RESUMEN
We previously reported a chromatography system for purifying immunoglobulin M (IgM) using N,N,N',N'-ethylenediaminetetrakis(methylenephosphonic acid)-modified zirconia particles that selectively absorb immunoglobulins. Here, we report a simple procedure for preparing biotinylated IgM from hybridoma culture medium using this zirconia-based chromatography system. The culture medium of an IgM-producing hybridoma cell line was used as the starting sample solution, and the IgM in the medium was concentrated and partially purified by zirconia chromatography. Next, 9-(biotinamido)-4,7-dioxanonanoic acid N-succinimidyl ester was added to react with the proteins in the sample. Subsequently, only the biotinylated IgM was isolated by Capto Core 400 polishing column chromatography. The entire process was easy to perform, could be completed within 2 h, and provided highly pure biotin-labeled IgM. This procedure is expected to be applicable to the labeling of IgM with various compounds and drugs.
Asunto(s)
Biotinilación , Medios de Cultivo , Hibridomas , Inmunoglobulina M , Inmunoglobulina M/química , Inmunoglobulina M/aislamiento & purificación , Animales , Medios de Cultivo/química , Ratones , Circonio/química , Biotina/químicaRESUMEN
At present, biopharmaceuticals have received extensive attention from the society, among which recombinant proteins have a good growth trend and a large market share. Chinese hamster ovary (CHO) cells are the preferred mammalian system to produce glycosylated recombinant protein drugs. A highly efficient and stable cell screening method needs to be developed to obtain more and useful recombinant proteins. Limited dilution method, cell sorting, and semi-solid medium screening are currently the commonly used cell cloning methods. These methods are time-consuming and labor-intensive, and they have the disadvantage of low clone survival rate. Here, a method based on semi-solid medium was developed to screen out high-yielding and stable cell line within 3 weeks to improve the screening efficiency. The semi-solid medium was combined with an expression vector containing red fluorescent protein (RFP) for early cell line development. In accordance with the fluorescence intensity of RFP, the expression of upstream target gene could be indicated, and the fluorescence intensity was in direct proportion to the expression of upstream target gene. In conclusion, semi-solid medium combined with bicistronic expression vector provides an efficient method for screening stable and highly expressed cell lines.
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Cricetulus , Proteínas Recombinantes , Células CHO , Animales , Proteínas Recombinantes/genética , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/metabolismo , Vectores Genéticos/genética , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Cricetinae , Proteína Fluorescente Roja , Medios de Cultivo/químicaRESUMEN
The extensive use of various chemicals in synthetic plastics is toxic and threatens the biosphere. To address this, the study aimed to isolate, screen, characterize, optimize, and quantify polyhydroxybutyrate (PHB)-producing bacteria using cost-effective residues. Isolated from a landfill site, the Gram-positive, rod-shaped, spore-forming, motile bacterium with intracellular PHB granules was identified as Bacillus pacificus based on phenotypic and genotypic characteristics. Optimal PHB production parameters included a nutrient broth medium, 72 h of incubation, a temperature of 37° C, a pH of 7.0, glucose as the carbon source, ammonium chloride as the nitrogen source, and a carbon-to-nitrogen ratio of 4:1, resulting in a 1.42-fold PHB production increase. B. pacificus was also cultured on various low-cost substrates. Among the oil wastes, feedstock showed the highest PHB production (1.983 ± 0.005 g/L) and among agricultural residues, the maximum PHB was obtained from rice bran (1.626 ± 0.01 g/L). UV-visible spectrophotometric, FT-IR, and HR-LCMS analysis of extracted PHB confirmed characteristics of PHB molecules (Ê-max at 210 nm, functional groups between 1152 and 2925 cm-1). The 1H NMR analysis revealed distinct signals for protons resonating at aliphatic CH3 proton groups, bridged CH protons, and shielding CH2 proton regions that matched PHBs. Thermal gravimetric analysis (TGA) and direct scanning colorimetric (DSC) analysis revealed 89.4% degradation and melting temperature at 124.1 °C for the extracted PHB compound.
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Bacillus , Hidroxibutiratos , Bacillus/metabolismo , Bacillus/genética , Bacillus/clasificación , Hidroxibutiratos/metabolismo , Poliésteres/metabolismo , Poliésteres/química , Nitrógeno/metabolismo , Medios de Cultivo/química , Carbono/metabolismo , Temperatura , Concentración de Iones de HidrógenoRESUMEN
Two strains of Yarrowia lipolytica (CBS 2075 and DSM 8218) were first studied in bioreactor batch cultures, under different controlled dissolved oxygen concentrations (DOC), to assess their ability to assimilate aliphatic hydrocarbons (HC) as a carbon source in a mixture containing 2 g·L-1 of each alkane (dodecane and hexadecane), and 2 g·L-1 hexadecene. Both strains grew in the HC mixture without a lag phase, and for both strains, 30 % DOC was sufficient to reach the maximum values of biomass and lipids. To enhance lipid-rich biomass and enzyme production, a pulse fed-batch strategy was tested, for the first time, with the addition of one or three pulses of concentrated HC medium. The addition of three pulses of the HC mixture (total of 24 g·L-1 HC) did not hinder cell proliferation, and high protease (> 3000 U·L-1) and lipids concentrations of 3.4 g·L-1 and 4.3 g·L-1 were achieved in Y. lipolytica CBS 2075 and DSM 8218 cultures, respectively. Lipids from the CBS 2075 strain are rich in C16:0 and C18:1, resembling the composition of palm oil, considered suitable for the biodiesel industry. Lipids from the DSM 8218 strain were predominantly composed of C16:0 and C16:1, the latter being a valuable monounsaturated fatty acid used in the pharmaceutical industry. Y. lipolytica cells exhibited high intrinsic surface hydrophobicity (> 69 %), which increased in the presence of HC. A reduction in surface tension was observed in both Y. lipolytica cultures, suggesting the production of extracellular biosurfactants, even at low amounts. This study marks a significant advancement in the valorization of HC for producing high-value products by exploring the hydrophobic compounds metabolism of Y. lipolytica.
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Alcanos , Alquenos , Técnicas de Cultivo Celular por Lotes , Biomasa , Reactores Biológicos , Medios de Cultivo , Yarrowia , Yarrowia/crecimiento & desarrollo , Yarrowia/metabolismo , Alcanos/metabolismo , Reactores Biológicos/microbiología , Medios de Cultivo/química , Alquenos/metabolismo , Ácidos Grasos/metabolismo , Ácidos Grasos/análisis , Lípidos/biosíntesis , Lípidos/análisis , Oxígeno/metabolismo , Metabolismo de los LípidosRESUMEN
Mechanical stress is a measure of internal resistance exhibited by a body or material when external forces, such as compression, tension, bending, etc. are applied. The study of mechanical stress on health and aging is a continuously growing field, as major changes to the extracellular matrix and cell-to-cell adhesions can result in dramatic changes to tissue stiffness during aging and diseased conditions. For example, during normal aging, many tissues including the ovaries, skin, blood vessels, and heart exhibit increased stiffness, which can result in a significant reduction in function of that organ. As such, numerous model systems have recently emerged to study the impact of mechanical and physical stress on cell and tissue health, including cell-culture conditions with matrigels and other surfaces that alter substrate stiffness and ex vivo tissue models that can apply stress directly to organs like muscle or tendons. Here, we sought to develop a novel method in an in vivo model organism setting to study the impact of altering substrate stiffness on aging by changing the stiffness of solid agar medium used for growth of C. elegans. We found that greater substrate stiffness had limited effects on cellular health, gene expression, organismal health, stress resilience, and longevity. Overall, our study reveals that altering substrate stiffness of growth medium for C. elegans has only mild impact on animal health and longevity; however, these impacts were not nominal and open up important considerations for C. elegans biologists in standardizing agar medium choice for experimental assays.
Asunto(s)
Caenorhabditis elegans , Longevidad , Animales , Caenorhabditis elegans/fisiología , Caenorhabditis elegans/crecimiento & desarrollo , Estrés Mecánico , Medios de CultivoRESUMEN
BACKGROUND: Poplar canker caused by Botryosphaeria dothidea is one of the most severe plant disease of poplars worldwide. In our study, we aimed to investigate the modes of antagonism by fermentation broth supernatant (FBS) of Streptomyces spiroverticillatus HS1 against B. dothidea. RESULTS: In vitro, the strain and FBS of S. spiroverticillatus HS1 significantly inhibited mycelial growth and biomass accumulation, and also disrupted the mycelium morphology of B. dothidea. On the 3rd day after treatment, the inhibition rates of colony growth and dry weight were 80.72% and 52.53%, respectively. In addition, FBS treatment damaged the plasma membrane of B. dothidea based on increased electrical conductivity in the culture medium, and malondialdehyde content of B. dothidea mycelia. Notably, the analysis of key enzymes in glycolysis pathway showed that the activity of hexokinase (HK), phosphofructokinase (PFK), and pyruvate kinase (PK), Ca2+Mg2+-ATPase were significantly increased after FBS treatment. But the glucose contents were significantly reduced, and pyruvate contents were significantly increased in B. dothidea after treatment with FBS. CONCLUSIONS: The inhibitory mechanism of S. spiroverticillatus HS1 against B. dothidea was a complex process, which was associated with multiple levels of mycelial growth, cell membrane structure, material and energy metabolism. The FBS of S. spiroverticillatus HS1 could provide an alternative approach to biological control strategies against B. dothidea.
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Ascomicetos , Micelio , Enfermedades de las Plantas , Populus , Streptomyces , Ascomicetos/crecimiento & desarrollo , Ascomicetos/efectos de los fármacos , Enfermedades de las Plantas/microbiología , Enfermedades de las Plantas/prevención & control , Streptomyces/fisiología , Populus/microbiología , Micelio/crecimiento & desarrollo , Micelio/efectos de los fármacos , Antibiosis , Fermentación , Medios de Cultivo/químicaRESUMEN
This study investigated the ability of lactic acid bacteria (LAB) isolated from oranges to use fish by-products (FB) and chicken by-products (CB) as nitrogen sources alternative to yeast extract for lactic acid (LA) production in a papaya by-product medium as a carbon source. Once the fermentation agents had been isolated, they were subjected to biochemical and molecular characterization. Inexpensive nitrogen sources, precisely CB and FB, were prepared, freeze-dried, and yield evaluated. Also, before to the fermentation experiments, the Total Kjehdahl Nitrogen (TKN) of these by-products and that of the yeast extract were determined. Then, three production media differing in terms of nitrogen source were formulated from these nitrogen sources. From the 22 LAB isolated from orange, two isolates of interest (NGO25 and NGO23) were obtained; all belonging to the Lactiplantibacillus plantarum species based on 16S rRNA gene sequencing. Furthermore, the production yield powder obtained after lyophilization of 1 L of CB and FB surpernatant were, respectively, 16.6 g and 12.933 g. The TKN of different nitrogen sources powder were 71.4 ± 0.000% DM (FB), 86.145 ± 0.001% DM (CB), and 87.5 ± 0.99% DM (yeast extract). The best kinetic parameters of LA production (LA (g/L): 31.945 ± 0.078; volumetric productivity (g/L.h): 1.331 ± 0.003; LA yield (mg/g) 63.89 ± 0.156; biomass (g/L) 7.925 ± 0.035; cell growth rate (g/L.h): 0.330 ± 0.001) were recorded by Lactiplantibacillus plantarum NGO25 after 24 h of fermentation. The latter data were obtained in the production medium containing CB as nitrogen sources. In addition, this production medium cost only $0.152 to formulate, compared to yeast extract which required $1.692 to formulate. Thus, freeze-dried CB can be used as an alternative to yeast extract in large-scale production of LA.
Asunto(s)
Carbono , Fermentación , Ácido Láctico , Nitrógeno , Nitrógeno/metabolismo , Ácido Láctico/metabolismo , Carbono/metabolismo , Lactobacillales/metabolismo , Animales , ARN Ribosómico 16S/genética , Citrus/microbiología , Pollos/microbiología , Medios de CultivoRESUMEN
Cordycepin (3'-deoxyadenosine) is a bioactive nucleoside analog synthesized by Cordyceps militaris. Liquid fermentation of C. militaris by addition in different concentrations of five additives singly was evaluated. Glycine at 15.00 g/L after 20 d enhanced the cordycepin of 1773.33 mg/L (15-fold increment over control). Adenine at 4.00 g/L and 6.00 g/L in the liquid media showed significantly higher cordycepin i.e.1596.66 mg/L and 1550.00 mg/L (3-fold increment over control) after 40 d. Tryptone supplementation 14.00 g/L significantly higher cordycepin 784.33 mg/L (6.70-fold increment over control) and 912.66 mg/L production after 20 and 40 d of inoculation. Peanut oil at 10.00 g/L produced 585.66 mg/L (5-fold increment over control) cordycepin after 20 d and after 40 d, also addition of peanut oil at 20.00 g/L and 30.00 g/L in the media showed 631.66 and 624.31 mg/L cordycepin content. Supplementation of mono-sodium glutamate at 0.30 g/L produced significantly highest cordycepin i.e. 614 mg/L and 635.00 mg/L cordycepin after 20 and 40 d, respectively.
Asunto(s)
Cordyceps , Medios de Cultivo , Desoxiadenosinas , Fermentación , Desoxiadenosinas/biosíntesis , Desoxiadenosinas/metabolismo , Cordyceps/metabolismo , Cordyceps/química , Cordyceps/crecimiento & desarrollo , Medios de Cultivo/química , Aceite de Cacahuete , Adenina/metabolismo , Peptonas/metabolismoRESUMEN
The present study was carried out to optimize the strain and evaluate the effect of amendment of growth media with different hormone concentrations for enhancing mycelium growth of lion's mane mushroom Hericium erinaceus under in vitro conditions. Among the five strains of H. erinaceus, He-04 strain showed maximum average GR (GRavr) of 4.78 mm d-1. Five different media, potato dextrose agar (PDA), malt extract agar, sawdust extract agar, wheat straw extract agar, and rice straw extract agar, amended with four concentrations (10, 20, 30, and 40 ppm) of gibberellic acid, kinetin, and indole acetic acid, were evaluated for promotion of mycelial growth of H. erinaceus. PDA was observed to be the best media promoting the mycelial growth of H. erinaceus. The highest mycelial GRavr 8.47 mm d-1 was observed in PDA amended with indole acetic acid (10 ppm) followed by gibberellic acid and kinetin (30 ppm) decreasing mycelial GRav to 8.15 and 7.75mm d-1, respectively. Temperature of 25°C and pH 7.0 was found to be the best for mycelium growth of H. erinaceus.
Asunto(s)
Medios de Cultivo , Giberelinas , Hericium , Ácidos Indolacéticos , Micelio , Micelio/crecimiento & desarrollo , Micelio/efectos de los fármacos , Ácidos Indolacéticos/farmacología , Giberelinas/farmacología , Medios de Cultivo/química , Hericium/crecimiento & desarrollo , Hericium/química , Cinetina/farmacología , Temperatura , Reguladores del Crecimiento de las Plantas/farmacología , Concentración de Iones de HidrógenoRESUMEN
BACKGROUND: Mycobacterium abscessus complex (MABSc) causes chronic infection in patients with concomitant structural changes in the respiratory tract, which is especially important for patients with cystic fibrosis. To isolate an MABSc culture from clinical material, a variety of nutrient media are used. For species determination of microorganisms isolated on these media, additional identification methods are used, for example, polymerase chain reaction, sequencing, or mass spectrometry. The latter method is relatively easy to implement but requires improvement, due to the identification inaccuracy of nontuberculosis mycobacterias in general. Consequently, a set of nutrient media may be important for subsequent identification by mass spectrometry. METHODS: The study was conducted on 64 strains of MABSc representatives: 56 strains were obtained from patients with cystic fibrosis and 8 strains from patients with pulmonary pathology unrelated to cystic fibrosis. The obtained MABSc strains were transplanted to the universal chromogenic medium and the selective medium for the Burkholderia cepacia complex (BCC) isolation. Species identification was carried out by mass spectrometry based on matrix-activated laser time-of-flight desorption/ionization (MALDI-ToF MS). Microbial identification is based on a comparison of the obtained mass spectra with reference spectra from the database. Microorganisms were identified based on the coincidence degree (Score value). Sample preparation for microbial identification by mass spectrometry was carried out by an extended direct application method. Fragments of the rpoB and hsp65 genes with lengths of 752 bp and 441 bp, respectively, were used as molecular markers for subspecific identification of MABSc strains. RESULTS: A comparison of the peaks obtained after mass spectrometry of MABSc strains isolated on the studied nutrient media showed significant differences between these indicators selective medium for the BCC isolation with the supplement of iron polymaltose hydroxide (III) and universal chromogenic medium (P < 0.001) and selective medium for the BCC isolation with universal chromogenic medium (P < 0.001). Twenty-five strains of MABSc representatives were sequenced: results of subspecies determination in strains isolated on the universal chromogenic medium coincided with the results sequencing in 13 (86.6%) strains out of 15. CONCLUSION: MALDI-ToF mass spectrometry allows microbial identification in a short time and with minimal cost, but it does not yet allow the proper identification of the subspecies of certain microbial groups, such as MABSc. Cultivation methods need optimization and new approaches to the extraction process of the bacterial protein fraction.
Asunto(s)
Medios de Cultivo , Fibrosis Quística , Infecciones por Mycobacterium no Tuberculosas , Mycobacterium abscessus , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Mycobacterium abscessus/aislamiento & purificación , Mycobacterium abscessus/clasificación , Mycobacterium abscessus/genética , Humanos , Medios de Cultivo/química , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Fibrosis Quística/microbiología , Infecciones por Mycobacterium no Tuberculosas/microbiología , Proteínas Bacterianas/genética , Proteínas Bacterianas/análisis , Chaperonina 60/genéticaRESUMEN
The fecal microbiome is identical to the gut microbial communities and provides an easy access to the gut microbiome. Therefore, fecal microbial transplantation (FMT) strategies have been used to alter dysbiotic gut microbiomes with healthy fecal microbiota, successfully alleviating various metabolic disorders, such as obesity, type 2 diabetes, and inflammatory bowel disease (IBD). However, the success of FMT treatment is donor-dependent and variations in gut microbes cannot be avoided. This problem may be overcome by using a cultured fecal microbiome. In this study, a human fecal microbiome was cultured using five different media; growth in brain heart infusion (BHI) media resulted in the highest microbial community cell count. The microbiome (16S rRNA) data demonstrated that the cultured microbial communities were similar to that of the original fecal sample. Therefore, the BHI-cultured fecal microbiome was selected for cultured FMT (cFMT). Furthermore, a dextran sodium sulfate (DSS)-induced mice-IBD model was used to confirm the impact of cFMT. Results showed that cFMT effectively alleviated IBD-associated symptoms, including improved gut permeability, restoration of the inflamed gut epithelium, decreased expression of pro-inflammatory cytokines (IFN-γ, TNF-α, IL-1, IL-6, IL-12, and IL-17), and increased expression of anti-inflammatory cytokines (IL-4 and IL-10). Thus, study's findings suggest that cFMT can be a potential alternative to nFMT. KEY POINTS: ⢠In vitro fecal microbial communities were grown in a batch culture using five different media. ⢠Fecal microbial transplantation was performed on DSS-treated mice using cultured and normal fecal microbes. ⢠Cultured fecal microbes effectively alleviated IBD-associated symptoms.
Asunto(s)
Citocinas , Modelos Animales de Enfermedad , Trasplante de Microbiota Fecal , Heces , Microbioma Gastrointestinal , Enfermedades Inflamatorias del Intestino , ARN Ribosómico 16S , Trasplante de Microbiota Fecal/métodos , Animales , Heces/microbiología , Ratones , Humanos , Enfermedades Inflamatorias del Intestino/terapia , Enfermedades Inflamatorias del Intestino/microbiología , Citocinas/metabolismo , ARN Ribosómico 16S/genética , Ratones Endogámicos C57BL , Sulfato de Dextran , Masculino , Medios de Cultivo/química , Bacterias/clasificación , Bacterias/genética , Bacterias/aislamiento & purificaciónRESUMEN
The present study was designed to investigate the effects of amino acid (histidine and L-Tyrosine) on in vitro maturation (IVM), in vitro fertilization (IVF), cleavage (CR) rates, and in vitro embryonic cultivation (IVC; Morula and Blastocyst stage) in buffaloes. Within two hours of buffalo slaughter, the ovaries were collected and transported to the laboratory. Follicles with a diameter of 2 to 8 mm were aspirated to recover the cumulus oocyte complexes (COCs). Histidine (0.5, 1, and 3 mg/ml) or L-Tyrosine (1, 5, and 10 mg/ml) were added to the synthetic oviductal fluid (SOF) and Ferticult media. The IVM, IVF, CR, and IVC (Morula and Blastocyst) rates were evaluated. The results showed that SOF maturation media containing histidine at 0.5 mg/ml significantly (P ≤ 0.01) improved the oocyte maturation when compared to control and other concentrations. The addition of histidine to FertiCult media at 0.5, 1, and 3 mg/ml did not improve the IVM, IVF, CR, or IVC percentages. However, the embryos in the control group were unable to grow into a morula or blastocyst in the SOF or Ferticult, while addition of L-Tyrosine to the SOF or Ferticult at various concentrations improved IVC (morula and blastocyst rates). There was a significant (P ≤ 0.01) increase in IVM when histidine was added to SOF medium at a concentration of 0.5 mg/ml compared with L-Tyrosine. Also, there were significant (P ≤ 0.01) increases in IVC when L-Tyrosine was added to SOF medium at concentrations of 1 and 10 mg/ml compared with histidine. In conclusion, the supplementation of the SOF and FertiCult with the amino acids histidine and L-Tyrosine improve the maturation rate of oocytes and development of in vitro-produced buffalo embryos.
Asunto(s)
Búfalos , Medios de Cultivo , Fertilización In Vitro , Histidina , Técnicas de Maduración In Vitro de los Oocitos , Oocitos , Tirosina , Animales , Tirosina/farmacología , Tirosina/administración & dosificación , Histidina/farmacología , Histidina/administración & dosificación , Oocitos/efectos de los fármacos , Femenino , Técnicas de Maduración In Vitro de los Oocitos/veterinaria , Técnicas de Maduración In Vitro de los Oocitos/métodos , Fertilización In Vitro/veterinaria , Medios de Cultivo/farmacología , Técnicas de Cultivo de Embriones/veterinaria , Desarrollo Embrionario/efectos de los fármacosRESUMEN
Riboflavin, an essential vitamin for humans, is extensively used in various industries, with its global demand being met through fermentative processes. Hyphopichia wangnamkhiaoensis is a novel dimorphic yeast species capable of producing riboflavin. However, the nutritional factors affecting riboflavin production in this yeast species remain unknown. Therefore, we conducted a kinetic study on the effects of various nutritional factors-carbon and energy sources, nitrogen sources, vitamins, and amino acids-on batch riboflavin production by H. wangnamkhiaoensis. Batch experiments were performed in a bubble column bioreactor to evaluate cell growth, substrate consumption, and riboflavin production. The highest riboflavin production was obtained when the yeast growth medium was supplemented with glucose, ammonium sulfate, biotin, and glycine. Using these chemical components, along with the mineral salts from Castañeda-Agullo's culture medium, we formulated a novel, low-cost, and effective culture medium (the RGE medium) for riboflavin production by H. wangnamkhiaoensis. This medium resulted in the highest levels of riboflavin production and volumetric productivity, reaching 16.68 mg/L and 0.713 mg/L·h, respectively, within 21 h of incubation. These findings suggest that H. wangnamkhiaoensis, with its shorter incubation time, could improve the efficiency and cost-effectiveness of industrial riboflavin production, paving the way for more sustainable production methods.
Asunto(s)
Medios de Cultivo , Riboflavina , Riboflavina/biosíntesis , Riboflavina/metabolismo , Medios de Cultivo/química , Cinética , Reactores Biológicos , Fermentación , Nitrógeno/metabolismo , Saccharomycetales/metabolismo , Saccharomycetales/crecimiento & desarrollo , Vitaminas/metabolismo , Glucosa/metabolismoRESUMEN
Pseudomonas aeruginosa, a notable pathogen frequently associated with hospital-acquired infections, displays diverse intrinsic and acquired antibiotic resistance mechanisms, posing a significant challenge in infection management. Antimicrobial blue light (aBL) has been demonstrated as a potential alternative for treating P. aeruginosa infections. In this study, we investigated the impact of blue light wavelength, bacterial growth stage, and growth medium composition on the efficacy of aBL. First, we compared the efficacy of light wavelengths 405 nm, 415 nm, and 470 nm in killing three multidrug resistant clinical strains of P. aeruginosa. The findings indicated considerably higher antibacterial efficacy for 405 nm and 415 nm wavelength compared to 470 nm. We then evaluated the impact of the bacterial growth stage on the efficacy of 405 nm light in killing P. aeruginosa using a reference strain PAO1 in exponential, transitional, or stationary phase. We found that bacteria in the exponential phase were the most susceptible to aBL, followed by the transitional phase, while those in the stationary phase exhibited the highest tolerance. Additionally, we quantified the production of reactive oxygen species (ROS) in bacteria using the 2',7'-dichlorofluorescein diacetate (DCFH-DA) probe and flow cytometry, and observed a positive correlation between aBL efficacy and ROS production. Finally, we determined the influence of growth medium on aBL efficacy. PAO1 was cultivated in brain heart infusion (BHI), Luria-Bertani (LB) broth or Casamino acids (CAA) medium, before being irradiated with aBL at 405 nm. The CAA-grown bacteria exhibited the highest sensitivity to aBL, followed by those grown in LB broth, and the BHI-grown bacteria demonstrated the lowest sensitivity. By incorporating FeCl3, MnCl2, ZnCl2, or the iron chelator 2,2'-bipyridine (BIP) into specific media, we discovered that aBL efficacy was affected by the iron levels in culture media.
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Medios de Cultivo , Luz , Pseudomonas aeruginosa , Especies Reactivas de Oxígeno , Pseudomonas aeruginosa/efectos de los fármacos , Pseudomonas aeruginosa/efectos de la radiación , Especies Reactivas de Oxígeno/metabolismo , Medios de Cultivo/química , Antibacterianos/farmacología , Antibacterianos/química , Pruebas de Sensibilidad Microbiana , Luz AzulRESUMEN
BACKGROUND: The detection of causative pathogens plays a crucial role in the diagnosis and targeted treatment of periprosthetic joint infections (PJI). While there have been improvements in analytic methods in the past, pre-analytical procedures have not yet been sufficiently investigated. The objective of this study was to compare the culture yield of four different pre-analytical procedures. METHODS: Patients with perioperative diagnosis of PJI were included in a single center cross-sectional study (2021-2022). Tissue samples (n = 20) of each patient were randomly and equally distributed to each of the four study arms. Tissue samples were either send to the laboratory without culture medium (group A) or were transported in thioglycolate medium immediately after sampling at three different temperatures (room temperature, 4 °C, 37° for 24 h; group B-D). Culture media were investigated for growth on days 1, 3, 7, 12, 14. All organisms, the number of positive samples and the time to positivity were recorded and compared between the study arms. Single positive cultures were considered as contamination. RESULTS: In total, 71 patients were included. The proportions of culture negative samples (10-15%) and polymicrobial infections (51-54%) were comparable between the four arms. Seven patients (10%) were culture-negative in group A, but showed growth in thioglycolate media (group B-D). Furthermore, 13% of patients showed growth in all groups, but additional organisms were cultured in thioglycolate. There was growth beyond day 7 of culturing only in thioglycolate, but not in group A. A storage temperature of 4 °C showed a longer time to positivity compared to the other groups (p < 0.001). CONCLUSIONS: Pre-analytical storage of tissue samples in thioglycolate broth did not improve the culture yield and did not detect additional cases of infection compared to the standard (pre-analytical storage in sterile containers). However, including a thioglycolate medium to the sampling algorithm reduced the rate of culture-negative infections and helped to identify additional organisms.
Asunto(s)
Medios de Cultivo , Infecciones Relacionadas con Prótesis , Humanos , Infecciones Relacionadas con Prótesis/microbiología , Infecciones Relacionadas con Prótesis/diagnóstico , Femenino , Masculino , Anciano , Estudios Transversales , Persona de Mediana Edad , Medios de Cultivo/química , Manejo de Especímenes/métodos , Bacterias/aislamiento & purificación , Bacterias/crecimiento & desarrollo , Bacterias/clasificación , Anciano de 80 o más Años , Técnicas Microbiológicas/métodos , Técnicas Bacteriológicas/métodosRESUMEN
Mycobacterium abscessus (MABS) displays differential subspecies susceptibility to macrolides. Thus, identifying MABS's subspecies (M. abscessus, M. bolletii and M. massiliense) is a clinical necessity for guiding treatment decisions. We aimed to assess the potential of Machine Learning (ML)-based classifiers coupled to Matrix-Assisted Laser Desorption/Ionization Time-of-Flight (MALDI-TOF) MS to identify MABS subspecies. Two spectral databases were created by using 40 confirmed MABS strains. Spectra were obtained by using MALDI-TOF MS from strains cultivated on solid (Columbia Blood Agar, CBA) or liquid (MGIT®) media for 1 to 13 days. Each database was divided into a dataset for ML-based pipeline development and a dataset to assess the performance. An in-house programme was developed to identify discriminant peaks specific to each subspecies. The peak-based approach successfully distinguished M. massiliense from the other subspecies for strains grown on CBA. The ML approach achieved 100% accuracy for subspecies identification on CBA, falling to 77.5% on MGIT®. This study validates the usefulness of ML, in particular the Random Forest algorithm, to discriminate MABS subspecies by MALDI-TOF MS. However, identification in MGIT®, a medium largely used in mycobacteriology laboratories, is not yet reliable and should be a development priority.
Asunto(s)
Medios de Cultivo , Aprendizaje Automático , Mycobacterium abscessus , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Mycobacterium abscessus/clasificación , Mycobacterium abscessus/química , Mycobacterium abscessus/aislamiento & purificación , Medios de Cultivo/química , Humanos , Infecciones por Mycobacterium no Tuberculosas/microbiología , Infecciones por Mycobacterium no Tuberculosas/diagnósticoRESUMEN
The "duckweed-microbes co-cultivation method" is a microbial isolation technique that effectively recovers diverse microbes, including rarely cultivated bacterial phyla, from environmental samples. In this method, aseptic duckweed and microbes collected from an environmental sample are co-cultivated for several days, and duckweed-associated microbes are then isolated from its roots using a conventional agar plate-based cultivation method. We herein propose several improvements to the method in order to specifically obtain members of the rarely cultivated bacterial phylum, Verrucomicrobiota. In systems using river water as the inoculum, the marked enrichment of Verrucomicrobiota was observed after 10 days of co-cultivation, particularly in the roots and co-cultivated media. We also successfully isolated 44 strains belonging to subdivisions 1, 3, and 4 of the phylum Verrucomicrobiota from these systems. This was achieved by changing the concentration of nitrogen in the co-cultivation medium, which is known to affect duckweed growth and/or metabolism, and by subjecting the fronds and co-cultivated media as well as the roots after co-cultivation to microbial isolation.
Asunto(s)
Araceae , Bacterias , Técnicas de Cocultivo , Raíces de Plantas , ARN Ribosómico 16S , Raíces de Plantas/microbiología , Araceae/microbiología , Araceae/crecimiento & desarrollo , Bacterias/clasificación , Bacterias/aislamiento & purificación , Bacterias/genética , Bacterias/crecimiento & desarrollo , Bacterias/metabolismo , ARN Ribosómico 16S/genética , Filogenia , Medios de Cultivo/química , Ríos/microbiología , ADN Bacteriano/genética , Nitrógeno/metabolismo , Biodiversidad , Microbiología del AguaRESUMEN
The influence of talc microparticles on metabolism and morphology of S. rimosus at various initial organic nitrogen concentrations was investigated. The shake flask cultivations were conducted in the media with yeast extract (nitrogen source) concentration equal to 1 g YE L- 1 and 20 g YE L- 1. Two talc microparticle concentrations of 5 g TALC L- 1 and 10 g TALC L- 1 were tested in microparticle-enhanced cultivation (MPEC) runs. A high nitrogen concentration of 20 g YE L- 1 promoted the development of small agglomerates (pellets) of projected area lower than 105 µm2 and dispersed pseudohyphae. A low nitrogen concentration of 1 g YE L- 1 led to the limitation of S. rimosus growth and, in consequence, the development of the smaller number of large pseudohyphal agglomerates (pellets) of projected area higher than 105 µm2 compared to the culture containing a high amount of nitrogen source. In both cases talc microparticles were embedded into pellets and caused the decrease in their sizes. The lower amount of talc (5 g TALC L- 1) usually caused the weaker effect on S. rimosus morphology and metabolite production than the higher one. This correlation between the microparticles effect on morphology and metabolism of S. rimosus was especially noticeable in the biosynthesis of oxytetracycline, 2-acetyl-2-dicarboxamide oxytetracycline (ADOTC) and spinoxazine A. Compared to the control run, in MPEC their levels increased 4-fold, 5-fold and 1.6-fold respectively. The addition of talc also improved the production of 2-methylthio-cis-zeatin, lorneic acid J and milbemycin A3.
Asunto(s)
Nitrógeno , Streptomyces , Nitrógeno/metabolismo , Streptomyces/metabolismo , Streptomyces/crecimiento & desarrollo , Talco/metabolismo , Medios de Cultivo/química , Metabolismo SecundarioRESUMEN
Biophysical models can predict the behavior of cell cultures including 3D cell aggregates (3DCAs), thereby reducing the need for costly and time-consuming experiments. Specifically, mass transfer models enable studying the transport of nutrients, oxygen, signaling molecules, and drugs in 3DCA. These models require the defining of boundary conditions (BC) between the 3DCA and surrounding medium. However, accurately modeling the BC that relates the inner and outer boundary concentrations at the border between the 3DCA and the medium remains a challenge that this paper addresses using both theoretical and experimental methods. The provided biophysical analysis indicates that the concentration of molecules inside boundary is higher than that at the outer boundary, revealing an amplification factor that is confirmed by a particle-based simulator (PBS). Due to the amplification factor, the PBS confirms that when a 3DCA with a low concentration of target molecules is introduced to a culture medium with a higher concentration, the molecule concentration in the medium rapidly decreases. The theoretical model and PBS simulations were used to design a pilot experiment with liver spheroids as the 3DCA and glucose as the target molecule. Experimental results agree with the proposed theory and derived properties.
Asunto(s)
Agregación Celular , Esferoides Celulares , Esferoides Celulares/metabolismo , Esferoides Celulares/citología , Difusión , Humanos , Modelos Biológicos , Glucosa/metabolismo , Técnicas de Cultivo Tridimensional de Células/métodos , Medios de Cultivo/químicaRESUMEN
This research propounds an innovative technology focused on sustainability to increase the biomass yield of Akkermansia muciniphila, the next-generation probiotic, using prebiotic sources to replace or reduce animal mucin levels. A series of experimental design approaches were developed aiming to optimize the growth of Akkermansiamuciniphila by incorporating extracts of green leafy vegetables and edible mushroom into the cultivation media. Experiments using kale extract (KE), Brassica oleracea L., associated with lyophilized mushroom extract (LME) of Pleurotus ostreatus were the most promising, highlighting the assays with 0.376% KE and 0.423% LME or 1.05% KE and 0.5% LME, in which 3.5 × 1010 CFU (Colony Forming Units) mL- 1 was achieved - higher than in experiments in optimized synthetic media. Such results enhance the potential of using KE and LME not only as mucin substitutes, but also as a source to increase Akkermansia muciniphila biomass yields and release short-chain fatty acids. The work is relevant to the food and pharmaceutical industries in the preparation of the probiotic ingredient.