Vegetative Cells Research Articles

Hyperbaric inactivation at 150–250 MPa of Alicyclobacillus acidoterrestris spores at room temperature and effect of innovative technologies pre-treatments

This study evaluated hyperbaric inactivation (HI) at 150, 200 and 250 MPa, up to 24 h (at ambient temperatures,18–23 °C) to inactivate Alicyclobacillus acidoterrestris (ACB) spores in apple juice. The effects of pre-treatments on spores by thermal pasteurization (90 °C, 30 s), high pressure processing (600 MPa, 3 min, HPP), pulsed electric field (30 kV, 80 μs, 1400 Hz, PEF), and ultrasound (67 W, 20 kHz, 5 min, US) prior to HI was evaluated. The results were fitted to non-linear inactivation kinetic models, including Biphasic, Log-logistic, and Weibull. Without a previous pre-treatment, ACB spores were reduced ≈4.56 log units by HI after 24 h, regardless of the pressure level (initial load around 6 log CFU/mL). The pre-treatments did not affect ACB spores during HI (≈4–5 log units' inactivation after 24 h under pressure), except for PEF that resulted in a lower inactivation (≈3.5 log units after 24 h under pressure). Phase contrast microscopy revealed that the spores were unable to form a vegetative cell during HI. HI at 5 °C resulted in a lower inactivation level, with 3.86, 2.54 and 1.77 log units of inactivation, respectively, at 150, 200 and 250 MPa after 96 h. Industrial relevanceThe results of this work point to the possibility of using HI, before or after conventional thermal pasteurization and other nonthermal technologies (HPP, PEF, and US) to process apple juice, to inactivate ACB spores at room temperature and also under refrigeration. This methodology should be further studied in the context of ACB spores' high thermal stability and its industrial relevance.

A novel immunopharmacological biocompound

The publication is devoted to topical issues of experimental study of new biocompounds, based on the creation of a consortium of biological composite compounds – biological active substances (metabiotics) produced by strains 59T and 60T of saprophytic compounds of the genus Bacillus subtilis. These strains are very promising for the creation of hepatopicts, new medicinal substances, in the creation and development of a new pharmacological class of hepatoprotectors. Previous studies have shown the safety and hepatoprotective effect of biologically active drugs. It is worth noting that the very effective compounds are the produced biologically active substances – metabolites of bacterial origin, on the basis of which it seems appropriate to create a new class of drugs – metabiotics. The absence of vegetative probiotic cells in such compounds will reduce the additional immune load on the body. The combined use of these compounds provides a potentiated pharmacological effect. In this regard, it seemed appropriate, under the conditions of modeling toxic liver damage by carbon tetrachloride, to conduct an experimental assessment of the immunotropic effect of biologically active substances (BAS) on laboratory animals in order to confirm the effect on cellular factors of immunity. The purpose of the study was to study the effect of the combined use of dietary supplements produced by Bacillus subtilis microorganisms on indicators of cellular immunity in laboratory animals with toxic liver damage. Acute toxic hepatitis was reproduced in white laboratory rats with repeated intragastric administration of carbon tetrachloride. The comparison drug was a registered hepatoprotective drug – ursosan. The immunotropic effect was assessed using factors such as: phagocytic activity of macrophages and neutrophils (FA), NBT test, quantitative assessment of antibody-forming cells, T and B lymphocytes. As a result of the studies performed, reliable data were obtained on an increase in the number of T and B lymphocytes, antibody-forming cells, as well as an increase in FA, which confirms the activation of all parts of cellular immunity in conditions of acute toxic liver damage. The data obtained allow us to recommend the studied biocompound for the creation of new drug candidates of microbiological origin, hepatoprotective agents with immunotropic effects.

Fgk3, a Glycogen Synthase Kinase, Regulates Chitin Synthesis through the Carbon Catabolite Repressor FgCreA in Fusarium graminearum.

The glycogen synthase kinase-3 (GSK3) orthologs are well-conserved in eukaryotic organisms. However, their functions remain poorly characterized in filamentous fungi. In our previous study, we unveiled the function of Fgk3, the GSK3 ortholog, in glycogen metabolism in Fusarium graminearum, the causal agent of Fusarium head blight. Interestingly, the fgk3 mutant was unstable and tended to produce fast-growing suppressors, including secondary suppressors. Using whole-genome sequencing, we identified suppressor mutations in FgCHS5, FgFKS1, FgCREA, FgSSN6, FgRGR1, and FgPP2A in nine primary and four secondary suppressors. Subsequently, we validated that deletion of FgCHS5 or FgCREAΔH253 mutation partially suppressed the defects of fgk3 in vegetative growth and cell wall integrity, suggesting that Fgk3 may regulate the chitin synthesis through FgCreA-mediated transcriptional regulation in F. graminearum. Accordingly, the FGK3 deletion led to hyphal swelling with abnormal chitin deposition, and deletion of FGK3 or FgCREA caused the upregulation of the expression of chitin synthases FgCHS5 and FgCHS6. The interaction between Fgk3 and FgCreA was verified by Yeast two-hybrid and Co-Immunoprecipitation assays. More importantly, we verified that the nuclear localization and protein stability of FgCreA relies on the Fgk3 kinase, while the H253 deletion facilitated the re-localization of FgCreA to the nucleus in the fgk3 mutant background, potentially contributing to the suppression of the fgk3 mutant's defects. Intriguingly, the ΔH253 mutation of FgCreA, identified in suppressor mutant S3, is adjacent to a conserved phosphorylation site, S254, suggesting that this mutation may inhibit the S254 phosphorylation and promote the nuclear localization of FgCreA. Collectively, our findings indicate that the glycogen synthase kinase Fgk3 regulates the chitin synthesis through the carbon catabolite repressor FgCreA in F. graminearum.

Altruistic feeding and cell-cell signaling during bacterial differentiation actively enhance phenotypic heterogeneity.

Starvation triggers bacterial spore formation, a committed differentiation program that transforms a vegetative cell into a dormant spore. Cells in a population enter sporulation nonuniformly to secure against the possibility that favorable growth conditions, which put sporulation-committed cells at a disadvantage, may resume. This heterogeneous behavior is initiated by a passive mechanism: stochastic activation of a master transcriptional regulator. Here, we identify a cell-cell communication pathway containing the proteins ShfA (YabQ) and ShfP (YvnB) that actively promotes phenotypic heterogeneity, wherein Bacillus subtilis cells that start sporulating early use a calcineurin-like phosphoesterase to release glycerol, which simultaneously acts as a signaling molecule and a nutrient to delay nonsporulating cells from entering sporulation. This produced a more diverse population that was better poised to exploit a sudden influx of nutrients compared to those generating heterogeneity via stochastic gene expression alone. Although conflict systems are prevalent among microbes, genetically encoded cooperative behavior in unicellular organisms can evidently also boost inclusive fitness.

Microbial Food Safety of Sous Vide Cooking Processes of Chicken and Eggs.

Sous vide cooking implies cooking foods, packed under vacuum conditions, at controlled temperatures (<80 °C). Although this method opens a new window of culinary possibilities, it also involves a series of risks, mainly microbiologically related, that must be assessed. The aim of this work was to evaluate the effectiveness of SV processes to inactivate three important foodborne pathogens (Campylobacter, Salmonella, and Clostridium spores) in chicken breast and eggs (omelet). For this purpose, two levels of inoculation (102 and 106 CFU/g), two different recipes, and two distinct treatments (with and without storage) for each food were studied. After treatments and storage, the corresponding microbiological counts were performed with standard methods. Average inactivation rates observed were 1.70, 4.82, and 4.34 log for Clostridium spores, Campylobacter, and Salmonella, respectively. No significant differences in microbial inactivation were perceived between the different recipes (food composition) or treatments, except for Clostridium spores, which showed a higher inactivation rate (2.30 log) when samples were stored. In general, preliminary results showed that, although appropriate levels of inactivation are reached for vegetative pathogenic cells, in some cases (spores in breast and Salmonella in eggs), the remaining microbiological risks should be considered and further studied, especially if long-term storage is planned.

Myxococcus xanthus fruiting body morphology is important for spore recovery after exposure to environmental stress.

Environmental microorganisms have evolved a variety of strategies to survive fluctuations in environmental conditions, including the production of biofilms and differentiation into spores. Myxococcus xanthus are ubiquitous soil bacteria that produce starvation-induced multicellular fruiting bodies filled with environmentally resistant spores (a specialized biofilm). Isolated spores have been shown to be more resistant than vegetative cells to heat, ultraviolet radiation, and desiccation. The evolutionary advantage of producing spores inside fruiting bodies is not clear. Here, we examine a hypothesis that the fruiting body provides additional protection from environmental insults. We developed a high-throughput method to compare the recovery (outgrowth) of distinct cell types (vegetative cells, free spores, and spores within intact fruiting bodies) after exposure to ultraviolet radiation or desiccation. Our data indicate that haystack-shaped fruiting bodies protect spores from extended UV radiation but do not provide additional protection from desiccation. Perturbation of fruiting body morphology strongly impedes recovery from both UV exposure and desiccation. These results hint that the distinctive fruiting bodies produced by different myxobacterial species may have evolved to optimize their persistence in distinct ecological niches.IMPORTANCEEnvironmental microorganisms play an important role in the production of greenhouse gases that contribute to changing climate conditions. It is imperative to understand how changing climate conditions feedback to influence environmental microbial communities. The myxobacteria are environmentally ubiquitous social bacteria that influence the local microbial community composition. Defining how these bacteria are affected by environmental insults is a necessary component of predicting climatic feedback effects. When starved, myxobacteria produce multicellular fruiting bodies filled with spores. As spores are resistant to a variety of environmental insults, the evolutionary advantage of building a fruiting body is not clear. Using the model myxobacterium, Myxococcus xanthus, we demonstrate that the tall, haystack-shaped fruiting body morphology enables significantly more resistance to UV exposure than the free spores. In contrast, fruiting bodies are slightly detrimental to recovery from extended desiccation, an effect that is strongly exaggerated if fruiting body morphology is perturbed. These results suggest that the variety of fruiting body morphologies observed in the myxobacteria may dictate their relative resistance to changing climate conditions.

Microfluidic platform for microbial spore germination studies in multiple growth conditions

BackgroundSpores are highly resistant dormant cells, adapted for survival and dispersal, that can withstand unfavourable environmental conditions for extended periods of time and later reactivate. Understanding the germination process of microbial spores is important in numerous areas including agriculture, food safety and health, and other sectors of biotechnology. Microfluidics combined with high-resolution microscopy allows to study spore germination at the single-cell level, revealing behaviours that would be hidden in standard population-level studies.MethodsHere, we present a microfluidic platform – the so-called four-conditions microfluidic chemostat (4CMC) – for germination studies where spores are confined to monolayers inside microchambers, allowing the testing of four growth conditions in parallel. This platform can be used with multiple species, including non-model organisms, and is compatible with existing image analysis software.ResultsIn this study, we focused on three soil dwellers, two bacteria and one fungus, and revealed new insights into their germination. We studied endospores of the model bacterium Bacillus subtilis and demonstrated a correlation between spore density and germination in rich media. We then investigated the germination of the obligate-oxalotrophic environmental bacterium Ammoniphilus oxalaticus in a concentration gradient of potassium oxalate, showing that lower concentrations result in more spores germinating compared to higher concentrations. We also used this microfluidic platform to study the soil beneficial filamentous fungus Trichoderma rossicum, showing for the first time that the size of the spores and hyphae increase in response to increased nutrient availability, while germination times remain the same.DiscussionOur platform allows to better understand microbial behaviour at the single-cell level, under a variety of controlled conditions. While we used it to decipher the responsiveness of soil dwellers’ spores, it would also be suitable for other spores from bacteria or filamentous fungi, but also vegetative cells and yeast, and even microbial communities.

Characterization of the Clostridioides difficile 630Δerm putative Pro-Pro endopeptidase CD1597.

Clostridioides difficile is the leading cause of antibiotic-associated infections worldwide. Within the host, C. difficile can transition from a sessile to a motile state by secreting PPEP-1, which releases the cells from the intestinal epithelium by cleaving adhesion proteins. PPEP-1 belongs to the group of Pro-Pro endopeptidases (PPEPs), which are characterized by their unique ability to cleave proline-proline bonds. Interestingly, another putative member of this group, CD1597, is present in C. difficile. Although it possesses a domain similar to other PPEPs, CD1597 displays several distinct features that suggest a markedly different role for this protein. We investigated the proteolytic activity of CD1597 by testing various potential substrates. In addition, we investigated the effect of the absence of CD1597 by generating an insertional mutant of the cd1597 gene. Using the cd1597 mutant, we sought to identify phenotypic changes through a series of in vitro experiments and quantitative proteomic analyses. Furthermore, we aimed to study the localization of this protein using a fluorogenic fusion protein. Despite its similarities to PPEP-1, CD1597 did not show proteolytic activity. In addition, the absence of CD1597 caused an increase in various sporulation proteins during the stationary phase, yet we did not observe any alterations in the sporulation frequency of the cd1597 mutant. Furthermore, a promoter activity assay indicated a very low expression level of cd1597 in vegetative cells, which was independent of the culture medium and growth stage. The low expression was corroborated by our comprehensive proteomic analysis of the whole cell cultures, which failed to identify CD1597. However, an analysis of purified C. difficile spores identified CD1597 as part of the spore proteome. Hence, we predict that the protein is involved in sporulation, although we were unable to define a precise role for CD1597 in C. difficile.

Freezing stress tolerance of benthic freshwater diatoms from the genus Pinnularia: Comparison of strains from polar, alpine, and temperate habitats.

Diatoms are among the most important primary producers in alpine and polar freshwaters. Although temperate diatoms are sensitive to freezing, polar diatoms often exhibit more resistance. This is particularly true for members of the (predominantly terrestrial) Pinnularia borealis species complex. However, it remains unclear to what extent this resistance applies to other representatives of the genus. Here, we compare the freezing-stress tolerance of 11 freshwater, benthic strains representing different species of Pinnularia (including Caloneis) from polar, alpine, and temperate habitats. As vegetative cells, strains were exposed to freezing temperatures of -4, -10, -20, -40, -80, and -196°C. Survival was assessed by light microscopy and photosynthetic measurements. We observed vegetative cells to be sensitive to low freezing temperatures; only "mild" freezing was survived by all tested strains, and most tested strains did not survive treatments ≤-10°C. However, individual strain sensitivities appeared related to their original habitats. For example, polar and alpine strains better withstood "mild" and "moderate" freezing (-4 and -10°C, respectively); although temperate strains were significantly affected by the "mild" freezing treatment, polar and alpine strains were not. The -10°C treatment was survived exclusively by polar strains, and only P. catenaborealis survived all treatments. Interestingly, this species exhibited the lowest survival in the -10°C treatment, potentially implying some metabolic activity even at freezing temperatures. Thus, despite more extensive sampling throughout the genus and finer temperature scaling compared to previous studies, the remarkable freezing-stress tolerance of the P. borealis species complex remains unique within the genus.

Evaluation of in vitro probiotic properties and colonization of Brevibacillus Laterosporus S62-9 in the intestine of broiler chickens

Brevibacillus laterosporus has been used as a direct-fed microbial additive in chicken feed owing to its strain safety and the ability to produce broad-spectrum antimicrobial peptides. To assess the efficacy and persistence of Brevibacillus laterosporus, it is necessary to track the supplied strains throughout the gastrointestinal tract. This study aims to evaluate the in vitro probiotic properties of Brevibacillus laterosporus S62-9 and its capacity to colonize and persist in the intestines of broiler chickens. Results indicated that the spores of Brevibacillus laterosporus S62-9 exhibited higher tolerance to the digestive condition, and higher adhesion rate to mucus and intestinal cells compared to vegetative cells. The spores remained dormant during digestion in the crop and stomach but germinate in the small intestine with a germination rate exceeding 70%. Subsequently, the whole genome of S62-9 was sequenced, and specific primers were designed and validated based on comparative genome analysis. The quantitative PCR-based method had a detection limit ranging from 2.71 to 7.71 log10 CFU/mL in pure culture and from 3.25 to 9.25 log10 CFU/g in broiler cecal content. In vivo colonization assay indicated that the Brevibacillus laterosporus S62-9 can transiently colonize the broiler cecum. In summary, our results indicated that the Brevibacillus laterosporus S62-9 spores exhibited high tolerance to the digestive tract environment, germinated in the intestine, and establish colonization in the cecum of broilers. The findings provide a theoretical basis for the future application of Brevibacillus laterosporus S62-9 in broiler farming.

MRNA localization and thylakoid protein biogenesis in the filamentous heterocyst-forming cyanobacterium Anabaena sp. PCC 7120.

Heterocyst-forming cyanobacteria such as Anabaena (Nostoc) sp. PCC 7120 exhibit extensive remodeling of their thylakoid membranes during heterocyst differentiation. Here we investigate the sites of translation of thylakoid membrane proteins in Anabaena vegetative cells and developing heterocysts, using mRNA fluorescent in situ hybridization (FISH) to detect the location of specific mRNA species. We probed mRNAs encoding reaction center core components and the heterocyst-specific terminal oxidases Cox2 and Cox3. As in unicellular cyanobacteria, the mRNAs encoding membrane-integral thylakoid proteins are concentrated in patches at the inner face of the thylakoid membrane system, adjacent to the central cytoplasm. These patches mark the putative sites of translation and membrane insertion of these proteins. Oxidase activity in mature heterocysts is concentrated in the specialized "honeycomb" regions of the thylakoid membranes close to the cell poles. However, cox2 and cox3 mRNAs remain evenly distributed over the inner face of the thylakoids, implying that oxidase proteins migrate extensively after translation to reach their destination in the honeycomb membranes. The RNA-binding protein RbpG is the closest Anabaena homolog of Rbp3 in the unicellular cyanobacterium Synechocystis sp. PCC 6803, which we previously showed to be crucial for the correct location of photosynthetic mRNAs. An rbpG null mutant shows decreased cellular levels of photosynthetic mRNAs and photosynthetic complexes, coupled with perturbations to thylakoid membrane organization and lower efficiency of the Photosystem II repair cycle. This suggests that the chaperoning of photosynthetic mRNAs by RbpG is important for the correct coordination of thylakoid protein translation and assembly.IMPORTANCECyanobacteria have a complex thylakoid membrane system which is the site of the photosynthetic light reactions as well as most of the respiratory activity in the cell. Protein targeting to the thylakoids and the spatial organization of thylakoid protein biogenesis remain poorly understood. Further complexity is found in some filamentous cyanobacteria that produce heterocysts, specialized nitrogen-fixing cells in which the thylakoid membranes undergo extensive remodeling. Here we probe mRNA locations to reveal thylakoid translation sites in a heterocyst-forming cyanobacterium. We identify an RNA-binding protein important for the correct co-ordination of thylakoid protein translation and assembly, and we demonstrate the effectiveness of mRNA fluorescent in situ hybridization (FISH) as a way to probe cell-specific gene expression in multicellular cyanobacteria.

Influence of Chitosan on the Viability of Encapsulated and Dehydrated Formulations of Vegetative Cells of Actinomycetes.

This study focuses on developing an encapsulated and dehydrated formulation of vegetative actinobacteria cells for an efficient application in sustainable agriculture, both as a fungicidal agent in crop protection and as a growth-stimulating agent in plants. Three strains of actinobacteria were used: one from a collection (Streptomyces sp.) and two natives to agricultural soil, which were identified as S3 and S6. Vegetative cells propagated in a specific liquid medium for mycelium production were encapsulated in various alginate-chitosan composites produced by extrusion. Optimal conditions for cell encapsulation were determined, and cell damage from air-drying at room temperature was evaluated. The fresh and dehydrated composites were characterized by porosity, functional groups, size and shape, and their ability to protect the immobilized vegetative cells' viability. Actinomycetes were immobilized in capsules of 2.1-2.7 mm diameter with a sphericity index ranging from 0.058 to 0.112. Encapsulation efficiency ranged from 50% to 88%, and cell viability after drying varied between 44% and 96%, depending on the composite type, strain, and airflow. Among the three immobilized and dried strains, S3 and S6 showed greater resistance to encapsulation and drying with a 4 L·min-1 airflow when immobilized in coated and core-shell composites. Encapsulation in alginate-chitosan matrices effectively protects vegetative actinobacteria cells during dehydration, maintaining their viability and functionality for agricultural applications.

Underlying mechanism of the microwave non-thermal effect as additional microbial inactivation on Clostridium sporogenes at pasteurization temperature level

Microwave processing can induce non-thermal effects that can inactivate additional levels of microorganisms at pasteurization temperature level (80–100 °C). The objective of this study was to investigate the mechanism behind the non-thermal effects of microwaves on vegetative cells of Clostridium sporogenes. Cell membrane damage were assessed through nuclear fluorescent dyes and by measuring nuclear acid and protein leakage. The results showed that microwave non-thermal effects and conventional water bath treatment resulted in similar nuclear acid leakage. However, microwave non-thermal effects inflicted more severe damage to the cell membranes of Clostridium sporogenes, resulting in increased protein leakage from both the inner of the cell and the cell membrane (A260 = 0.25 vs. 0.18). Consequently, it can be inferred that the inactivation of additional Clostridium sporogenes by microwave non-thermal effects is primarily attributed to the disruption of cell membranes induced by alternating electromagnetic fields, and this phenomenon is further potentiated at elevated temperatures.

Agar lot-specific inhibition in the plating efficiency of yeast spores and cells

Abstract The fission yeast Schizosaccharomyces pombe and the budding yeast Saccharomyces cerevisiae are highly diverged (530 mya), single-celled, model eukaryotic organisms. Scientists employ mating, meiosis, and the plating of ascospores and cells to generate strains with novel genotypes and to discover biological processes. Our three laboratories encountered independently sudden-onset, major impediments to such research. Spore suspensions and vegetative cells no longer plated effectively on minimal media. By systematically analyzing multiple different media components from multiple different suppliers, we identified the source of the problem. Specific lots of agar were toxic. We report that this sporadic toxicity affects independently the agar stocks of multiple vendors, has occurred repeatedly over at least three decades, and extends to species in highly diverged taxa. Interestingly, the inhibitory effects displayed variable penetrance and were attenuated on rich media. Consequently, quality control checks that use only rich media can provide false assurances on the quality of the agar. Lastly, we describe likely sources of the toxicity and we provide specific guidance for quality control measures that should be applied by all vendors as preconditions for their sale of agar.

Heat resistance differences are common between both vegetative cells and spores of Clostridium perfringens type F isolates carrying a chromosomal vs plasmid-borne enterotoxin gene.

Clostridium perfringens type F isolates utilize C. perfringens enterotoxin (CPE) to cause food poisoning (FP) and nonfoodborne gastrointestinal diseases. The enterotoxin gene (cpe) can be located on either the chromosome or plasmids, but most FP isolates carry a chromosomal cpe (c-cpe) gene. Our 2000 article in Applied and Environmental Microbiology (66:3234-3240, 2000, https://doi.org/10.1128/aem.66.8.3234-3240.2000https://doi.org/10.1128/AEM.66.8.3234-3240.2000) determined that vegetative cells and spores of c-cpe isolates are more heat resistant than those of plasmid cpe (p-cpe) isolates, which is favorable for their survival in improperly cooked or held food. However, that 2000 article was recently retracted (90:e00249-24, 2024, https://doi.org/10.1128/aem.00249-24). To our knowledge, the 2000 article remains the only study reporting that heat resistance differences are common between both vegetative cells and spores of type F c-cpe isolates vs type F p-cpe isolates. To confirm and preserve this information in the literature, the heat resistance portion of the 2000 study has been repeated. The 2024 results reproduced the 2000 results by indicating that, relative to the surveyed type F p-cpe isolates, the vegetative cells of surveyed type F c-cpe isolates are ~2-fold more heat resistant and the spores of most surveyed c-cpe isolates are ~30-fold more heat resistant. However, consistent with several reports since our 2000 paper, one surveyed type F c-cpe isolate (which did not appreciably sporulate in 2000 but sporulated in 2024) produced spores with intermediate heat sensitivity, confirming that spores of some type F c-cpe isolates lack exceptional heat resistance.IMPORTANCEClostridium perfringens type F food poisoning (FP), which is the second most common bacterial cause of FP, involves the production of C. perfringens enterotoxin. While the enterotoxin gene (cpe) can be located on either the chromosome or plasmids in type F isolates, most FP cases are caused by chromosomal cpe isolates. The current results support the conclusion that the vegetative cells and spores of type F chromosomal cpe isolates are often more heat resistant than vegetative cells and spores of type F plasmid cpe isolates. Greater heat resistance should favor the survival of the spores and vegetative cells of those chromosomal cpe isolates in temperature-abused food, which may help explain the strong association of type F chromosomal cpe strains with FP.

Unveiling Microbial Diversity: Raman Spectroscopy's Discrimination of Clostridium and Related Genera.

In the clinical environment, the identification of phylogenetic closely related genera and species like Clostridium and Bacillus spp. is challenging. Both genera contain representatives of pathogenic and nonpathogenic species that need to be distinguished for a proper diagnostic read-out. Therefore, reliable and accurate detection methods must be employed for the correct identification of these genera and species. Despite their high pathogenicity, clostridial infections and food contaminations present significant challenges due to their unique cultivation conditions and developmental needs. Therefore, in many diagnostic protocols, the toxins are used for microbiological documentation. However, the applied laboratory methods suffer in accuracy, sometimes require large bacterial loads to provide reliable results, and cannot differentiate pathogenic from nonpathogenic strains. Here, Raman spectroscopy was employed to create an extensive Raman database consisting of pathogenic and nonpathogenic Bacillus and Clostridium species. These genera, as well as representatives of Paraclostridium and Clostridioides were specifically selected for their phylogenetic relation, cultivation conditions, and metabolic activity. A chemometric evaluation of the Raman spectra of single vegetative cells revealed a high discriminating power at the genus level. However, bacilli are considerably easier to classify at the species level than clostridia. The discrimination between the genera and species was based on their phylogeny and not their aerobic and anaerobic cultivation conditions. These encouraging results demonstrated that Raman spectroscopy coupled with chemometrics is a robust and helpful method for differentiating Clostridium species from Bacillus, Clostridioides, and Paraclostridium species. This approach has the potential to be a valuable tool in clinical diagnostics.

Unraveling the benefits of Bacillus subtilis DSM 29784 poultry probiotic through its secreted metabolites: an in vitro approach.

Probiotics provide beneficial metabolites to its host. Here, we describe the mode of action of a commonly used probiotic in poultry, Bs29784. By using in vitro cellular techniques and simulated chickens' intestinal model, we show the functional link between Bs29784 metabolites and the three lines of animal resilience. Indeed, both Bs29784 vegetative cells and its metabolites stimulate cellular anti-inflammatory responses, strengthen intestinal barrier, and positively modulate microbiota composition and fermentative profile. Taken together, these results strengthen our understanding of the effect of Bs29784 on its host and explain, at least partly, its positive effects on animal health, resilience, and performance.

PSXII-9 Effects of lauric acid- and hops-based products on select microbial populations of aerobically exposed silage

Abstract Air exposed silages can harbor spoilage and pathogenic microbes which can reduce the palatability, nutritive quality and microbial safety of these feedstuffs. Accordingly, effective, environmentally friendly interventions are sought to rescue the biological and economical value of spoiled silages. The objectives of this study were to test the effects of treating air exposed corn silage with LipoVital GL-45, a Berg + Schmidt GmbH &amp; Co. KG (Hamburg, Germany) product containing 45% monoglyceride of lauric acid (mixed within a 50% di/triglyceride base) alone or mixed with either Chinook or Galena hops or with an experimental extract containing hops β-acids. All treatments were administered as a warm water spray to deliver 1.6% product to the silage on a dry matter basis. Silage samples collected after 24 h ambient incubation were serially diluted in 0.4 M sodium phosphate buffer (pH 6.4) and plated to 3M Petrifilm for enumeration of yeast and molds and total aerobes as well as to Rogosa SL for enumeration of lactic acid bacteria and streptococci. Fluid samples from sampled silage were also inoculated in 10-fold increments to triplicate sets of tubes containing anaerobically prepared Reinforced Clostridial agar supplemented cellobiose and xylose (0.05% wt/vol each) for most probable number enumeration of total anaerobes. Populations of presumptive spore-forming aerobes and anaerobes were measured as described above except inocula were heated to 80 oC for 10 min to kill vegetative cells. An analysis of variance (Statistix Analytical Software, Tallahassee, FL, USA) revealed no treatment effects on yeasts and mold populations (P &amp;gt; 0.05) after 24 h air exposure of the silage, with counts averaging (± SD) 5.0 ± 0.7 log10 CFU/g. Silage treated with combinations of LipoVital GL-45 with Galena hops or the β-acid extract had 0.8 to 0.9 log10 units fewer (P &amp;lt; 0.05) lactic acid bacteria after 24 h air exposure than untreated controls (7.4 ± 0.1 log10 CFU/g). Similarly, silage treated with the LipoVital GL-45/Galena hops combination had 0.6 to 0.7 log10 units fewer (P &amp;lt; 0.05) heat-sensitive and presumptive spore-forming aerobes after 24 h air exposure when compared with controls (5.5 ± 0.2 and 5.2 ± 0.2 log10 CFU/g, respectively). Conversely, most probable numbers of nonheated anaerobe populations were lower (P &amp;lt; 0.05) in silage treated with LipoVital GL-45 combined with the β-acid extract when compared with controls (8.6 ± 0.x and 9.9 ± 0.7 log10 cells/g) with all other treatments being similar to controls. Populations of presumptive spore-forming anaerobes in the air exposed silage were unaffected by treatments, averaging 8.3 ± 0.7 log10 cells/g. Results warrant further research to investigate whether optimized dosages and formulations of LipoVital GL-45, Galena hops and β-acid extracts may ultimately yield treatments to prevent spoilage of air-exposed silages.

Nanoscale elemental and morphological imaging of nitrogen-fixing cyanobacteria.

Nitrogen-fixing cyanobacteria bind atmospheric nitrogen and carbon dioxide using sunlight. This experimental study focused on a laboratory-based model system, Anabaena sp., in nitrogen-depleted culture. When combined nitrogen is scarce, the filamentous prokaryotes reconcile photosynthesis and nitrogen fixation by cellular differentiation into heterocysts. To better understand the influence of micronutrients on cellular function, 2D and 3D synchrotron X-ray fluorescence mappings were acquired from whole biological cells in their frozen-hydrated state at the Bionanoprobe, Advanced Photon Source. To study elemental homeostasis within these chain-like organisms, biologically relevant elements were mapped using X-ray fluorescence spectroscopy and energy-dispersive X-ray microanalysis. Higher levels of cytosolic K+, Ca2+, and Fe2+ were measured in the heterocyst than in adjacent vegetative cells, supporting the notion of elevated micronutrient demand. P-rich clusters, identified as polyphosphate bodies involved in nutrient storage, metal detoxification, and osmotic regulation, were consistently co-localized with K+ and occasionally sequestered Mg2+, Ca2+, Fe2+, and Mn2+ ions. Machine-learning-based k-mean clustering revealed that P/K clusters were associated with either Fe or Ca, with Fe and Ca clusters also occurring individually. In accordance with XRF nanotomography, distinct P/K-containing clusters close to the cellular envelope were surrounded by larger Ca-rich clusters. The transition metal Fe, which is a part of nitrogenase enzyme, was detected as irregularly shaped clusters. The elemental composition and cellular morphology of diazotrophic Anabaena sp. was visualized by multimodal imaging using atomic force microscopy, scanning electron microscopy, and fluorescence microscopy. This paper discusses the first experimental results obtained with a combined in-line optical and X-ray fluorescence microscope at the Bionanoprobe.

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.