not sure whether the changing expressions of the two genes did regulate the transition of life cycle (i.e. cyst formation), or happened as parallels to the expressions of other truly regulating genes, our observations are surely inspirational for further investigations on the genetic regulation of life cycle transition in dinoflagellates. Our work will provide clues to probe the physiological and molecular mechanisms underlying the nutrient deficiency-induced alternation between life cycle stages in dinoflagellates.

Crystal structure of the pheromone Er-13 from the ciliate Euplotes raikovi, with implications for a protein–protein association model in pheromone/receptor interactions

In the ciliate Euplotes raikovi, water-borne protein pheromones promote the vegetative cell growth and mating by competitively binding as autocrine and heterologous signals to putative cell receptors represented by membrane-bound pheromone isoforms. A previously determined crystal structure of pheromone Er-1 supported a pheromone/receptor binding model in which strong protein–protein interactions result from the cooperative utilization of two distinct types of contact interfaces that arrange molecules into linear chains, and these into two-dimensional layers. We have now determined the crystal structure of a new pheromone, Er-13, isolated from cultures that are strongly mating reactive withculturessource of pheromone Er-1.The comparison between the Er-1 and Er-13 crystal structuresreinforces the fundamental of the cooperative model of pheromone/receptor binding, in that the molecules arrange into linear chains taking a rigorously alternate opposite orientation reflecting the presumed mutual orientation of pheromone and receptor molecules on the cell surface. In addition, the comparison provides two new lines of evidence for a univocal rationalization of observations on the differentbehaviourbetween the autocrine and heterologous pheromone/receptor complexes. (i) In the Er-13 crystal, chains do not form layers which thus appear to be an over-structureunique tothe Er-1 crystal, not essential for the pheromone signalling mechanisms. (ii) In both crystal structures, the intra-chain interfaces are equally derived from burying amino-acid side-chains mostly residing on helix-3 of the three-helical pheromonefold. This helix is thus identified as the key structural motif underlying the pheromone activity, in line with its tight intra- and interspecificstructuralconservation.

NanI Sialidase Contributes to the Growth and Adherence of Clostridium perfringens Type F Strain F4969 in the Presence of Adherent Mucus.

Clostridium perfringens type F strains causing nonfoodborne human gastrointestinal diseases (NFD) typically produce NanI sialidase as their major secreted sialidase. Type F NFDs can persist for several weeks, indicating their pathogenesis involves intestinal colonization, including vegetative cell growth and adherence, with subsequent sporulation that fosters enterotoxin production and release. We previously reported that NanI contributes to type F NFD strain adherence and growth using Caco-2 cells. However, Caco-2 cells make minimal amounts of mucus, which is significant because the intestines are coated with adherent mucus. Therefore, it was important to assess if NanI contributes to the growth and adherence of type F NFD strains in the presence of adherent mucus. Consequently, the current study first demonstrated greater growth of nanI-carrying versus non-nanI-carrying type F strains in the presence of HT29-MTX-E12 cells, which produce an adherent mucus layer, versus their parental HT29 cells, which make minimal mucus. Demonstrating the specific importance of NanI for this effect, type F NFD strain F4969 or a complementing strain grew and adhered better than an isogenic nanI null mutant in the presence of HT29-MTX-E12 cells versus HT29 cells. Those effects involved mucus production by HT29-MTX-E12 cells since mucus reduction using N-acetyl cysteine reduced F4969 growth and adherence. Consistent with those in vitro results, NanI contributed to growth of F4969 in the mouse small intestine. By demonstrating a growth and adherence role for NanI in the presence of adherent mucus, these results further support NanI as a potential virulence factor during type F NFDs.

The macronuclear genome of the Antarctic psychrophilic marine ciliate Euplotes focardii reveals new insights on molecular cold adaptation

The macronuclear (MAC) genomes of ciliates belonging to the genus Euplotes species are comprised of numerous small DNA molecules, nanochromosomes, each typically encoding a single gene. These genomes are responsible for all gene expression during vegetative cell growth. Here, we report the analysis of the MAC genome from the Antarctic psychrophile Euplotes focardii. Nanochromosomes containing bacterial sequences were not found, suggesting that phenomena of horizontal gene transfer did not occur recently, even though this ciliate species has a substantial associated bacterial consortium. As in other euplotid species, E. focardii MAC genes are characterized by a high frequency of translational frameshifting. Furthermore, in order to characterize differences that may be consequent to cold adaptation and defense to oxidative stress, the main constraints of the Antarctic marine microorganisms, we compared E. focardii MAC genome with those available from mesophilic Euplotes species. We focussed mainly on the comparison of tubulin, antioxidant enzymes and heat shock protein (HSP) 70 families, molecules which possess peculiar characteristic correlated with cold adaptation in E. focardii. We found that α-tubulin genes and those encoding SODs and CATs antioxidant enzymes are more numerous than in the mesophilic Euplotes species. Furthermore, the phylogenetic trees showed that these molecules are divergent in the Antarctic species. In contrast, there are fewer hsp70 genes in E. focardii compared to mesophilic Euplotes and these genes do not respond to thermal stress but only to oxidative stress. Our results suggest that molecular adaptation to cold and oxidative stress in the Antarctic environment may not only be due to particular amino acid substitutions but also due to duplication and divergence of paralogous genes.

Metformin reduces Clostridium difficile infection

Clostridium difficile (C. difficile) is the most common pathogen causing hospital acquired infections, linked with dramatic abnormal changes in gut microbiome. Antibiotic resistance is common in C. difficile and currently invasive procedures like fecal microbiota transplantation is only viable option, which also do not stop recurrence incidences. Thus, discovering non-invasive, safe, inexpensive and clinically adoptable strategies are needed. Here, we demonstrated that metformin- a safe, inexpensive and FDA approved antidiabetic drug, showed growth inhibitory activity against the C. difficile. We show that metformin reduces vegetative cell growth of C. difficile in-vitro, as well as ex-vivo in human microbiome culture system. Interestingly, metformin beneficially modified human gut microbiome by decreasing C. difficile growth while increasing the growth of non-pathogenic Clostridium strains. In addition, metformin inhibit colonization of C. difficile in mice and significantly reduces infection clinical score. Metformin reduced C. difficile colonization more than 10 times in caecum and 100 times in colon; and it also reduced bacterial translocation from gut to spleen, liver and mesenteric lymph nodes. Overall, our results demonstrated that metformin reduces the C. difficile infection and can be re-purposed for prevention and treatment of C. difficile infections in clinical settings.

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.

Heterocyst Development and Diazotrophic Growth of Anabaena variabilis under Different Nitrogen Availability.

Nitrogen is globally limiting primary production in the ocean, but some species of cyanobacteria can carry out nitrogen (N) fixation using specialized cells known as heterocysts. However, the effect of N sources and their availability on heterocyst development is not yet fully understood. This study aimed to evaluate the effect of various inorganic N sources on the heterocyst development and cellular growth in an N-fixing cyanobacterium, Anabaena variabilis. Growth rate, heterocyst development, and cellular N content of the cyanobacteria were examined under varying nitrate and ammonium concentrations. A. variabilis exhibited high growth rate both in the presence and absence of N sources regardless of their concentration. Ammonium was the primary source of N in A. variabilis. Even the highest concentrations of both nitrate (1.5 g L−1 as NaNO3) and ammonium (0.006 g L−1 as Fe-NH4-citrate) did not exhibit an inhibitory effect on heterocyst development. Heterocyst production positively correlated with the cell N quota and negatively correlated with vegetative cell growth, indicating that both of the processes were interdependent. Taken together, N deprivation triggers heterocyst production for N fixation. This study outlines the difference in heterocyst development and growth in A. variabilis under different N sources.

Effect of temperature and pH combination on vegetative cell growth of Bacillus megaterium

B. megaterium is a potential bacterial species to be recommended as a probiotic which has been tested to have beneficial effects for fish. Vegetative cells availability is not only influenced by culture media composition but is also affected by environmental conditions such as temperature and pH. The purpose of this study was to determine the effect of temperature and pH combination on vegetative cell production and growth rate of B. megaterium. This study used a combination of temperature treatment (37, 44 and 51 °C) and pH (4, 7 and 10) to produce B. megaterium planted in wheat flour (Triticum aestivum) media. Results showed that the highest vegetative density was found at 37 °C and pH of 10 as much as 11 x 108 cells. mL−1 with a growth rate of 0.65 hour−1. In addition, the morphology of bacteria from this study presented the length and width of B. megaterium at 2.44 – 3.82 µm and 1.11 – 1.31 µm, respectively.

Reuterin disrupts Clostridioides difficile metabolism and pathogenicity through reactive oxygen species generation

ABSTRACT Antibiotic resistance is one of the world’s greatest public health challenges and adjunct probiotic therapies are strategies that could lessen this burden. Clostridioides difficile infection (CDI) is a prime example where adjunct probiotic therapies could decrease disease incidence through prevention. Human-derived Lactobacillus reuteri is a probiotic that produces the antimicrobial compound reuterin known to prevent C. difficile colonization of antibiotic-treated fecal microbial communities. However, the mechanism of inhibition is unclear. We show that reuterin inhibits C. difficile outgrowth from spores and vegetative cell growth, however, no effect on C. difficile germination or sporulation was observed. Consistent with published studies, we found that exposure to reuterin stimulated reactive oxygen species (ROS) in C. difficile, resulting in a concentration-dependent reduction in cell viability that was rescued by the antioxidant glutathione. Sublethal concentrations of reuterin enhanced the susceptibility of vegetative C. difficile to vancomycin and metronidazole treatment and reduced toxin synthesis by C. difficile. We also demonstrate that reuterin is protective against C. difficile toxin-mediated cellular damage in the human intestinal enteroid model. Overall, our results indicate that ROS are essential mediators of reuterin activity and show that reuterin production by L. reuteri is compatible as a therapeutic in a clinically relevant model.

Schizosaccharomyces pombe DNA translocases Rrp1 and Rrp2 have distinct roles at centromeres and telomeres that ensure genome stability.

The regulation of telomere and centromere structure and function is essential for maintaining genome integrity. Schizosaccharomyces pombe Rrp1 and Rrp2 are orthologues of Saccharomyces cerevisiae Uls1, a SWI2/SNF2 DNA translocase and SUMO-targeted ubiquitin ligase. Here, we show that Rrp1 or Rrp2 overproduction leads to chromosome instability and growth defects, a reduction in global histone levels and mislocalisation of centromere-specific histone Cnp1. These phenotypes depend on putative DNA translocase activities of Rrp1 and Rrp2, suggesting that Rrp1 and Rrp2 may be involved in modulating nucleosome dynamics. Furthermore, we confirm that Rrp2, but not Rrp1, acts at telomeres, reflecting a previously described interaction between Rrp2 and Top2. In conclusion, we identify roles for Rrp1 and Rrp2 in maintaining centromere function by modulating histone dynamics, contributing to the preservation of genome stability during vegetative cell growth.

Drivers of Clostridioides difficile hypervirulent ribotype 027 spore germination, vegetative cell growth and toxin production in vitro

ObjectivesClostridioides difficile infection (CDI) is a considerable healthcare and economic burden worldwide. Faecal microbial transplant remains the most effective treatment for CDI, but is not at the present time the recommended standard of care. We hereby investigate which factors derived from a healthy gut microbiome might constitute the colonization resistance barrier (CRB) in the gut, inhibiting CDI. MethodsCRB drivers pH, short chain fatty acid (SCFA), and oxidation–reduction potential (ORP) were investigated in vitro using C. difficile NAP1/BI/027. Readouts for inhibitory mechanisms included germination, growth, toxin production and virulence gene expression. pH ranges (3–7.6), SCFA concentrations (25–200 mM) and ORP (–300 to 200 mV) were manipulated in brain heart infusion broth cultures under anaerobic conditions to assess the inhibitory action of these mechanisms. ResultsA pH < 5.3 completely inhibited C. difficile growth to optical density (OD) 0.019 vs. 1.19 for control pH 7.5. Toxin production was reduced to 25 units vs. 3125 units for pH 7.6 (1 in 5 dilutions). Virulence gene expression reduced by 150-fold compared with pH 7.6 (p < 0.05). Germination and proliferation of spores below pH 6.13 yielded an average OD of 0.006 vs. 0.99 for control. SCFA were potent regulators of toxin production at 25 mM and above (p < 0.05). Acetate significantly inhibited toxin production to 25 units independent of OD (0.8733) vs. control (OD 0.6 and toxin titre 3125) (p < 0.05). ORP did not impact C. difficile growth. ConclusionsThis study highlights the critical role that pH has in the CRB, regulating CDI in vitro and that SCFA can regulate C. difficile function independent of pH.

Repurposing auranofin as a Clostridioides difficile therapeutic.

Clostridioides difficile (previously Clostridium difficile) is the leading cause of nosocomial, antibiotic-associated diarrhoea worldwide. Currently, the gold standard of treatment for C. difficile infection (CDI) is vancomycin or metronidazole, although these antibiotics also perturb the protective resident microbiota, often resulting in disease relapse. Thus, an urgent need remains for the development of new treatment strategies. Auranofin is an FDA-approved oral antirheumatic drug that was previously shown to inhibit C. difficile vegetative cell growth, toxin production and spore production in vitro. To determine the efficacy of auranofin as a CDI therapeutic by examining the effect of treatment on toxin and spore production in vitro and in vivo, and on disease outcomes in mice. C. difficile cultures were treated with auranofin and examined for effects on sporulation and toxin production by sporulation assay and ELISA, respectively. Mice were pretreated with auranofin prior to infection with C. difficile and monitored for physiological conditions, survival and gut damage compared with control animals. Faeces from mice were analysed to determine whether auranofin reduces sporulation and toxin production in vivo. Auranofin significantly reduces sporulation and toxin production under in vitro conditions and in infected mice in vivo. Mice treated with auranofin lost less weight, displayed a significant increase in survival rates and had significantly less toxin-mediated damage in their colon and caecum compared with control mice. Auranofin shows promise as a prospective therapeutic option for C. difficile infections.

Investigating the effect of supplementation on Clostridioides (Clostridium) difficile spore recovery in two solid agars

BackgroundA variety of supplemented solid media are used within Clostridium difficile research to optimally recover spores. Our study sought to investigate different media and additives, providing a method of optimised C. difficile spore recovery. Additionally, due to the results observed in the initial experiments, the inhibitory effects of three amino acids (glycine, l-histidine &l-phenylalanine) on C. difficile spore outgrowth were investigated. MethodsSpores of five C. difficile strains (PCR ribotypes 001,015,020,027,078) were recovered on two commonly used solid media (BHI & CCEY, or cycloserine-cefoxitin egg yolk) supplemented with various concentrations of germinants (taurocholate, glycine & lysozyme). Agar-incorporation minimum inhibitory concentration (MIC) testing was carried out for glycine and taurocholate on vegetative cells and spores of all five strains. Additionally a BHI broth microassay method was utilised to test the growth of C. difficile in the presence of increasing concentrations (0,1,2,3,4%) of three amino acids (glycine,l-histidine,l-phenyalanine). ResultsCCEY agar alone and BHI supplemented with taurocholate (0.1/1%) provided optimal recovery for C. difficile spores. Glycine was inhibitory to spore recovery at higher concentrations, although these varied between the two media used. In agar-incorporated MIC testing, glycine concentrations higher than 2% (20 g/L) were inhibitory to both C. difficile spore and vegetative cell growth versus the control (mean absorbance = 0.33 ± 0.02 vs 0.12 ± 0.01) (P < 0.001). This indicates a potential mechanism whereby glycine interferes with vegetative cell growth. Further microbroth testing provided evidence of inhibition by two amino acids other than glycine, l-histidine and l-phenylalanine. ConclusionsWe provide two media for optimal recovery of C. difficile spores (CCEY alone and BHI supplemented with 0.1/1% taurocholate). CCEY is preferred for isolation from faecal samples. For pure cultures, either CCEY or supplemented BHI agar are appropriate. The inhibitory nature of three amino acids (glycine,l-histidine,l-phenylalanine) to C. difficile vegetative cell proliferation is also highlighted.

Stability of the antimicrobial activity of acetogenins from avocado seed, under common food processing conditions, against Clostridium sporogenes vegetative cell growth and endospore germination

SummaryAnticlostridial activity of acetogenins from avocado seed was further characterised, and their stability and effectiveness under food processing conditions, and in a model food system, studied for the first time. Isolated AcO‐avocadenyne (1) and AcO‐avocadene (2) showed anticlostridial potential, particularly the latter molecule. Enriched acetogenins extracts, obtained at laboratory scale (EAE) and semi‐commercially (Avosafe®), presented similar MIC values (3.9–9.8 ppm) and a bacteriostatic effect. Extracts bioactivity showed resistance to heat (≤120 °C), high hydrostatic pressure (HHP; 300–600 MPa, 3–6 min, 25 °C) and salt (≤3% w/v). In addition, the extract was most stable at pH ≥ 7.0 and potency against endospores increased after HHP treatment and exposure to pH 9.5, suggesting a positive effect on solubility or structure of particular acetogenins. In a model food system processed by HHP, acetogenins were retained; however, initial quantities gradually declined by 63% and 32% at 25 and 4 °C, respectively, at the end of the storage period (42 days). Most stable molecules (persediene (4) &gt; persenones &gt; AcO‐avocadene (2)) possess a keto or trans‐enone group at C‐4 in the aliphatic chain, which could support hydrogen donation to surrounding carbon atoms and confer antioxidant activity. Active endospores were completely inhibited by 5000 ppm Avosafe® in the model food system (37 °C, 72 h) and lower concentrations (500–1000 ppm) resulted in 1–2 log reduction of a 3 log inoculum target. Efficacy information generated in the present work is considered crucial to improve scientific knowledge on spore inhibition properties of avocado acetogenins.

An Amino Acid Substitution in RNA Polymerase That Inhibits the Utilization of an Alternative Sigma Factor.

Sigma (σ) factors direct gene transcription by binding to and determining the promoter recognition specificity of RNA polymerase (RNAP) in bacteria. Genes transcribed under the control of alternative sigma factors allow cells to respond to stress and undergo developmental processes, such as sporulation in Bacillus subtilis, in which gene expression is controlled by a cascade of alternative sigma factors. Binding of sigma factors to RNA polymerase depends on the coiled-coil (or clamp helices) motif of the β' subunit. We have identified an amino acid substitution (L257P) in the coiled coil that markedly inhibits the function of σH, the earliest-acting alternative sigma factor in the sporulation cascade. Cells with this mutant RNAP exhibited an early and severe block in sporulation but not in growth. The mutant was strongly impaired in σH-directed gene expression but not in the activity of the stress-response sigma factor σB Pulldown experiments showed that the mutant RNAP was defective in associating with σH but could still associate with σA and σB The differential effects of the L257P substitution on sigma factor binding to RNAP are likely due to a conformational change in the β' coiled coil that is specifically detrimental for interaction with σH This is the first example, to our knowledge, of an amino acid substitution in RNAP that exhibits a strong differential effect on a particular alternative sigma factor.IMPORTANCE In bacteria, all transcription is mediated by a single multisubunit RNA polymerase (RNAP) enzyme. However, promoter-specific transcription initiation necessitates that RNAP associates with a σ factor. Bacteria contain a primary σ factor that directs transcription of housekeeping genes and alternative σ factors that direct transcription in response to environmental or developmental cues. We identified an amino acid substitution (L257P) in the B. subtilis β' subunit whereby RNAPL257P associates with some σ factors (σA and σB) and enables vegetative cell growth but is defective in utilization of σH and is consequently blocked for sporulation. To our knowledge, this is the first identification of an amino acid substitution within the core enzyme that affects utilization of a specific sigma factor.