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1.
Int J Food Microbiol ; 337: 108915, 2021 Jan 16.
Artigo em Inglês | MEDLINE | ID: mdl-33152569

RESUMO

Spore-forming bacteria are implicated in cases of food spoilage or food poisoning. In their sporulated form, they are resistant to physical and chemical treatments applied in the food industry and can persist throughout the food chain. The sporulation leads to an increase in the concentration of resistant forms in final products or food processing equipment. In order to identify sporulation environments in the food industry, it is necessary to be able to predict bacterial sporulation according to environmental factors. As sporulation occurs after bacterial growth, a kinetic model of growth-sporulation was used to describe the evolution of vegetative cells and spores through time. The effects of temperature, pH and water activity on the growth and the sporulation abilities of Bacillus subtilis BSB1 were modelled. The values of the growth boundaries were used as inputs to predict these effects. The good description of the sporulation kinetics by growth parameters suggests that the impact of the studied environmental factors is the same on both physiological process. Suboptimal conditions for growth delay the appearance of the first spores, and spores appear more synchronously in suboptimal conditions for growth. The developed model was also applicable to describe the growth and sporulation curves in changing temperature and pH conditions over time.


Assuntos
Bacillus subtilis/crescimento & desenvolvimento , Microbiologia de Alimentos , Esporos Bacterianos/crescimento & desenvolvimento , Temperatura , Água/química , Manipulação de Alimentos , Concentração de Íons de Hidrogênio , Cinética
2.
PLoS One ; 15(10): e0241200, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33104721

RESUMO

Quantification of phenotypic heterogeneity present amongst bacterial cells can be a challenging task. Conventionally, classification and counting of bacteria sub-populations is achieved with manual microscopy, due to the lack of alternative, high-throughput, autonomous approaches. In this work, we apply classification-type convolutional neural networks (cCNN) to classify and enumerate bacterial cell sub-populations (B. subtilis clusters). Here, we demonstrate that the accuracy of the cCNN developed in this study can be as high as 86% when trained on a relatively small dataset (81 images). We also developed a new image preprocessing algorithm, specific to fluorescent microscope images, which increases the amount of training data available for the neural network by 72 times. By summing the classified cells together, the algorithm provides a total cell count which is on parity with manual counting, but is 10.2 times more consistent and 3.8 times faster. Finally, this work presents a complete solution framework for those wishing to learn and implement cCNN in their synthetic biology work.


Assuntos
Carga Bacteriana/métodos , Processamento de Imagem Assistida por Computador , Microscopia de Fluorescência/métodos , Redes Neurais de Computação , Algoritmos , Bacillus subtilis/crescimento & desenvolvimento
3.
Nat Commun ; 11(1): 5078, 2020 10 08.
Artigo em Inglês | MEDLINE | ID: mdl-33033266

RESUMO

Metabolic engineering facilitates chemical biosynthesis by rewiring cellular resources to produce target compounds. However, an imbalance between cell growth and bioproduction often reduces production efficiency. Genetic code expansion (GCE)-based orthogonal translation systems incorporating non-canonical amino acids (ncAAs) into proteins by reassigning non-canonical codons to ncAAs qualify for balancing cellular metabolism. Here, GCE-based cell growth and biosynthesis balance engineering (GCE-CGBBE) is developed, which is based on titrating expression of cell growth and metabolic flux determinant genes by constructing ncAA-dependent expression patterns. We demonstrate GCE-CGBBE in genome-recoded Escherichia coli Δ321AM by precisely balancing glycolysis and N-acetylglucosamine production, resulting in a 4.54-fold increase in titer. GCE-CGBBE is further expanded to non-genome-recoded Bacillus subtilis to balance growth and N-acetylneuraminic acid bioproduction by titrating essential gene expression, yielding a 2.34-fold increase in titer. Moreover, the development of ncAA-dependent essential gene expression regulation shows efficient biocontainment of engineered B. subtilis to avoid unintended proliferation in nature.


Assuntos
Acetilglucosamina/metabolismo , Bacillus subtilis/crescimento & desenvolvimento , Vias Biossintéticas , Escherichia coli/crescimento & desenvolvimento , Ácido N-Acetilneuramínico/metabolismo , Bacillus subtilis/metabolismo , Proliferação de Células , Escherichia coli/metabolismo , Código Genético , Proteínas de Fluorescência Verde/metabolismo , Engenharia Metabólica , Análise do Fluxo Metabólico , Regiões Promotoras Genéticas/genética , RNA de Transferência/genética , Tirosina/metabolismo
4.
J Dairy Sci ; 103(11): 9992-10000, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32921470

RESUMO

Current cleaning and sanitation protocols may not be adequately effective in cleaning separation membranes and can result in the formation of resilient multispecies biofilms. The matured biofilms may result in a bacterial predominance with resilient strains on membranes with a prolonged use. In our previous study, we isolated organisms such as Bacillus subtilis, Bacillus licheniformis, Exiguobacterium aurantiacum, and Acinetobacter radioresistens from an 18-mo-old reverse osmosis membrane. The competitive exclusion studies revealed the predominance of B. subtilis within the membrane biofilm microflora. This study investigated the antimicrobial activity of the B. subtilis isolate as a potential cause of its predominance. The culture isolate was propagated in tryptic soy broth at 37°C, and microfiltered to prepare cell-free extracts (CFE) at 8-, 10-, 12-, 14-, 16-, and 18-h intervals. The CFE were freeze-dried and suspended in minimum quantities of HPLC-grade water to prepare concentrated solutions. The antimicrobial activities of CFE were tested using the agar-well assay against the biofilm constitutive microflora. The experiments were conducted in triplicates and means were compared for significant differences using a general linear mixed model procedure. The results indicated the highest antimicrobial activity of 12-h CFE of B. subtilis against other constitutive microflora such as Exiguobacterium sp., E. auranticum, and A. radioresistens, with average inhibition zone sizes of 16.5 ± 0.00, 16.25 ± 0.66, and 20.6 ± 0.00 mm, respectively. Upon treatment with proteinase K, the CFE completely lost its antimicrobial activity, establishing it to be a proteinaceous compound. The AA profiling revealed the total crude protein in CFE to be 51% (wt/wt), with its major constituent as glutamic acid (11.30% wt/wt). The freeze-dried CFE was thermally stable on exposure to the common temperature used for sanitizer applications (23.8°C for 5 and 10 min) and over a pH range of 3.0 to 6.3. The study helped us understand the role of the antimicrobial compound produced by B. subtilis as a potential cause of its predominance within the biofilm constitutive microflora.


Assuntos
Anti-Infecciosos/farmacologia , Bacillus subtilis/química , Biofilmes/crescimento & desenvolvimento , Soro do Leite/microbiologia , Acinetobacter/crescimento & desenvolvimento , Acinetobacter/isolamento & purificação , Anti-Infecciosos/isolamento & purificação , Bacillus licheniformis/crescimento & desenvolvimento , Bacillus licheniformis/isolamento & purificação , Bacillus subtilis/crescimento & desenvolvimento , Bacillus subtilis/isolamento & purificação , Bacillus subtilis/fisiologia , Biofilmes/efeitos dos fármacos , Caseínas , Filtros Microporos/microbiologia , Osmose , Hidrolisados de Proteína
5.
Appl Environ Microbiol ; 86(22)2020 10 28.
Artigo em Inglês | MEDLINE | ID: mdl-32917750

RESUMO

Iron (Fe) is one of the most important micronutrients for most life forms on earth. While abundant in soil, Fe bioavailability in oxic soil is very low. Under environmental conditions, bacteria need to acquire sufficient Fe to sustain growth while limiting the energy cost of siderophore synthesis. Biofilm formation might mitigate this Fe stress, since it was shown to accumulate Fe in certain Gram-negative bacteria and that this Fe could be mobilized for uptake. However, it is still unclear if, and to what extent, the amount of Fe accumulated in the biofilm can sustain growth and if the mobilization of this local Fe pool is modulated by the availability of environmental Fe (i.e., Fe outside the biofilm matrix). Here, we use a nondomesticated strain of the ubiquitous biofilm-forming soil bacterium Bacillus subtilis and stable Fe isotopes to precisely evaluate the origin of Fe during growth in the presence of tannic acid and hydroxides, used as proxies for different environmental conditions. We report that this B. subtilis strain can accumulate a large quantity of Fe in the biofilm, largely exceeding Fe associated with cells. We also report that only a fraction of biofilm-bound Fe is available for uptake in the absence of other sources of Fe in the vicinity of the biofilm. We observed that the availability of environmental Fe modulates the usage of this pool of biofilm-bound Fe. Finally, our data suggest that consumption of biofilm-bound Fe relates to the efficacy of B. subtilis to transport Fe from the environment to the biofilm, possibly through siderophores.IMPORTANCE Recent pieces of evidence suggest that Fe bound to the biofilm could assume at least two important functions, a local source of Fe for uptake and a support to extracellular metabolism, such as extracellular electron transfer. Our results show that B. subtilis can use biofilm-bound Fe for uptake only if it does not compromise Fe homeostasis of the biofilm, i.e., maintains a minimum Fe concentration in the biofilm for extracellular purposes. We propose a theoretical framework based on our results and recent literature to explain how B. subtilis manages biofilm-bound Fe and Fe uptake in response to environmental Fe availability. These results provide important insights into the management of biofilm-bound and environmental Fe by B. subtilis in response to Fe stress.


Assuntos
Bacillus subtilis/fisiologia , Biofilmes , Ferro/metabolismo , Bacillus subtilis/crescimento & desenvolvimento , Transporte Biológico
6.
PLoS One ; 15(9): e0238689, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32903284

RESUMO

MOTIVATION: Determining intracellular metabolic flux through isotope labeling techniques such as 13C metabolic flux analysis (13C-MFA) incurs significant cost and effort. Previous studies have shown transcriptomic data coupled with constraint-based metabolic modeling can determine intracellular fluxes that correlate highly with 13C-MFA measured fluxes and can achieve higher accuracy than constraint-based metabolic modeling alone. These studies, however, used validation data limited to E. coli and S. cerevisiae grown on glucose, with significantly similar flux distribution for central metabolism. It is unclear whether those results apply to more diverse metabolisms, and therefore further, extensive validation is needed. RESULTS: In this paper, we formed a dataset of transcriptomic data coupled with corresponding 13C-MFA flux data for 21 experimental conditions in different unicellular organisms grown on varying carbon substrates and conditions. Three computational flux-balance analysis (FBA) methods were comparatively assessed. The results show when uptake rates of carbon sources and key metabolites are known, transcriptomic data provides no significant advantage over constraint-based metabolic modeling (average correlation coefficients, transcriptomic E-Flux2 0.725 and SPOT 0.650 vs non-transcriptomic pFBA 0.768). When uptake rates are unknown, however, predictions obtained utilizing transcriptomic data are generally good and significantly better than those obtained using constraint-based metabolic modeling alone (E-Flux2 0.385 and SPOT 0.583 vs pFBA 0.237). Thus, transcriptomic data coupled with constraint-based metabolic modeling is a promising method to obtain intracellular flux estimates in microorganisms, particularly in cases where uptake rates of key metabolites cannot be easily determined, such as for growth in complex media or in vivo conditions.


Assuntos
Bactérias/genética , Ciclo do Carbono/genética , Transcriptoma/genética , Bacillus subtilis/efeitos dos fármacos , Bacillus subtilis/genética , Bacillus subtilis/crescimento & desenvolvimento , Bactérias/efeitos dos fármacos , Bactérias/crescimento & desenvolvimento , Carbono/farmacologia , Ciclo do Carbono/efeitos dos fármacos , Árvores de Decisões , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Synechococcus/efeitos dos fármacos , Synechococcus/genética , Synechococcus/crescimento & desenvolvimento , Synechocystis/efeitos dos fármacos , Synechocystis/genética , Synechocystis/crescimento & desenvolvimento
7.
Nat Commun ; 11(1): 4149, 2020 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-32811832

RESUMO

Many bacteria can form wall-deficient variants, or L-forms, that divide by a simple mechanism that does not require the FtsZ-based cell division machinery. Here, we use microfluidic systems to probe the growth, chromosome cycle and division mechanism of Bacillus subtilis L-forms. We find that forcing cells into a narrow linear configuration greatly improves the efficiency of cell growth and chromosome segregation. This reinforces the view that L-form division is driven by an excess accumulation of surface area over volume. Cell geometry also plays a dominant role in controlling the relative positions and movement of segregating chromosomes. Furthermore, the presence of the nucleoid appears to influence division both via a cell volume effect and by nucleoid occlusion, even in the absence of FtsZ. Our results emphasise the importance of geometric effects for a range of crucial cell functions, and are of relevance for efforts to develop artificial or minimal cell systems.


Assuntos
Bacillus subtilis/crescimento & desenvolvimento , Divisão Celular/fisiologia , Segregação de Cromossomos/fisiologia , Formas L/crescimento & desenvolvimento , Dispositivos Lab-On-A-Chip/microbiologia , Bacillus subtilis/citologia , Bacillus subtilis/fisiologia , Parede Celular/fisiologia , Cromossomos Bacterianos/metabolismo , Cromossomos Bacterianos/fisiologia , Formas L/citologia , Formas L/fisiologia , Modelos Biológicos
8.
Nature ; 584(7821): 470-474, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32669712

RESUMO

The rate of cell growth is crucial for bacterial fitness and drives the allocation of bacterial resources, affecting, for example, the expression levels of proteins dedicated to metabolism and biosynthesis1,2. It is unclear, however, what ultimately determines growth rates in different environmental conditions. Moreover, increasing evidence suggests that other objectives are also important3-7, such as the rate of physiological adaptation to changing environments8,9. A common challenge for cells is that these objectives cannot be independently optimized, and maximizing one often reduces another. Many such trade-offs have indeed been hypothesized on the basis of qualitative correlative studies8-11. Here we report a trade-off between steady-state growth rate and physiological adaptability in Escherichia coli, observed when a growing culture is abruptly shifted from a preferred carbon source such as glucose to fermentation products such as acetate. These metabolic transitions, common for enteric bacteria, are often accompanied by multi-hour lags before growth resumes. Metabolomic analysis reveals that long lags result from the depletion of key metabolites that follows the sudden reversal in the central carbon flux owing to the imposed nutrient shifts. A model of sequential flux limitation not only explains the observed trade-off between growth and adaptability, but also allows quantitative predictions regarding the universal occurrence of such tradeoffs, based on the opposing enzyme requirements of glycolysis versus gluconeogenesis. We validate these predictions experimentally for many different nutrient shifts in E. coli, as well as for other respiro-fermentative microorganisms, including Bacillus subtilis and Saccharomyces cerevisiae.


Assuntos
Adaptação Fisiológica , Meio Ambiente , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/metabolismo , Acetatos/metabolismo , Bacillus subtilis/citologia , Bacillus subtilis/crescimento & desenvolvimento , Bacillus subtilis/metabolismo , Divisão Celular , Escherichia coli/enzimologia , Escherichia coli/genética , Fermentação , Gluconeogênese , Glucose/metabolismo , Glicólise , Metabolômica , Modelos Biológicos , Mutação , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo
9.
Can J Microbiol ; 66(11): 664-669, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32497447

RESUMO

Bacillus subtilis and Bacillus velezensis are used in organic agriculture as an alternative to chemical pesticides to fight against phytopathogen organisms. These Gram-positive soil-dwelling bacteria are able to resist harsh conditions and survive by differentiating into endospores. Few studies have examined how bacterial populations change on plants over time, and whether they remain active or enter a dormant state. Nonetheless, these characteristics are strikingly important to determine the usage of B. subtilis and B. velezensis and their efficacy in environmental conditions. Here, we investigated the population dynamics of B. subtilis NCIB3610 and B. velezensis QST713 when applied as spores on different ornamental plants. We report that on all the plants studied (Echinacea purpurea 'Salsa red', Echinacea purpurea 'Fatal attraction', and Lavandula angustifolia 'Hidecote blue'), spores rapidly germinated and colonized the rhizoplane, maintaining a relatively low proportion of spores in the population over time, whereas the bacterial population on the leaves rapidly declined. Bacteria in the surrounding soil did not germinate and persisted as spores. Taken together, these results suggest that only cells found at the rhizosphere remain metabolically active to allow the formation of a lasting relationship with the plant, making possible beneficial effects from the inoculated bacteria.


Assuntos
Bacillus subtilis/crescimento & desenvolvimento , Bacillus/crescimento & desenvolvimento , Plantas/microbiologia , Contagem de Colônia Microbiana , Folhas de Planta/microbiologia , Rizosfera , Esporos Bacterianos/crescimento & desenvolvimento
10.
J Food Sci ; 85(7): 2164-2170, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32572963

RESUMO

Sanguisorba officinalis L. (family Rosaceae, subfamily Rosoideae) is a plant found throughout Southern Europe, Northern Africa, and Eastern Asia. This study demonstrated the antibacterial activity of a purified polyphenolic extract (PPE) from S. officinalis L. against Bacillus subtilis using growth inhibitory and apoptosis assays, and investigated the antibacterial mechanism responsible for changes in cell membrane properties. Fourier transform infrared spectroscopy suggested that PPE altered the cell wall and membrane properties of B. subtilis. Further determination of cell membrane integrity and permeability revealed that B. subtilis membrane integrity was more severely damaged by PPE at the minimum inhibitory concentration (MIC) than at the minimum bactericidal concentrati on (MBC). Instead, PPE at the MBC reduced cell membrane fluidity by significantly decreasing the proportion of anteiso- and iso-branched phospholipid fatty acids (PLFAs) from 64.17 ± 0.28% and 27.23 ± 0.03% in the control to 5.57 ± 1.06% and 6.00 ± 1.40%, respectively (P < 0.001). Scanning electron microscopy revealed different effects of PPE on cell morphology, demonstrating that, at the MIC and MBC, PPE exerted antibacterial activity by disrupting the cell membrane and reducing cell membrane fluidity, respectively. Consequently, this study elucidated changes in the bacterial membrane due to exposure to PPE and its potential use as an antimicrobial agent. PRACTICAL APPLICATION: The abuse of synthetic chemical preservatives raises food safety concerns; however, plant-derived polyphenolic compounds may be a safe and effective alternative. This study demonstrated the strong antibacterial activity of a purified polyphenolic extract (PPE) of Sanguisorba officinalis L. and revealed its antibacterial mechanism against Bacillus subtilis, suggesting that it may provide a useful antimicrobial agent in food industry applications.


Assuntos
Antibacterianos/farmacologia , Bacillus subtilis/efeitos dos fármacos , Membrana Celular/metabolismo , Ácidos Graxos/metabolismo , Fosfolipídeos/metabolismo , Extratos Vegetais/farmacologia , Polifenóis/farmacologia , Sanguisorba/química , Bacillus subtilis/crescimento & desenvolvimento , Bacillus subtilis/metabolismo , Membrana Celular/efeitos dos fármacos , Parede Celular/efeitos dos fármacos , Parede Celular/metabolismo , Ácidos Graxos/química , Conservantes de Alimentos/farmacologia , Testes de Sensibilidade Microbiana , Fosfolipídeos/química
11.
J Food Sci ; 85(6): 1799-1804, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32458576

RESUMO

Demand for antimicrobial packaging films is growing due to public attention to food safety. The structures and properties of gellan gum-guar gum blend films incorporated with nisin were investigated in this paper. Fourier transform infrared spectroscopy, rheological analyses showed intermolecular interactions among gellan gum, guar gum, and nisin. Furthermore, scanning electron microscopy and thermogravimetric analysis also indicated higher compatibility of the blend film components and better thermal stability than the gellan gum film. Tensile strength (TS), elongation at break (EAB) and water vapor permeability (WVP) of the blend films were enhanced with the addition of guar gum. The TS of the blend film reached 2.89 × 103  MPa, the EAB increased to 67.99%, and the WVP increased to 1.80 × 10-5  g/mm·s·Pa. Additionally, the film with nisin had antibacterial activity for Bacillus subtilis. The results demonstrated that a homogenous and smooth antimicrobial film with gellan gum, guar gum, and nisin could be a good option of antimicrobial packaging film for food preservation. PRACTICAL APPLICATION: This work investigated blend package films of gellan gum and guar gum incorporated with nisin. The results showed compatibility and thermal stability of the film were improved with adding a certain amount of guar gum, and also antibacterial activity for Bacillus subtilis of the blend film with nisin. Therefore, it can be used to the development of antimicrobial packaging films.


Assuntos
Embalagem de Alimentos/instrumentação , Galactanos/química , Mananas/química , Nisina/química , Gomas Vegetais/química , Polissacarídeos Bacterianos/química , Antibacterianos/química , Antibacterianos/farmacologia , Bacillus subtilis/efeitos dos fármacos , Bacillus subtilis/crescimento & desenvolvimento , Plásticos Biodegradáveis/química , Plásticos Biodegradáveis/farmacologia , Microscopia Eletrônica de Varredura , Nisina/farmacologia , Permeabilidade , Espectroscopia de Infravermelho com Transformada de Fourier , Vapor/análise , Resistência à Tração
12.
BMC Mol Cell Biol ; 21(1): 35, 2020 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-32357828

RESUMO

BACKGROUND: Fluorescence microscopy is a powerful tool in cell biology, especially for the study of dynamic processes. Intensive irradiation of bacteria with UV, blue and violet light has been shown to be able to kill cells, but very little information is available on the effect of blue or violet light during live-cell imaging. RESULTS: We show here that in the model bacterium Bacillus subtilis chromosome segregation and cell growth are rapidly halted by standard violet (405 nm) and blue light (CFP) (445-457 nm) excitation, whereas they are largely unaffected by green light (YFP). The stress sigma factor σB and the blue-light receptor YtvA are not involved in growth arrest. Using synchronized B. subtilis cells, we show that the use of blue light for fluorescence microscopy likely induces non-specific toxic effects, rather than a specific cell cycle arrest. Escherichia coli and Caulobacter crescentus cells also stop to grow after 15 one-second exposures to blue light (CFP), but continue growth when imaged under similar conditions in the YFP channel. In the case of E. coli, YFP excitation slows growth relative to white light excitation, whereas CFP excitation leads to cell death in a majority of cells. Thus, even mild violet/blue light excitation interferes with bacterial growth. Analyzing the dose-dependent effects of violet light in B. subtilis, we show that short exposures to low-intensity violet light allow for continued cell growth, while longer exposures do not. CONCLUSIONS: Our experiments show that care must be taken in the design of live-cell imaging experiments in that violet or blue excitation effects must be closely controlled during and after imaging. Violet excitation during sptPALM or other imaging studies involving photoactivation has a threshold, below which little effects can be seen, but above which a sharp transition into cell death occurs. YFP imaging proves to be better suited for time-lapse studies, especially when cell cycle or cell growth parameters are to be examined.


Assuntos
Bacillus subtilis/crescimento & desenvolvimento , Bacillus subtilis/efeitos da radiação , Caulobacter crescentus/efeitos da radiação , Escherichia coli/efeitos da radiação , Microscopia de Fluorescência , Imagem com Lapso de Tempo , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Caulobacter crescentus/crescimento & desenvolvimento , Pontos de Checagem do Ciclo Celular/efeitos da radiação , Cor , Escherichia coli/crescimento & desenvolvimento , Luz , Proteínas Luminescentes/toxicidade , Fator sigma/metabolismo , Fatores de Tempo
13.
Int J Food Microbiol ; 326: 108650, 2020 Aug 02.
Artigo em Inglês | MEDLINE | ID: mdl-32402916

RESUMO

Use of carbon dots (CDs) in combination with aqueous chitosan solution to extend shelf life and improve stability of soy milk was investigated. Soy milk samples with chitosan solution (0.00%, 0.08%, 0.12%, 0.16% and 0.20%) and banana-based CDs (4%, 6% and 8%) were prepared and stored at room temperature (25-30 °C) for shelf life evaluation. Soy milk with 0.16% chitosan solution exhibited improved stability as evident by increased viscosity, stability coefficient, zeta potential and decreased centrifugation rate compared with soy milk without chitosan. The suitable amount of carbon dots could effectively inhibit the growth of Escherichia coli, Staphylococcus aureus and Bacillus subtilis. Soy milk with 0.16% chitosan and 8% CDs exhibited longer shelf life and significantly lower total bacterial count after storage at room temperature for up to 4 days. Electronic nose-based flavor characteristics of all treated soy milk samples were not far from that of the control sample.


Assuntos
Bacillus subtilis/crescimento & desenvolvimento , Quitosana/farmacologia , Escherichia coli/crescimento & desenvolvimento , Armazenamento de Alimentos/estatística & dados numéricos , Leite de Soja/metabolismo , Staphylococcus aureus/crescimento & desenvolvimento , Bacillus subtilis/efeitos dos fármacos , Carbono/farmacologia , Escherichia coli/efeitos dos fármacos , Contaminação de Alimentos/análise , Pontos Quânticos , Staphylococcus aureus/efeitos dos fármacos , Paladar , Água/farmacologia
14.
Sci Rep ; 10(1): 8265, 2020 05 19.
Artigo em Inglês | MEDLINE | ID: mdl-32427943

RESUMO

Bacterial spores are among the most resistant forms of life on Earth. Their exceptional resistance properties rely on various strategies, among them the core singular structure, organization and hydration. By using elastic incoherent neutron scattering, we probed the dynamics of Bacillus subtilis spores to determine whether core macromolecular motions at the sub-nanosecond timescale could also contribute to their resistance to physical stresses. In addition, in order to better specify the role of the various spore components, we used different mutants lacking essential structure such as the coat (PS4150 mutant), or the calcium dipicolinic acid complex (CaDPA) located in the core (FB122 mutant). PS4150 allows to better probe the core's dynamics, as proteins of the coat represent an important part of spore proteins, and FB122 gives information about the role of the large CaDPA depot for the mobility of core's components. We show that core's macromolecular mobility is not particularly constrained at the sub-nanosecond timescale in spite of its low water content as some dynamical characteristics as force constants are very close to those of vegetative bacteria such as Escherichia coli or to those of fully hydrated proteins. Although the force constants of the coatless mutant are similar to the wild-type's ones, it has lower mean square displacements (MSDs) at high Q showing that core macromolecules are somewhat more constrained than the rest of spore components. However, no behavior reflecting the glassy state regularly evoked in the literature could be drawn from our data. As hydration and macromolecules' mobility are highly correlated, the previous assumption, that core low water content might explain spores' exceptional resistance properties seems unlikely. Thus, we confirm recent theories, suggesting that core water is mostly as free as bulk water and proteins/macromolecules are fully hydrated. The germination of spores leads to a much less stable system with a force constant of 0.1 N/m and MSDs ~2.5 times higher at low Q than in the dormant state. DPA has also an influence on core mobility with a slightly lower force constant for the DPA-less mutant than for the wild-type, and MSDs that are ~ 1.8 times higher on average than for the wild-type at low Q. At high Q, germinated and DPA-less spores were very similar to the wild-type ones, showing that DPA and core compact structure might influence large amplitude motions rather than local dynamics of macromolecules.


Assuntos
Bacillus subtilis/crescimento & desenvolvimento , Ácidos Picolínicos/farmacologia , Esporos Bacterianos/efeitos dos fármacos , Bacillus subtilis/química , Bacillus subtilis/efeitos dos fármacos , Bacillus subtilis/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Cinética , Mutação , Esporos Bacterianos/química , Esporos Bacterianos/crescimento & desenvolvimento
15.
J Dairy Sci ; 103(6): 4991-5002, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32307173

RESUMO

This study aims to characterize Bacillus subtilis complex group from raw, pasteurized, and packaged extended shelf-life (ESL) milk samples, to determine their biofilm potential and source-track the microbial contaminants to control their presence during processing. Isolates were characterized using multi-locus sequence typing (MLST) with 7 housekeeping genes. The primers used were designed from the coding regions with the highest number of polymorphic sites. The heat resistance profile indicated that all 12 isolates are psychrotolerant as well as thermophilic, with temperature ranges of 6°C to 55°C (B43, B44, B52, B54, B55, B56, B57), 6°C to 60°C (B46, B47, B48), and 15°C to 60°C (B49, B50). A general linear model 2-way repeated-measure ANOVA of the biofilm-forming potential of the isolates shows a statistically significant difference across the time of incubation (6, 12, 18, and 24 h). All isolates except 2 formed moderate to strong biofilms, with B44 having the most robust biofilm formation (3.14 ± 0.60). Scanning electron and confocal microscopy images reveal the strain specificity of the biofilm structure. The MLST analysis identified all isolates as belonging to either B. subtilis or Bacillus velezensis. All the isolates were novel sequence types (ST) when compared with the PubMLST database (https://pubmlst.org/) but showed relatedness to isolates in the raw milk that was processed. The closest ST are 96 for B. velezensis and 128 for B. subtilis, mostly isolated from soil. This study presents the significance of biofilms of thermophilic B. subtilis and B. velezensis and their possible perpetuation in the dairy processing plant. The information provided is a call for an innovative food contact surface or any other intervention that can minimize or prevent microbial adhesion in the processing plant, to prevent negative effects in ESL milk.


Assuntos
Bacillus subtilis/crescimento & desenvolvimento , Bacillus/crescimento & desenvolvimento , Biofilmes , Indústria de Laticínios , Leite/microbiologia , Animais , Bacillus/isolamento & purificação , Tipagem de Sequências Multilocus , Pasteurização , Esporos Bacterianos/isolamento & purificação
16.
J Food Sci ; 85(5): 1536-1541, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32249415

RESUMO

The aim of this study was to investigate the influence of surface hydrophobicity and roughness of carrier materials on the inactivation of bacterial spores with gaseous hydrogen peroxide whereas condensate formation is prevented. Spores of Bacillus subtilis and Bacillus atrophaeus were applied either as single spore culture or as a mixed spore population to simulate natural contamination with microorganisms of different characteristics. Inactivation with gaseous hydrogen peroxide was carried out at 5200 ppm hydrogen peroxide without condensate formation. The inactivation results of B. subtilis and B. atrophaeus spores on carrier materials with varying surface hydrophobicity differed significantly. However, inactivation of the mixed spore populations resulted in similar resistance compared to the single spore batches. The results of this study indicate that surface hydrophobicity most probably has an impact on the inactivation with gaseous hydrogen peroxide whereas surface roughness only plays a minor role.


Assuntos
Bacillus subtilis/efeitos dos fármacos , Peróxido de Hidrogênio/química , Peróxido de Hidrogênio/farmacologia , Esporos Bacterianos/química , Bacillus/efeitos dos fármacos , Bacillus/crescimento & desenvolvimento , Bacillus subtilis/crescimento & desenvolvimento , Bacillus subtilis/fisiologia , Gases/química , Gases/farmacologia , Interações Hidrofóbicas e Hidrofílicas , Solo/química , Microbiologia do Solo , Esporos Bacterianos/efeitos dos fármacos , Esporos Bacterianos/crescimento & desenvolvimento , Propriedades de Superfície , Volatilização
17.
PLoS One ; 15(4): e0231274, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32271828

RESUMO

We evaluated the minimum inhibitory concentrations of clindamycin and erythromycin toward 98 Bacillus licheniformis strains isolated from several types of fermented soybean foods manufactured in several districts of Korea. First, based on recent taxonomic standards for bacteria, the 98 strains were separated into 74 B. licheniformis strains and 24 B. paralicheniformis strains. Both species exhibited profiles of erythromycin resistance as an acquired characteristic. B. licheniformis strains exhibited acquired clindamycin resistance, while B. paralicheniformis strains showed unimodal clindamycin resistance, indicating an intrinsic characteristic. Comparative genomic analysis of five strains showing three different patterns of clindamycin and erythromycin resistance identified 23S rRNA (adenine 2058-N6)-dimethyltransferase gene ermC and spermidine acetyltransferase gene speG as candidates potentially involved in clindamycin resistance. Functional analysis of these genes using B. subtilis as a host showed that ermC contributes to cross-resistance to clindamycin and erythromycin, and speG confers resistance to clindamycin. ermC is located in the chromosomes of strains showing clindamycin and erythromycin resistance and no transposable element was identified in its flanking regions. The acquisition of ermC might be attributable to a homologous recombination. speG was identified in not only the five genome-analyzed strains but also eight strains randomly selected from the 98 test strains, and deletions in the structural gene or putative promoter region caused clindamycin sensitivity, which supports the finding that the clindamycin resistance of Bacillus species is an intrinsic property.


Assuntos
Bacillus licheniformis/genética , Bacillus/genética , Clindamicina/farmacologia , Farmacorresistência Bacteriana/genética , Genes Bacterianos , Genômica , Bacillus/efeitos dos fármacos , Bacillus/crescimento & desenvolvimento , Bacillus licheniformis/classificação , Bacillus licheniformis/efeitos dos fármacos , Bacillus licheniformis/crescimento & desenvolvimento , Bacillus subtilis/efeitos dos fármacos , Bacillus subtilis/genética , Bacillus subtilis/crescimento & desenvolvimento , Sequência de Bases , Farmacorresistência Bacteriana/efeitos dos fármacos , Eritromicina/farmacologia , Testes de Sensibilidade Microbiana
18.
PLoS One ; 15(4): e0232096, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32339210

RESUMO

Endophytic bacteria, which are common in plant tissues, may help to control plant pathogens and enhance plant growth. Camellia oleifera, an oil-producing plant, is widely grown in warm, subtropical, hilly regions in China. However, C. oleifera is strongly negatively affected by C. oleifera anthracnose, which is caused by Colletetrichum fructicola. To find a suitable biocontrol agent for C. oleifera anthracnose, 41 endophytes were isolated from the stems, leaves, and roots of C. oleifera. Bacterial cultures were identified based on analyses of 16S rDNA sequences; most strains belonged to the genus Bacillus. The antagonistic effects of these strains on C. fructicola were tested in vitro. In total, 16 strains inhibited C. fructicola growth, with B. subtilis strain 1-L-29 being the most efficient. Strain 1-L-29 demonstrated antagonistic activity against C. siamense, C. asianum, Fusarium proliferatum, Agaricodochium camellia, and Pseudomonas syringae. In addition, this strain produced indole acetic acid, solubilized phosphate, grew on N-free media, and produced siderophores. To facilitate further microecological studies of this strain, a rifampicin-resistant, green fluorescent protein (GFP)-labeled strain, 1-L-29gfpr, was created using protoplast transformation. This plasmid had good segregational stability. Strain 1-L-29gfpr was re-introduced into C. oleifera and successfully colonized root, stem, and leaf tissues. This strain remained at a stable concentration in the root more than 20 d after inoculation. Fluorescence microscopic analysis showed that strain 1-L-29gfpr thoroughly colonized the root surfaces of C. fructicola as well as the root vascular tissues of Arabidopsis thaliana.


Assuntos
Antibacterianos/metabolismo , Bacillus subtilis/metabolismo , Camellia/crescimento & desenvolvimento , Endófitos/metabolismo , Doenças das Plantas/prevenção & controle , Raízes de Plantas/crescimento & desenvolvimento , Bacillus subtilis/classificação , Bacillus subtilis/crescimento & desenvolvimento , Bacillus subtilis/isolamento & purificação , Camellia/metabolismo , Camellia/microbiologia , Endófitos/crescimento & desenvolvimento , Endófitos/isolamento & purificação , Controle Biológico de Vetores , Desenvolvimento Vegetal , Doenças das Plantas/microbiologia , Raízes de Plantas/metabolismo , Raízes de Plantas/microbiologia
19.
Sci Rep ; 10(1): 5744, 2020 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-32238880

RESUMO

Bacillus widely exists in wet natural environment such as soil, water and air, and is often studied as one of representative microorganisms for microbiologically influenced corrosion(MIC) research. In this paper, the growth curve of Bacillus subtilis isolated from marine environment was determined by turbidimetry and its effect on corrosion behavior of 10MnNiCrCu steel was studied by open circuit potential, AC impedance, polarization curve and scanning electron microscopy(SEM). The results showed that with the change of the growth curve of Bacillus subtilis(BS), the open circuit potential(Eocp) shifted positively and then negatively, and the charge transfer resistance shown by AC impedance was much lower than that of the sterile system, increasing first and then decreasing. The polarization curves showed that the corrosion current density in BS medium was obviously higher than that in sterile system. The corrosion morphology observation showed that although a biofilm by BS developed on the steel surface, the localized corrosion of 10MnNiCrCu steel was aggravated due to the acidness of the metabolite itself and the biofilm with access for electrolyte ions.


Assuntos
Bacillus subtilis/fisiologia , Biofilmes , Aço/química , Bacillus subtilis/crescimento & desenvolvimento , Biofilmes/crescimento & desenvolvimento , Cromo/química , Cobre/química , Corrosão , Manganês/química , Níquel/química , Microbiologia da Água
20.
Sci Adv ; 6(10): eaaz5108, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32181369

RESUMO

Much is known about the effects of antibiotics on isolated bacterial species, but their influence on polybacterial communities is less understood. Here, we study the joint response of a mixed community of nonresistant Bacillus subtilis and Escherichia coli bacteria to moderate concentrations of the ß-lactam antibiotic ampicillin. We show that when the two organisms coexist, their population response to the antibiotic is opposite to that in isolation: Whereas in monoculture B. subtilis is tolerant and E. coli is sensitive to ampicillin, in coculture it is E. coli who can proliferate in the presence of the antibiotic, while B. subtilis cannot. This antithetic behavior is predicted by a mathematical model constrained only by the responses of the two species in isolation. Our results thus show that the collective response of mixed bacterial ecosystems to antibiotics can run counter to what single-species potency studies tell us about their efficacy.


Assuntos
Ampicilina/farmacologia , Antibacterianos/farmacologia , Bacillus subtilis/crescimento & desenvolvimento , Escherichia coli/crescimento & desenvolvimento , Consórcios Microbianos/efeitos dos fármacos , Modelos Biológicos , Resistência beta-Lactâmica , Técnicas de Cocultura
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