Microplate Research Articles

Evaluation of Antibacterial and Antibiofilm Activity of Essential Oils Against Isolated Bacteria from Milk Samples

ABSTRACT: The consumption of food contaminated with bacteria or their toxins can result in foodborne infections or illnesses. This study investigated the antibacterial and antibiofilm activity of essential oils against bacteria isolated from milk samples collected in Pune, Maharashtra, India. Twenty-five raw milk samples were collected from local vendors and examined for the presence of biofilm-forming microorganisms. The isolated bacteria were identified and characterized using morphological and biochemical tests, revealing that 20% of the samples were contaminated with Acinetobacter spp. and 28% with E. coli. The antibacterial activity of the essential oils was evaluated using the disc diffusion method, and the minimum inhibitory concentration (MIC) was determined using 96-well plates. The minimum bactericidal concentration (MBC) was also assessed by inoculating assay mixtures from wells exhibiting no microbial growth onto sterile nutrient agar medium. Biofilm formation and disruption were evaluated using crystal violet assay and biofilm disruption assay, respectively. The results demonstrated that the Cinnamon Bark and Oregano essential oils exhibited significant antibacterial and antibiofilm activity at the lowest MIC value of 0.02-0.04 μg/ml against the isolated bacteria. The findings suggest that essential oils could be potential natural alternatives to conventional antibiotics for controlling bacterial contamination and biofilm formation in milk and dairy products. Further research is needed to explore the practical applications of essential oils in the dairy industry and to ensure their safety and efficacy as natural antimicrobial agents.

Design and development of spectrophotometric enzymatic cyanide assays.

Determination of free cyanide (fCN) is required for various industrial, environmental, food, and clinical samples. Enzymatic methods are not widely used in this field despite their selectivity and mild conditions. Therefore, we present here a proof of concept for new spectrophotometric enzymatic assays of fCN. These are based on the hydrolysis of fCN affording the readily detectable NADH. fCN is hydrolyzed either in one step by cyanide dihydratase (CynD) or in two steps by cyanide hydratase (CynH) and formamidase (AmiF). An advantage of the latter route is the higher activity of CynH and AmiF compared to CynD. In both cases, the resulting formate is then transformed by an NAD-dependent formate dehydrogenase (FDH). The NADH thus formed is quantified colorimetrically using a known method based on a reduction of a tetrazolium salt (WST-8) with NADH. The developed assays of fCN are selective except for formic acid interference, proceed under mild conditions, and, moreover, fCN is detoxified during the reactions. The assays proceeded in a microtiter plate format. The limit of detection (LOD) and the limit of quantification (LOQ) were lower for the three-enzyme (CynH-AmiF-FDH) method (7.00 and 21.2µmol/L, respectively) than for the two-enzyme (CynD-FDH) method (10.7 and 32.4µmol/L, respectively). In conclusion, the new fCN assays presented in this work are selective, high-throughput, do not require harsh conditions, and use only small amounts of chemicals and enzymes.

Antibacterial, antibiofilm, and gene expression assessment of ajwain (Trachyspermum ammi) essential oil on drug-resistant gastrointestinal pathogens and its combination effect with ampicillin.

Essential oils are natural substances used as therapeutic agents and food preservatives to inhibit harmful microorganisms. This study aimed to assess the synergistic effect of Trachyspermum ammi essential oil and ampicillin on antibiotic-resistant gastrointestinal pathogens, including Escherichia coli, Enterococcus faecalis, Shigella flexneri, and Salmonella serotype Typhimurium. Using gas chromatography-mass spectrometry (GC-MS), the main components of T. ammi essential oil were identified as thymol, gamma terpenes, and cymene. The antibacterial and antibiofilm properties were evaluated by minimum inhibitory concentration (MIC), disk diffusion, and microtiter plate methods, revealing MIC values of 2, 1, 4, and 4mg ml-1 for E. coli, E. faecalis, S. flexneri, and S. Typhimurium, respectively, and inhibition zones between 10 and 14mm. Pathogens were examined for their biofilm-related virulence genes, including aggR, esp, icsA, and fliC, using real-time polymerase chain reaction (RT-PCR) in E. coli, E. faecalis, S. flexneri, and S. Typhimurium, respectively. The methyl thiazole tetrazolium (MTT) assay was used to evaluate the essential oil's effect on the viability of human embryonic kidney 293 (HEK293) cells, which showed cell viability of over 80%. The combination of T. ammi oil and ampicillin demonstrated a synergistic effect, and biofilm formation was inhibited. E. faecalis exhibited the greatest sensitivity, while S. flexneri exhibited the lowest sensitivity.

Efficient in vitro assay for evaluating drug efficacy and synergy against emerging SARS-CoV-2 strains.

Novel and repurposed antiviral drugs are available for the treatment of coronavirus disease 2019 (COVID-19). However, antiviral combinations may be more potent and lead to faster viral clearance, but the methods for screening antiviral combinations against respiratory viruses are not well established and labor-intensive. Here, we describe a time-efficient (72-96 h) and simple in vitro drug-sensitivity assay for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) using standard 96-well plates. We employ different synergy models (zero interaction potency, highest single agent, Loewe, Bliss) to determine the efficacy of antiviral therapies and synergistic combinations against ancestral and emerging clinical SARS-CoV-2 strains. We found that monotherapy of remdesivir, nirmatrelvir, and active metabolite of molnupiravir (EIDD-1931) demonstrated baseline EC50s within clinically achievable levels of 4.34 mg/L (CI: 3.74-4.94 mg/L), 1.25 mg/L (CI: 1.10-1.45 mg/L), and 0.25 mg/L (CI: 0.20-0.30 mg/L), respectively, against the ancestral SARS-CoV-2 strain. However, their efficacy varied against newer Omicron variants BA.1.1.15 and BA.2, particularly with the protease inhibitor nirmatrelvir. We also found that remdesivir and nirmatrelvir have a consistent, strong synergistic effect (Bliss synergy score >10) at clinically relevant drug concentrations (nirmatrelvir 0.25-1 mg/L with remdesivir 1-4 mg/L) across all SARS-CoV-2 strains tested. This method offers a practical tool that streamlines the identification of effective combination therapies and the detection of antiviral resistance. Our findings support the use of antiviral drug combinations targeting multiple viral components to enhance COVID-19 treatment efficacy, particularly in the context of emerging viral strains.

Optimizing Lasing Performance of CsPbBr3 Microplates by Regulating Exciton Recombination Dynamics with Pressure.

This study aims to achieve an ultralow lasing threshold in CsPbBr3 microplates (MPs), a crucial step toward developing electrically driven micro/nanolasers for optics integrated chips. We investigate the lasing behavior of CsPbBr3 MPs under varying pressures by using static-state photoluminescence (PL), time-resolved PL (TRPL), and first-principles theory calculations based on density functional theory (DFT). Our results reveal that the lasing threshold initially decreases and then increases, with a critical turning point at 0.44 GPa. Notably, we achieve an optimal lasing threshold of 20.87 μJ/cm2 after releasing pressure from 1.87 GPa, highlighting the potential of pressure modulation to optimize the lasing performance. At low pressure, pressure-induced phonon hardening enhances the barrier, preventing excitons decay from free states to trapping states. Conversely, at higher pressure, the increased density of surface defects, due to pressure-induced anisotropic contraction of lattice constants along the c-axis, leads to excitons decay from free states to trapping states. For CsPbBr3 MPs, it is evident that only free excitons contribute to lasing, while both free and trapped excitons contribute to luminescence. These findings offer a novel strategy to optimize the lasing performance of perovskite micro/nanolasers, significantly advancing their potential for practical applications in optoelectronic devices.

Assessment of synergistic and antagonistic interactions between volatile compounds thymol, carvacrol, and eugenol diluted in solvents against Rhipicephalus microplus in in vitro tests.

The cattle tick Rhipicephalus microplus is prevalent in tropical and subtropical regions, causing substantial economic losses due to its resistance to conventional acaricides. There is an urgent need to identify safe and effective new acaricidal agents. Essential oils and their volatile compounds are promising alternatives. Ensuring the use of optimal solvents or surfactants that do not compromise the acaricidal activity of these compounds during testing is crucial. This study aims to evaluate how compounds thymol, carvacrol and eugenol interact with xylol, methanol, ethanol, acetone, isopropyl alcohol, glycerol, dimethyl sulfoxide, castor oil, propylene glycol, vaseline, and Tween 80® to enhance (or to worse) their acaricidal efficacy against R. microplus. Larval mortality time were compared against one negative control (soybean oil) and two positive controls (commercial pour-on products). The experiments were conducted in 48-well polyethylene plates, with around 100 larvae immersed in 200 μl of each solvent at 100, 50, 25, 12.5, 6.25, 3.125 and 1.56% and diluted in soybean oil or water, according to solubility. Each volatile compound (Thymol, carvacrol and eugenol) was diluted in the tested solvents to assess larval mortality time. Xylol demonstrated the shortest larval mortality time, even at a minimum concentration (p < 0.05). In contrast, liquid vaseline exhibited the longest larval mortality time. When thymol, carvacrol, and eugenol were combined with xylol, they achieved the shortest larval mortality time. Conversely, when diluted in liquid vaseline they exhibited synergistic effects decreasing the mortality time. Tween 80® worsen the efficacy of thymol, carvacrol, and eugenol, resulting in prolonged larval mortality times. These findings emphasize the critical role of solvent selection, indicating the choice of solvent profoundly affects the formulation's effectiveness, directly influencing the activity of the active compounds.

Expedited SARS-CoV-2 Main Protease Inhibitor Discovery through Modular 'Direct-to-Biology' Screening.

Reactive fragment (RF) screening has emerged as an efficient method for ligand discovery across the proteome, irrespective of a target's perceived tractability. To date, however, the efficiency of subsequent optimisation campaigns has largely been low-throughput, constrained by the need for synthesis and purification of target compounds. We report an efficient platform for 'direct-to-biology' (D2B) screening of cysteine-targeting chloroacetamide RFs, wherein synthesis is performed in 384-well plates allowing direct assessment in downstream biological assays without purification. Here, the developed platform was used to optimise inhibitors of SARS-CoV-2 main protease (MPro), an established drug target for the treatment of COVID-19. An initial RF hit was developed into a series of potent inhibitors, and further exploration using D2B screening enabled a 'switch' to a reversible inhibitor series. This example of ligand discovery for MPro illustrates the acceleration that D2B chemistry can offer for optimising RFs towards covalent inhibitor candidates, as well as providing future impetus to explore the evolution of RFs into non-covalent ligands.

Unveiling the Potency: Synthesis and Assessment of Antimicrobial Potential of Some Bis(indolyl)methane Derivatives

Background: The urgent need for new antimicrobial compounds arises from the growing threat of multidrug-resistant human pathogens responsible for infectious diseases. The indole moiety, a prevalent heterocyclic ring system found in nature, is a key structural element in many pharmaceutical agents due to its wide range of biological activities. Bis(in-dolyl)methanes, in particular, have emerged as promising candidates for antibacterial activ-ity. Aim: This study aimed to evaluate the antibacterial activity of nine bis(indolyl)methane derivatives against a range of pathogenic bacterial strains responsible for various human diseases. Methods: The compounds were synthesized using a solvent-free method, and their antibac-terial activity was evaluated using the disk diffusion assay. The minimum inhibitory con-centration (MIC) of the active compounds identified in the disk diffusion assay was deter-mined by the microtiter broth dilution method in 96-well microtiter plates. Bacterial strains in the mid-log phase of growth were utilized. Bacterial suspensions equivalent to 0.5 McFar-land standards were prepared by suspending the bacterial inoculum in sterile water. A work-ing concentration of 100 μg/mL was achieved by diluting the test compounds in 100% DMSO. Results: The antimicrobial activity of nine synthetic compounds was evaluated against nine medically significant pathogenic strains. These include Pseudomonas aeruginosa, Bacillus cereus, and Shigella flexneri, known for producing toxins that cause acute foodborne ill-nesses, as well as Escherichia coli K12 and Enterococcus faecalis, which can disrupt the intestinal barrier in immunocompromised individuals. The results suggest that these com-pounds have the potential to be effective antimicrobial agents. Conclusion: Our findings demonstrate the promising antimicrobial activity of the synthe-sized compounds, with 1-ethyl-3-((1-ethyl-1H-indol-3-yl)(phenyl)methyl)-1H-indole emerging as the most potent, significantly inhibiting most tested bacterial strains. These results highlight the potential for developing novel compounds for antibacterial treatment.

Assessing the efficacy of postbiotics derived from Lactobacillus plantarum on antibiotic resistance genes in nosocomial pathogens such as Enterococcus faecalis and Pseudomonas aeruginosa.

This study investigated the antibacterial and anti-biofilm properties of postbiotics derived from Lactobacillus plantarum and their effect on the expression of antibiotic resistance genes (ermB and blaKPC) in Enterococcus faecalis and Pseudomonas aeruginosa, respectively. Cell-free supernatants (CFSs) were analyzed through gas chromatography-mass spectrometry (GC-MS), which showed that butyric acid (14.31%) was the major compound, other metabolites present in CFSs included lactic acid (5.94%), hdroxyacetone (5,21%), benzoic acid (3.12%), Pyrrolo[1,2-a] pyrazine-1,4-dione (1.91%), 2,3-Butanediol (1.04%), and 2,3-dihydro-3,5-dihydroxy-6-methyl-4H-pyran-4-one (0.73.%). To investigate the effect of postbiotics on bacterial growth and biofilm formation, minimal inhibitory concentration (MIC) and microtiter plate assays were used. MIC results showed that resistant En. faecalis and P. aeruginosa can grow at concentrations of 2.5and 5mg/ml, respectively, after exposure to postbiotics. Furthermore, the microtiter plate results showed that postbiotics significantly reduced biofilm formation: 51%, 45%, and 39% in En. faecalis and 46%, 38%, and 27% in P. aeruginosa at different concentrations. Real-time polymerase chain reaction also confirmed the reduction of resistance genes (ermB; P=0.007 and blaKPC; P=0.02) expression. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay showed that the cell survival rate was 80%. These findings suggest that postbiotics from L. plantarum may be a promising approach for combating bacterial growth, biofilm formation, and antibiotic resistance.

Efficient, Low-Cost, and High-Throughput Sodium Dodecyl Sulfate (SDS) Removal from Protein Digests Using Weak-Anion Exchange.

Sodium dodecyl sulfate (SDS) plays a pivotal role in protein denaturation, tissue extraction, and protein mass-based electrophoretic separations. However, even modest concentrations of SDS can cause column overpressure, retention time shifts, and ionization signal suppression during liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses. Thus, SDS removal is a critical step for LC-MS/MS analysis of protein digests containing SDS. This study describes an inexpensive and high-throughput method to remove SDS from protein digests using weak-anion exchange (WAX) resins in 96-well filter plates. Requiring less than 3 min, this method can reduce SDS concentrations from 0.1-0.4% to less than 5 ppm and from 0.6-1% to less than 100 ppm. After SDS removal, the recoveries of unmodified tryptic peptides and phosphorylated peptides (at 94.3 nM) were ∼90% and ∼70%, respectively. Additionally, when using aqueous 1% SDS to solubilize trastuzumab-spiked mouse serum and subsequently removing the SDS using the WAX resin, quantitation of trastuzumab exhibited excellent linearity (R2 = 0.9996) together with a low coefficient of variation (<10%). Calculated concentrations were within 20% of the expected value for spiked standard samples (0.5, 1, and 2 μg/mL trastuzumab in mouse serum). The method is about 20× more cost-effective versus commercialized SDS removal kits and both the resin and filter plate are readily available, so the method should easily transfer to other laboratories.

Antibacterial and anti-biofilm efficacy of 1,4-naphthoquinone against Chromobacterium violaceum: an in vitro and in silico investigation.

Antimicrobial resistance (AMR) is an urgent worldwide health concern, requiring the exploration for novel antimicrobial interventions. A Gram-negative bacterium, Chromobacterium violaceum, synthesizes quorum-sensing-regulated violacein pigment, develops resilient biofilms, and is often used for the screening of anti-infective drugs. The aim of this work is to assess the antibacterial and antibiofilm properties of three polyphenols: 1,4-naphthoquinone, caffeic acid, and piperine. The determination of antibacterial activity was conducted by the agar overlay and broth microdilution techniques. Analysis of membrane rupture was conducted by crystal violet uptake and β-galactosidase assay. Inhibition of biofilm was evaluated using a 96-well microtiter plate assay. Biofilm structures were visualized using light, scanning electron microscopy (SEM), and confocal laser scanning electron microscopy (CLSM). Among the phytochemicals, 1,4-naphthoquinone exhibited the highest antibacterial action (25mm zone of inhibition). The minimum inhibitory concentration of 1,4-naphthoquinone was determined to be 405µM. Outer and inner membrane permeability was enhanced by 52.01% and 1.28 absorbance, respectively. Violacein production was reduced by 74.85%, and biofilm formation was suppressed by 63.25% at sub-MIC levels (202.5µM). Microscopic analyses confirmed reduced adhesion on surfaces. Hemolytic activity of 1,4-naphthoquinone showed a concentration-dependent effect, with 32.16% haemolysis at 202.5µM. Molecular docking revealed significant interactions of 1,4-naphthoquinone with DNA gyrase followed by CviR. These findings highlight 1,4-naphthoquinone's potent antibacterial efficacy against C. violaceum, proposing its use as a surface coating agent to prevent biofilm formation on medical devices, thereby offering a promising strategy to combat bacterial infections.

Upconversion-Linked Immunosorbent Assay for the Biomimetic Detection of the Mycotoxin Cyclopiazonic Acid.

The neurotoxin α-cyclopiazonic acid (CPA) is an emerging mycotoxin produced as a secondary metabolite by several fungi species (i.e., Penicillium spp. and Aspergillus spp.). CPA commonly contaminates maize, crops, cheese, and wine. In this work, CPA detection in foodstuff was accomplished by the innovative integration of two strategies: upconversion nanoparticles (UCNPs) and epitope-mimicking peptides, to develop a competitive upconversion-linked immunosorbent assay (ULISA). We have applied UCNPs (type NaYF4:Yb3+, Er3+) as background-free optical labels due to their anti-Stokes shift with excitation in the near-infrared region and emission in the ultraviolet-visible region. Moreover, a CPA epitope-mimicking cyclic peptide (A2) was used as a substitute for the toxin-conjugates traditionally applied to competitive assays. UCNPs were decorated with an anti-CPA fragment antigen-binding antibody (UCNP-Fab), and CPA detection was achieved through competition with a biotinylated CPA epitope-mimicking cyclic peptide (A2-biotin, ACNWWDLTLC-GGGSK (Biotin)-NH2), anchored to a streptavidin-coated microtiter plate, for antibody binding. The ULISA platform offers ultrasensitive detection of CPA (limit of detection of 1.3 pg mL-1 and IC50 value of 15 pg mL-1), and no cross-reactivity was observed with other coproduced mycotoxins. These results substantially outperformed the analytical features of conventional heterogeneous immunoassays based on enzymatic detection. Additionally, the use of advanced computational tools, such as MOE and Alphafold AI, proved advantageous in elucidating the molecular interactions between the antibody and the epitope, providing insights that enhance the rational design of immunoassays. The proposed ULISA was applied to detect CPA in spiked maize samples, and the results were validated by high-performance liquid chromatography coupled to a tandem mass spectrometry detector (HPLC-MS/MS).

AutoMEA: machine learning-based burst detection for multi-electrode array datasets.

Neuronal activity in the highly organized networks of the central nervous system is the vital basis for various functional processes, such as perception, motor control, and cognition. Understanding interneuronal connectivity and how activity is regulated in the neuronal circuits is crucial for interpreting how the brain works. Multi-electrode arrays (MEAs) are particularly useful for studying the dynamics of neuronal network activity and their development as they allow for real-time, high-throughput measurements of neural activity. At present, the key challenge in the utilization of MEA data is the sheer complexity of the measured datasets. Available software offers semi-automated analysis for a fixed set of parameters that allow for the definition of spikes, bursts and network bursts. However, this analysis remains time-consuming, user-biased, and limited by pre-defined parameters. Here, we present autoMEA, software for machine learning-based automated burst detection in MEA datasets. We exemplify autoMEA efficacy on neuronal network activity of primary hippocampal neurons from wild-type mice monitored using 24-well multi-well MEA plates. To validate and benchmark the software, we showcase its application using wild-type neuronal networks and two different neuronal networks modeling neurodevelopmental disorders to assess network phenotype detection. Detection of network characteristics typically reported in literature, such as synchronicity and rhythmicity, could be accurately detected compared to manual analysis using the autoMEA software. Additionally, autoMEA could detect reverberations, a more complex burst dynamic present in hippocampal cultures. Furthermore, autoMEA burst detection was sufficiently sensitive to detect changes in the synchronicity and rhythmicity of networks modeling neurodevelopmental disorders as well as detecting changes in their network burst dynamics. Thus, we show that autoMEA reliably analyses neural networks measured with the multi-well MEA setup with the precision and accuracy compared to that of a human expert.

The Impact of Using Laser and Milling Techniques to Create Zirconia Patterns on Streptococcus oralis Biofilm Formation

This study aimed to evaluate zirconia dental implant surfaces patterned using Nd:YAG laser or conventional milling techniques against Streptococcus oralis adhesion and biofilm formation. Zirconia dental implant discs were subjected to surface patterning treatments and categorized into four groups: groove texturing by conventional milling (GM), pore texturing by conventional milling (PM), groove texturing by Nd:YAG laser (GL), and pore texturing by Nd: YAG laser (PL). Streptococcus oralis CECT 907T was cultivated on enriched blood agar plates and then transferred to a brain–heart infusion modified medium and incubated at 37 °C under anaerobic conditions until reaching the exponential growth phase. The bacterial suspension was then seeded on 24-well plates containing the treated discs. The viability of bacteria within the biofilm was determined based on colony-forming unit (CFU) counts, while the total biofilm was quantified by measuring its biomass. A qualitative analysis was conducted using scanning electron microscopy (SEM) images to evaluate the bacterial morphology. The statistical analysis of multigroup comparisons was performed using Kruskal–Wallis test with post hoc pairwise comparison, as well as Mann Whiney U test, with significance set at p &lt; 0.05. After both 1 h and 24 h of incubation of Streptococcus oralis on the discs, all groups showed similar results, with no statistically significant differences (p &gt; 0.05). A comparison of the Nd: YAG laser-treated surfaces with conventionally milled surfaces, as well as grooves versus pores for CFU counts, also revealed no statistically significant differences (p &gt; 0.05) for both 1 h and 24 h of culture. Biomass quantification at both the 1 h and 24-h time points showed similar results across the groups, without statistical differences. When comparing the conventionally machined surfaces to Nd: YAG laser-treated surfaces in terms of biomass, no significant differences were observed (p &gt; 0.05). Similarly, the comparison between groove-patterned surfaces and pore-patterned surfaces showed no statistically significant difference. The groove and pore patterns on zirconia surfaces with Nd: YAG laser or conventional milling did not change the Streptococcus oralis adhesion and biofilm formation behavior. Additional studies are recommended to expand our knowledge in this area.

Sensitivity of Septoria lycopersici Speg. isolates to fungicides in Brazil

Disease management using synthetic fungicides is the main strategy used by tomato growers to prevent tomato yield reductions caused by Septoria leaf spot (SLS). Despite the importance of this plant disease, there is little information about the sensitivity of Septoria lycopersici isolates to fungicides. The sensitivity of 94 isolates of S. lycopersici to azoxystrobin, chlorothalonil, tebuconazole, and thiophanate-methyl was assessed in experiments using fungicide-amended 96-well microtiter plates. The highest EC 50 values above 100 mg/L were detected for azoxystrobin, thiophanate-methyl, and tebuconazole. Based on these results, five isolates classified as resistant and five as sensitive to each fungicide were selected for the experiments. Tomato plants were inoculated with sensitive (S) and resistant (R) isolates and sprayed by the fungicides in three greenhouse assays. The differences in the values of the area under the progress curve of Septoria leaf spot (AUDPC), disease severity progress, and progress rates were not so pronounced between tomato plants sprayed and non-sprayed with fungicides. The AUDPC values for plants inoculated with S and R isolates and treated with chlorothalonil, tebuconazole, and thiophanate-methyl ranged from 10 to 320, 0 to 80, and 50 to 150, respectively. For azoxystrobin, the AUDPC values were around 200 in plants inoculated with S and R isolates. There is evidence of reduced sensitivity of individuals of S. lycopersici to azoxystrobin, tebuconazole, and thiophanate-methyl in the population in Brazil. Therefore, tomato growers should rotate and combine fungicides with different mechanisms of action and low-risk of resistance to control SLS.

Cyst hatching and nauplii vitality of &lt;em&gt;Artemia salina&lt;/em&gt; treated with different potencies of Silicea terra

This study is a preliminary step of the Terra Project, a multicentric study aiming to develop methods for improving agriculture and husbandry production through potentized high dilutions (dynamized products), popularly known as “agro-homeopathy.” The present data reflect the characterization of the biological effects of different potencies of Silicea terra on the Artemia salina model concerning cyst hatching, nauplii vitality, and heat shock proteins (HSPs) gene expression, following the standards previously established to study bio-resilience processes [1]. Later, a similar protocol will be tested in rice germination in greenhouses to compose the whole data framework. Herein, the following potencies of Silicea terra were used: 6, 9, 12, 15, 18, 21, 24, 27, and 30cH, all prepared in purified sterile water (PSW). PSW and succussed PSW were used as controls. The potencies and controls were poured into the seawater (10% of the volume) at the moment of cyst seeding using 96-well plates kept at room temperature (average 25oC). All experiment steps were run during the full moon phase due to the high sensitivity of Artemia spp to the circalunar cycle. Two protocols were tested: (a) the constant temperature for 48 hours of incubation and (b) the exposition to one hour of thermic stress (40oC) after 24 hours of incubation. The samples were tested in octuplicate, and each data point was defined as a well containing 5 to 8 cysts. After 24 and 48 hours, the following parameters were evaluated: cyst hatching rate, nauplii vitality (number of live nauplii/total of cysts per well), and nauplii lethality (number of dead nauplii/total of cysts per well). After 48 hours, the remaining biological material was pooled according to each treatment, including a supplementary series of no-treated wells (baseline control), and the mRNA was extracted for further assay of HSP gene expression (HSP40, HSP90, HSP26, and p26). The statistical analysis was made by two-way ANOVA and Tukey test to compare potencies and controls in the function of time, being α=0.05. Lagrangian statistics was used to evaluate the linear time-dependent response in the function of the potencies. Among the different potencies, the 24cH showed a significant reduction in the lethality rate and a significant increase in the hatching and vitality rates when exposed to thermic stress. These data were similar to the baseline standards. The Lagrangian statistics showed a linear relation between time and potencies. The evaluation of HSP gene expression is still in course. This step concludes that Silicea terra 24cH is a good model for the embryo development of Artemia salina and bio-resilience. Both statistical approaches are valuable tools to characterize the mode of action of Silicea terra on biological systems, giving scientific plausibility for its further use in food production chains. Since Artemia salina are non-sentient animals, there is no need for ethical committee approval.

Biological and physicochemical effects of magnetic field on &lt;em&gt;Artemia franciscana&lt;/em&gt; exposed to cyanobacteria extract and treated with isotherapeutic 200cH

This study proposes an experimental model to analyze homeopathic potencies' biological and physicochemical effects on intense magnetic fields. The proposed model is based on the exposition of Artemia franciscana cysts to saxitoxin (cyanobacteria extract) and the treatment with R. raciborskii extract isotherapic 200cH (named saxitoxin 200cH), followed by the hatching and nauplii (larvae) vitality rate (live nauplii/total cysts), and the gene expression of heat shock proteins (HSP 26, 40, 90, and p26), as previously described ad bio-resilience indicators [1,2]. In a second experiment, pH, conductivity, and temperature temporal curves were built, and different treatments were compared before and after being poured into the seawater. The method details are as follows: Artemia franciscana cysts were seeded into artificial seawater in 96-well plates in triplicate, exposed immediately to 25ppm of R. raciborskii extract, and treated with sterile purified water (SPW), succussed SPW (vehicle controls), or saxitoxin 200cH prepared in SPW, submitted or not to a 2400 Gauss static magnetic field for 15 minutes. Plates were kept in a Faraday cage under stable lightening (300 mini-led lamps), temperature (26.5%), humidity (65%), and magnetic field (0.7 µT). The hatching and vitality rate were analyzed after 48 hours. In the second experiment, the physicochemical parameters were analyzed before and after one minute, two, and 24 hours after pouring the samples into seawater (100µL/100mL), kept at the same environmental conditions set for the first experiment. The statistical analysis was made by two-way ANOVA / Tukey, with α=0.05. Treatment of cysts with SPW alone reduced the hatching and nauplii vitality rate in relation to the other treatments (p≤0.05); however, the treatment with saxitoxin 200cH exhibited smaller variance and the highest statistical hatching (p=0.0009) and vitality (p=0.0007) levels. The previous submission of samples to the magnetic field reverted this result by canceling the statistical differences and increasing the variance of saxitoxin 200cH datapoints. The analyses of HSP gene expression are still in course. In the second experiment, there is no specific effect of saxitoxin 200cH on temperature, conductivity, and pH, independent of the exposition of samples to the magnetic field. The results corroborate the hypothesis that a possible interference on dipolar dynamics of saxitoxin 200cH caused by the intense magnetic field could negatively interfere with its biological effect independently of eventual changes in temperature, conductivity, and pH in the seawater, although its clear capacity to improve tracking homeopathic potencies in water when using the method of solvatochromic dyes, as previously shown [3].

Optical density assay to determine the sensitivity of Globisporangium and Pythium isolates to mefenoxam

Several species of Globisporangium and Pythium cause cavity spot of carrot. Disease management in conventional production relies primarily on applications of the fungicide mefenoxam. Growers’ reports in Ontario, Canada of severe cavity spot, even when using mefenoxam applications, suggests mefenoxam resistance may be prevalent in pathogen populations in this region. The objectives of the study were to develop a rapid and accurate optical density (OD) assay to assess mefenoxam sensitivity of 188 isolates of Globisporangium and Pythium species collected from 13 carrot fields in Ontario from 2020 to 2023. The isolates belonged to seven species: G. intermedium, G. irregulare, G. rostratifingens, G. sylvaticum, G. ultimum, G. violae, and P. sulcatum. The OD was measured at 600 nm in broth amended at different concentrations of mefenoxam, with OD readings at nine positions per well in a 96-well plate. The OD assay was validated by comparing EC50 values with a standard fungicide-amended agar medium assay using 20 representative isolates. There was a significant linear relationship (R2 = 0.97, P = &lt;0.0001) between the two assays. Of the 188 isolates tested, 24% exhibited &lt;60% inhibition of mycelial growth at 5 μg mefenoxam mL-1, 50% were inhibited by &gt;60% at 5 μg mL-1, 19% were inhibited by &gt;60% at 1 μg mL-1, and 7% were inhibited &gt;60% even at 0.1 μg mL-1. The OD assay proved to be efficient, rapid, and accurate for monitoring mefenoxam sensitivity of these oomycetes.

Effects of Phenolic Compounds on Biofilm Formation by Table Olive-Related Microorganisms.

The process of biofilm formation during table olive fermentation is crucial to turning this fermented vegetable into a probiotic food. Some phenolic compounds have been described as important quorum-sensing molecules during biofilm development. The present invitro study examined the effects of three phenolic compounds widely found in table olive fermentations (Oleuropein 0-3000 ppm, Hydroxytyrosol 0-3000 ppm, and Tyrosol 0-300 ppm) on the development of single biofilm by diverse microorganisms isolated from table olives (Lactiplantibacillus pentosus 13B4, Lp119, and LPG1; Lactiplantibacillus plantarum Lp15 and LAB23; and yeasts Wickerhamomyces anomalus Y12, Candida boidinii Y13, and Saccharomyces cerevisiae Y18). Biofilm formation was quantified invitro by crystal violet staining in microtiter plates after incubation at 30°C for 96 h. A clear tendency to decrease the biofilm production was observed for the L. plantarum strains when any of the three phenolic compounds were added to the medium, which was statistically significant (p ≤ 0.05) for certain concentrations and phenols. In the case of yeasts, no statistical influence on biofilm formation was noticed when the phenolic compounds were dosed to the culture medium. Finally, the effects of the phenolic compounds on the L. pentosus strains were dependent on the strain assayed. Thereby, addition of phenolic compounds on 13B4 or Lp119 strains did not have statistical influence on biofilm production. On the contrary, the probiotic LPG1 strain noticed a statistical increase in biofilm production when a low concentration of tyrosol (50 ppm) was added to the medium. Results obtained in this work could be useful to control the biofilm formation process on olive epidermis during table olive fermentation to include beneficial microorganisms.

Evaluating efflux pump inhibition in Staphylococcus aureus 1199B strain using thiadiazine-derived compounds: Invitro and in silico approaches.

Thiadiazines are heterocyclic compounds known for some pharmacological activities. However, the ability of these compounds and their derivatives to act as antibacterial agents and inhibitors of the efflux system in resistant bacteria remains unknown. This study aims to evaluate the antibacterial and NorA efflux pump inhibitory activities of thiadiazine-derived compounds (IJ14, IJ15, IJ16, IJ17, IJ18, IJ19, and IJ20) against the Staphylococcus aureus 1199B strain. Minimum Inhibitory Concentration (MIC) tests and antibacterial activity assessment through NorA efflux system inhibition were performed using microdilution assays in 96-well plates. Additionally, ethidium bromide (EtBr) fluorescence emission assays were conducted to evaluate efflux system inhibition. The methodology revealed that the IJ17 and IJ20 compounds presented MIC values of 256 and 597.3μg/mL, respectively. The efflux pump inhibition assessment using the microdilution method showed significant results for all compounds, which also increased the fluorescence rates emitted by EtBr. Consequently, thiadiazine-derived compounds exhibit promising results in targeting a key bacterial resistance mechanism, underscoring the need for further studies, such as molecular tests, to evaluate their mechanism of action and clarify the feasibility and efficacy of these compounds as antibacterial agents.