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Objective: The study aims at the derivatization of “Phthalides” and synthesizes 3- arylaminophthalides & 3-indolyl-phthalides compounds, and evaluates their anti-tubercular and antioxidant activities. The study has also intended to employ the in silico methods for the identification of possible drug targets in Mycobacterium and evaluate the binding affinities of synthesized compounds. Methods: This report briefly explains the synthesis of phthalide derivatives using ammonium chloride. The synthesized compounds were characterized using spectral analysis. Resazurin Microtiter Assay (REMA) plate method was used to demonstrate the anti-mycobacterial activity of the synthesized compounds. An in-silico pharmacophore probing approach was used for target identification in Mycobacterium. The structural level interaction between the identified putative drug target and synthesized phthalides was studied using Lamarckian genetic algorithm-based software. Results and Discussion: In the present study, we report an effective, environmentally benign scheme for the synthesis of phthalide derivatives. Compounds 5c and 5d from the current series appear to possess good anti-mycobacterial activity. dCTP: deaminasedUTPase was identified as a putative drug target in Mycobacterium. The docking results clearly showed the interactive involvement of conserved residues of dCTP with the synthesized phthalide compounds. Conclusion: On the eve of evolving anti-TB drug resistance, the data on anti-tubercular and allied activities of the compounds in the present study demonstrates the enormous significance of these newly synthesized derivatives as possible candidate leads in the development of novel anti-tubercular agents. The docking results from the current report provide a structural rationale for the promising anti-tubercular activity demonstrated by 3-arylaminophthalides and 3-indolyl-phthalides compounds.

Biofilm formation and antimicrobial resistance at surgical implant sites result in high morbidity and mortality. Identifying novel molecules that inhibit biofilm formation to coat surgical biomaterials is essential. One such compound is N-acetylcysteine (NAC), a potent antioxidant precursor for glutathione, necessary in mammalian cells and known to disrupt/prevent biofilms. In this study, NAC was covalently immobilized onto functionalized polyvinyl chloride surfaces using plasma immersion ion implantation (PIII) treatment that achieves covalent binding without the need for linker groups. NAC immobilization was characterized using water contact angles, Fourier-transform infrared, and X-ray photoelectron spectroscopy techniques. Bacterial viability and biofilm formation on NAC surfaces were assessed using resazurin assays, phase contrast microscopy, and colony counting experiments. Effect of NAC on bacterial polysaccharide production and DNA cleaving was investigated using the phenol-sulfuric acid method and the Qubit fluorometer. Surface thermodynamics between the NAC coating and bacterial cells were measured using the Lewis acid-base method. Surface characterization techniques demonstrated superficial changes after PIII treatment and subsequent covalent NAC immobilization. NAC-coated surfaces significantly reduced biofilm viability and the presence of Gram-negative and Gram-positive bacteria. NAC also decreased polysaccharide production and degraded DNA. This led to unfavorable conditions for biofilm formation on NAC-coated surfaces, as demonstrated by surface thermodynamic analysis. NAC-coated surfaces showed no cytotoxicity to human fibroblast cells. This study has successfully utilized NAC as an antibiofilm coating, which may pave the way for improved prophylactic coatings on medical implant devices in the future.

Objective This study tests the elution capabilities of a magnesium phosphate cement (MPC). Study objectives were to quantify the passive release of magnesium ions from MPC and to assess the effects of antibiotic-loaded MPC on bacterial growth and osteoblast viability. Methods MPC constructs were created and incubated in fetal bovine serum (FBS). At 2, 4, and 17 weeks, a sample was collected for magnesium ion concentration analysis. Control and vancomycin-loaded (vanc) MPC beads were also created. Zone of inhibition was measured after incubating beads on Staphylococcus aureus agar plates for 24 h. Osteoblasts were seeded onto control and vanc beads and cultured for 9 days. Metabolic activity was measured via a resazurin assay. ANOVA with Tukey HSD post-hoc tests and t-tests were performed. Results Magnesium ions were eluted at 2 and 4-week time points without significant difference, but demonstrated a significant spike at the 17-week time point. Zones of inhibition for the bacterial species was observed for Vanc-MPC beads, but not control beads. No cytotoxic effects on osteoblasts were noted. Conclusion MPC has potential to improve bone regeneration based on its ability to passively elute magnesium. Additionally, antibiotic-loaded MPC inhibits bacterial growth while avoiding osteoblast cytotoxicity.

Recently, there has been an increasing interest in using the photoreduction of resazurin by aliphatic amines such as hydroxylamine as a model reaction to assess the efficiency of metal nanoparticles as photocatalysts. However, some features of this reaction are still not understood well. In particular, one of the most important still misconstrued feature of resazurin photoreduction reaction is the lag time in the beginning of the reaction, when no turnover of resazurin is observed. This lag time is often known as the induction time. In order to understand the origin of the induction time, we investigate the factors governing the induction time associated with the reaction. We found that the concentrations of the hydroxylamine and the dissolved oxygen are most important factors influencing the induction time. In the ambient condition, the induction time for resazurin reduction reaction can be as long as 30 min in the absence of any catalyst. Decreasing the dissolved oxygen concentration or increasing the hydroxylamine concentration can significantly reduce the induction time. We attributed the induction time to a side reaction, autoxidation of hydroxylamine by dissolved oxygen. Interestingly, we found that the presence of metal nanocatalyst such as gold can also reduce the induction time. We concluded that in addition to catalyzing the photoreduction of resazurin, gold nanocatalyst can also accelerate the autoxidation reaction of hydroxylamine.

Objectives LAY-FOMM is a promising material for FDA-approved Fused Deposition Modeling (FDM) applications in drug delivery. Here we investigated the impact on oral cells. Materials and methods We evaluated the impact of 3D-printed LAY-FOMM 40, LAY-FOMM 60, and biocompatible polylactic acid (PLA) on the activity of murine L929 cells, gingival fibroblasts (GF), and periodontal ligament fibroblasts (PDLF) using indirect (samples on cells), direct monolayer culture models (cells on samples), and direct spheroid cultures with resazurin-based toxicity assay, confirmed by MTT and Live-dead staining. The surface topography was evaluated with scanning electron microscopy. Results The materials LAY-FOMM 40 and LAY-FOMM 60 led to a reduction in resazurin conversion in L929 cells, GF, and PDLF, higher than the impact of PLA in indirect and direct culture models. Fewer vital cells were found in the presence of LAY-FOMM 40 and 60 than PLA, in the staining in both models. In the direct model, LAY-FOMM 40 and PLA showed less impact on viability in the resazurin-based toxicity assay than in the indirect model. Spheroid microtissues showed a reduction of cell activity of GF and PDLF with LAY-FOMM 40 and 60. Conclusion Overall, we found that LAY-FOMM 40 and LAY-FOMM 60 can reduce the activity of L292 and oral cells. Based on the results from the PLA samples, the direct model seems more reliable than the indirect model. Clinical relevance A material modification is desired in terms of biocompatibility as it can mask the effect of drugs and interfere with the function of the 3D-printed device.

Assessment of specific activity of Russian and foreign-made filgrastim products by biological in vitro methods is performed using different types of dyes. It is important to choose one cell staining dye in order to align the procedure of filgrastim specific activity assessment using cell culture. The aim of this study was to perform comparative assessment of tetrazolium and resazurin dyes in tests determining filgrastim ability to activate proliferation of sensitive cells. Materials and methods: NFS-60 (mouse myelogenous leukemia) cell line, 2nd International Standard for Granulocyte Colony Stimulating Factor (IS), as well as МТТ, MTS, WST-1, and alamarBlue dyes were used in the study. Proliferative activity of cells was assessed in vitro. The level of cell proliferation was assessed by fluorescence or absorbance intensity. Origin Pro 9.1. and Microsoft Excel applications were used for statistical processing of the obtained results. Results: the paper compares characteristics of the most widely used dyes. It describes the procedure for choosing optimal test conditions for some of the studied dyes. The authors analysed the potential of some factors, such as duration of cell suspension incubation with IS and with a dye, composition of the lysis buffer (for MTT staining), and different readout modes, to influence the final results. Despite the fact that all the studied dyes gave reproducible dose–response curves under the given test conditions, 50% effective concentrations showed no statistically significant differences in tests with only three dyes: МТТ, MTS, and alamarBlue (р > 0.05). Conclusions: better reproducibility of results was obtained in tests using МТТ and alamarBlue. The test procedure using alamarBlue is easier to perform and less time-consuming, it does not include the cell lysis stage and does not require additional reagents, therefore this dye may be recommended for harmonisation of the test procedure to be elaborated for the Russian Pharmacopoeia.

The goal of the present study was to select a Gram-positive (Gram+) and Gram-negative (Gram−) strain to measure antimicrobial activity in environmental samples, allowing high-throughput environmental screening. The sensitivity of eight pre-selected bacterial strains were tested to a training set of ten antibiotics, i.e. three Gram+ Bacillus subtilis strains with different read-outs, and five Gram− strains. The latter group consisted of a bioluminescent Allivibrio fischeri strain and four Escherichia coli strains, i.e. a wild type (WT) and three strains with a modified cell envelope to increase their sensitivity. The WT B. subtilis and an E. coli strain newly developed in this study, were most sensitive to the training set. This E. coli strain carries an open variant of an outer membrane protein combined with an inactivated multidrug efflux transport system. The assay conditions of these two strains were optimized and validated by exposure to a validation set of thirteen antibiotics with clinical and environmental relevance. The assay sensitivity ranged from the ng/mL to μg/mL range. The applicability of the assays for toxicological characterization of aquatic environmental samples was demonstrated for hospital effluent extract. A future application includes effect-directed analysis to identify yet unknown antibiotic contaminants or their transformation products.

BackgroundDue to the side effects of chemical and synthetic antibiotics and the increase in bacterial resistance, extensive research has been conducted to obtain natural compounds without side effects from natural sources such as insects, especially Diptera order, because these insects require to live and survive on rotting food and be in direct contact with pathogenic and dangerous microorganisms because of their special diet.Materials & MethodsIn this study, hemolymphs were extracted from Musca domestica, Drosophila melanogaster, and Sarcophaga nodosa, and then the anti-bactericidal activity of these extracts against important pathogenic bacteria was investigated separately by the minimum inhibitory concentration (MIC) method using resazurin indicator.FindingsS. nodosa and M. domestica larvae were not able to prevent the growth of any of the bacteria. D. melanogaster larvae extract completely inhibited the growth of Escherichia coli and Pseudomonas aeruginosa bacteria at all densities, while Staphylococcus aureus was completely resistant to all concentrations. The minimum inhibitory concentration of D. melanogaster larvae extract against two bacteria of Listeria monocytogenes and Salmonella typhimurium was determined as 125 and 500 μL/mL, respectively.D. melanogaster pupae extract was unable to inhibit the growth of E. coli and S. typhimurium but prevented the growth of P. aeruginosa at all concentrations. Also, the minimum inhibitory concentration of this extract against both S. aureus and L. monocytogenes was determined as 1000 μL/mL.ConclusionThese outcomes show that D. melanogaster holds a high potential of antibacterial effects, and the purification and evaluation of this extract active substances are recommended for future utilization as antibacterial agents and food preservatives to fight pathogenic and toxigenic microorganisms.

Silver nanoparticles (Ag NP) were synthesized using rice leaf extract and optimized synthetic conditions were found to be 0.4 % leaf extract, 0.6 mM AgNO3 and 30 min of autoclaving. Produced NP were characterized using UV–vis, DLS, zeta potential, XRD, TEM and FTIR. Ag NP formation was established from UV–vis spectra and NP showed zeta potential value of −27.4 mV. NP were spherical, polydisperse and average size was 16.5 ± 6.2 nm. Antifungal activity of Ag NP was assessed by poisoned food technique and resazurin broth dilution against mycelium and sclerotia of fungus R. solani, the causative agent of sheath blight disease in rice. Results confirmed effective hyphal growth inhibition and % growth inhibition was dose dependent (2.5–10 μg/mL). Ag NP showed enhanced mycelial inhibition (81.7–96.7 %) at 10 μg/mL. MIC values of Ag NP were in the range of 5–10 and 15–20 μg/mL towards fungal mycelium and sclerotia, respectively. Ag NP treatment (20 μg/mL) completely inhibited the disease incidence at 20 μg/mL. Ag NP treatment (10 μg/mL) caused 1.3 and 1.5 times enhancement in seedling vigor index. Hence, Ag NP can be utilized towards management and control of various fungal diseases of crops.

Silver nanoparticles (AgNPs) were biosynthesized using the cell free supernatant of putative probiotic Lactobacillus paracasei A26. Several biological activities of biogenic AgNPs were investigated in respect to in vitro anti-oxidant and anti-tumor potentials. Anti-oxidant potentials were screened in terms of free radical scavenging activity against two free radicals, 2, 2-Diphenyl-1-picrylhydrazyl (DPPH) and resazurin dye. AgNPs exhibited a potent scavenging activity against resazurin dye (91±0.046%) with an EC50 concentration of 146.823 µg/ml, while scavenging of DPPH was significantly (P≤0.05) reduced to 72.330±0.114% using a higher EC50 concentration of 176.12 µg/ml. The anti-tumor potentials of biogenic AgNPs were studied in relation to the cytotoxicity against two human breast cancer cell lines (CAL-51 and MCF7), using crystal violet dye assay. The viability of AgNPs-treated cancerous cells was significantly decreased in a time- and concentration manner, as compared to insignificant cytotoxic effects against the normal cell line. However, the anti-proliferative activity of AgNPs did not exceed the value of 63.85±0.019% in both cancer cell lines. CAL-51 cells were the most sensitive to the introduced AgNPs, with a maximum decrease in viability of 49.889±0.021% being reached using an IC50 value of 98.65µg/ml for 48h exposure time. The inhibition percentage was increased to 60.13±0.005% when the used IC50 value was significantly declined to 40.73µg/ml with an exposure time expanded to 72h. MCF7 cells showed lower sensitivity than CAL-51 cells, but with a similar inhibition trend of 59.523±0.01% with an IC50 concentration of 66.54 µg/ml for 48h which was increased to 63.857±0.019% when the IC50 was reduced to 62.63 µg/ml and the exposure time expanded to 72h. The morphological changes of AgNPs-treated cells were apparent at 72h exposure time, with cells showing apoptotic-like features such as shrinkage and losing of regular fusiform shape. Moreover, cells became detached to surfaces and from each other.

Salmonella, one of the most common foodborne pathogens, poses a serious threat to human health. In recent years, the antibiotic resistance of Salmonella has become serious as well, which has strengthened the harm of Salmonella to human health. In this paper, a simple and effective electrochemical approach was developed to obtain profile of the antimicrobial susceptibility. Screen-printed carbon electrodes (SPCEs) were used to detect various concentrations of target bacteria at the same time, which have good stability, low cost, and easy mass production. The electroactive redox, resazurin, was used to monitor levels of metabolically active bacteria. As a demonstration, the antibacterial effect of ofloxacin and penicillin on Salmonella gallinarum (S. gallinarum) isolates was evaluated, and the minimum inhibitory concentration (MIC) was measured as well. In the real sample measurement, the MIC obtained was similar to that obtained by conventional antimicrobial susceptibility testing (AST). In this assay, bacterial activity was quantified sensitively and accurately, and the detection time was greatly reduced compared with conventional AST (16–20 h), which means that metabolic capacity of live bacteria could be observed after 1 h of incubation. We were able to clearly detect bacteria above 102 CFU/m. This method aims to nonspecific and can be widely applied to the detection of a variety of drug-resistant bacteria, providing an experimental basis for the rational use of antibiotics and bacterial resistance mechanisms.

Made available in DSpace on 2021-06-25T11:46:31Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-06-01 Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) INTRODUCTION: Resistance to first-line anti-tuberculosis drugs is a major concern in the treatment of the disease. New strategies, such as the use of efflux pump inhibitors (EPIs), are being investigated to improve the outcome of the treatment. Verapamil (VP), one such inhibitor, was shown to inhibit several efflux pump (EP) Mycobacterium tuberculosis proteins and demonstrate synergic activity with anti-TB drugs. OBJECTIVE: To evaluate the combinatory effect of isoniazid (INH) and VP in M. tuberculosis. METHODS: Minimal inhibitory concentrations and combinatory effects of INH+VP were determined using respectively resazurin microtitre assay plate (REMA) and resazurin drugs combination microtitre assay (REDCA). From the results, we selected three bacilli with different susceptibility profiles and assessed their expression of 10 EP genes through quantitative reverse transcription polymerase chain reaction after exposure to INH, VP and INH+VP for 48 h. RESULTS: A significant reduction of INH MIC was observed in INH-susceptible isolates upon combination with VP. In brief, gene expression assays revealed expression patterns that could be correlated with each resistance profile, presence or absence of gene mutations and combinatory effect with VP. CONCLUSION: Combining VP with INH showed important results in drug-susceptible strains, and clinical trials on combined VP + anti-TB drugs should be discussed. Univ Estadual Maringa, Lab Bacteriol Med, Maringa, PR, Brazil Univ Estadual Paulista, Dept Ciencias Biol, Fac Ciencias Farmaceut, Sao Paulo, SP, Brazil Univ Estadual Paulista, Dept Ciencias Biol, Fac Ciencias Farmaceut, Sao Paulo, SP, Brazil CNPq: 2016/14957-5

BackgroundThe WHO declared Staphylococcus aureus as a ‘pathogen of high importance’ in 2017. One-fifth of all bloodstream-related infections in Australia and 12 000 cases of bacteraemia in the UK (2017–18) were caused by the MRSA variant. To address the need for novel therapies, we investigated several permutations of an innovative combination therapy containing N-acetylcysteine (NAC), an antibiotic and an enzyme of choice in eradicating MRSA and MSSA biofilms.MethodsBiofilm viability (resazurin assay) and colony count methods were used to investigate the effect of NAC, antibiotics and enzymes on S. aureus biofilm disruption and killing. The effects of NAC and enzymes on the polysaccharide content of biofilm matrices were analysed using the phenol/sulphuric acid method and the effect of NAC on DNA cleavage was determined using the Qubit fluorometer technique. Changes in biofilm architecture when subjected to NAC and enzymes were visualized using confocal laser scanning microscopy (CLSM).ResultsNAC alone displayed bacteriostatic effects when tested on planktonic bacterial growth. Combination treatments containing 30 mM NAC resulted in ≥90% disruption of biofilms across all MRSA and MSSA strains with a 2–3 log10 decrease in cfu/mL in treated biofilms. CLSM showed that NAC treatment drastically disrupted S. aureus biofilm architecture. There was also reduced polysaccharide production in MRSA biofilms in the presence of NAC.ConclusionsOur results indicate that inclusion of NAC in a combination treatment is a promising strategy for S. aureus biofilm eradication. The intrinsic acidity of NAC was identified as key to maximum biofilm disruption and degradation of matrix components.

The assay of Anti T lymphocyte immunoglobulin for final drug product testing is carried out using flow cytometry on Peripheral Blood Mononuclear Cells (PBMCs) as specified in European and British Pharmacopeia. An alternate assay was developed wherein the potency based quality control evaluation of Anti T lymphocyte immunoglobulin is carried out by measuring complement dependent cytotoxicity (CDC) using fluorescent resazurin dye. The reported bioassay was specific, linear (R2 = 0.98), precise (%GCV for repeatability was 3.54% and intermediate precision was 4.27%) and accurate with relative bias of −5.54%. On the basis of results obtained from the repeated performances on single available product, system suitability criteria and sample acceptance criteria were proposed wherein Slope from 4 PL curve fit results for Reference Standard (RS) should be > 0.9, EC50 for RS should lie between 0.264 and 1.131 μg/ml and fold response should be > 2. Confidence interval range and estimated relative potency range obtained from the method validation were narrower than those mentioned for compendial method.

Investigations of the root extract of Dracaena usambarensis Engl. for anticancer principles led to the characterization of one new homoisoflavonoid, (3S)-3,4ʹ,5,6-tetrahydroxy-7-methoxyhomoisoflavanone (1) and a new retrodihydrochalcone, 4ʹ,4-dihydroxy-2,3-dimethoxydihydrochalcone (2) along with six previously reported compounds, including two homoisoflavonoids, 7-O-methyl-8-demethoxy-3-hydroxy-3,9-dihydropunctatin (3) and loureiriol (4); a phenolic amide, 3-(4ʹʹʹ-hydroxyphenyl)-N-[2ʹ-(4ʹʹ-hydroxyphenyl)-2ʹ-methoxyethyl]acrylamide (5); a spirostane, 25S-spirosta-1,4-dien-3-one (6) and two steroids, stigmasterol (7) and stigmasterol 3-O-β-D-glucopyranoside (8). The structures of 1-8 were determined using spectroscopic and spectrometric techniques. The absolute configurations of compounds 1 and 3 were achieved using circular dichroism spectroscopy. Using the resazurin reduction assay and doxorubicin as reference anticancer drug, 1 showed moderate cytotoxicity against drug sensitive CCRF-CEM but was inactive against all the other tested drug sensitive, resistance phenotypes and normal cells. The crude extract and 2-8 were inactive in the preliminary screening against CCRF-CEM and drug resistant CEM/ADR5000 cell lines. Interestingly, the activity of the standard drug, doxorubicin was comparable to those of inactive compounds against CEM/ADR5000 cells. Future studies should focus on structure modifications of 1-3, in order to obtain more potent analogues.

Development of viable cell estimation method without sacrificing proliferation and functions of cells cultured on regenerative biomaterials is essential for regenerative engineering. Cytotoxicity and depletion of resazurin are critical but often overlooked limitations that hindered applications of resazurin in viable cell estimation. The present work found that cytotoxicity and depletion of resazurin depended on cell concentration, resazurin concentration and resazurin incubation time. A simple strategy which only allowed cells to incubate with resazurin during each measurement was developed to eliminate negative effects of resazurin. This strategy was verified by monitoring proliferation of MC3T3-E1 preosteoblasts on poly(d,l-lactic acid) scaffold during a continuous 3D culture process for up to 21 days, comparing the accuracy with MTT assay which is a destructive assay with high sensitivity and accuracy and commonly used in regenerative engineering and comparing viability, proliferation and differentiation functions of MC3T3-E1, which were treated with/without this strategy for nondestructive evaluation. This method showed comparable linearity of standard curve and characteristics of growth curve to MTT assay. No major negative effects of this method on MC3T3-E1 viability and functions were found. Our work highlighted the importance of the concentration and incubation time of resazurin in designing application-specific nondestructive viability assay and would be helpful in improving the implanted medical devices as well as in regenerative engineering.

Background: In addition to nutrients, plant raw materials for food production should also contain substances with beneficial biological properties, which unquestionably include antioxidant compounds. Among the numerous methods of determining the antioxidant properties of samples of plant material, biological methods that provide information about not only the in vivo antioxidant potential of samples but also their metabolism and bioavailability are increasingly valued. Objective: The aim of the study was to assess the antioxidant properties of extracts from large cranberry (Vaccinium macrocarpon) obtained from different producers. Methods: Biologically active compounds were extracted from cranberry fruits using water alone and ethyl alcohol–water in proportions of 1+1 and 4+1 (v/v) as solvents. The following were determined in the extracts: content of phenolic compounds and anthocyanins, total antioxidant capacity based on reduction of the ABTS+• [2,20-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid)] radical cation, and antioxidant properties as reflected by the growth of a Saccharomyces cerevisiae Δsod1 mutant in a liquid hypertonic environment. The growth parameters of this Δsod1 mutant, monitored by a method exploiting a color reaction with resazurin, reflected the antioxidant properties of the extracts. Results: The ethanol–water cranberry extracts showed higher content of polyphenols, anthocyanins, and total antioxidants expressed as Trolox equivalent, determined on the basis of ABTS+• reduction. Conclusions: The antioxidant properties determined by the bioassay did not respond strongly to the data obtained in the in vitro chemical and biochemical assays, because they were more closely associated with the batch of fruit than with the type of solvent used to extract phytochemicals.

Background and Aim: Channa striatus extract, a freshwater snakehead fish known as Haruan, is popular in Southeast Asia for consumption and as a traditional therapeutic remedy for wound healing. C. striatus is also used in osteoarthritic for its anti-inflammatory. The aim of this study was to determine the presence of antibacterial properties of C. striatus extract against oral bacteria and to investigate the cytotoxic activity against Vero cells. Materials and Methods: The authors prepared C. striatus extract in chloroform-methanol solvents. Next, the authors took subgingival microbiological samples from 16 cats that had periodontal disease. The authors determined the antibacterial properties of C. striatus extract against the isolated bacteria using the disk diffusion method and a broth microdilution-based resazurin microtiter assay. Finally, the authors used the Vero cell line to evaluate the cytotoxic activity, and they assessed the cell availability using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Results: The results showed weak antibacterial activity of C. striatus extract against Pseudomonas spp. and Escherichia coli. In addition, the authors found that minimum inhibition concentration values ranged between 400 and 500 mg/mL, and minimum bactericidal concentration values ranged between 650 and 550 mg/mL. However, the cytotoxic results were promising, showing that C. striatus extract increased the cell viability and growth when it was at a higher concentration. The extract also promotes growth and cell proliferation. Conclusion: These findings suggest that C. striatus extract promoted cell proliferation in vitro and could be a plausible therapeutic wound healing alternative for periodontal disease in cats.

Subject of Research. The paper deals with theoretical justification of moving from an organoleptic analysis of milk quality in accordance with the requirements of the national standard to objective control based on quantitative assessment by an instrumental method. We propose a theoretical model for changes in color of milk reductase sample depending on its bacterial ontamination. Methods. The proposed model was based on the known patterns of the chemical process of resazurin, resofurin and hydroresofurin mutual transformation, as well as the impact of the concentration of coloring matters on the resulting solution color for homogeneous and inhomogeneous media. Real transmission spectra of the mentioned substances were measured for model development. For theoretical model verification, experimental studies of quality changes during the storage of a milk sample, which was delivered to a shop, were carried out by reductase method. The experiment duration was 3 days, until the souring of milk. The milk was stored between the measurements at the temperature of +4 °C. A theoretical dependence of color changes for the reductase sample of milk was built during the experiment taking into account real ransmission spectrum of milk. Real transmission spectra of the reductase sample were measured. The hromaticity coordinates (x, y) of the sample were calculated from the transmission spectra obtained through the modeling and measurement. To visualize the color change of the sample, the color coordinates in the sRGB color space were calculated for the case of a D65 light source, which is equivalent to daylight. Main Results. The simulation results are completely consistent with the results of experimental studies. The discrepancy between the results of theoretical modeling and experimental data does not exceed, on the average, 0.01 for the chromaticity coordinates x and y. The proposed model gives the possibility to calculate the concentration of enzymes for reductase sample with resazurin at any time, and, as a result, determine its color coordinates and color in accordance with the standards of the International Commission on Illumination for transparent and translucent media. Practical Relevance. The obtained temporal dependence of the chromaticity coordinates and the color of the reductase sample with resazurin can be used to control milk quality by spectrophotometric method and the method of technical vision.

The risk of infection of skin and soft tissue chronic wounds by gram-negative and gram-positive pathogens growing in biofilms is a major health-care concern. In this study we test a formulation of silver sulfadiazine, vitamin A and lidocaine (AF-SSD) for aerosol administration against biofilms of Pseudomonas aeruginosa and biofilms of methicillin-resistant (MRSA) and methicillin-sensitive (MSSA) strains of Staphylococcus aureus. The aerosol allows the administration of AF-SSD without the direct contact with the wound and avoids contamination of the product after reiterative usage. We evaluated in vitro the anti-biofilm activity of AF-SSD by carrying out different technical approaches such as resazurin assays to measure metabolic activity/viability, crystal violet staining assays to determine biofilm biomass, counting of CFUs and live/dead staining for confocal microscopy analysis. AF-SSD clearly affected biofilm viability, biomass and structure, in the three bacterial strains tested. AF-SSD displayed a strong anti-biofilm effect, showing total bactericidal activity on biofilms of P. aeruginosa at a 400-fold dilution of the product, and after a 100-fold and 10-fold dilution for MRSA and MSSA, respectively. Considering the benefits of aerosol administration, our results support this kind of formulation as a potential improvement over conventional treatments with silver sulfadiazine.

The article published the results of experiments on determining the sensitivity of the GOST 23454-2016 method to antibiotics from the group of aminoglycosides and lincosamides. Since the method itself indicates that only the following antibiotics are determined: penicillin in a mass concentration of 0,004 mg/dm3, streptomycin – 10 mg/dm3, tetracycline – 1 mg/dm3, levomycetin– 5 mg/dm3, and at the present stage, a wider range of medications are used to treat animals, it is important to expand information on sensitivity and to other groups of antibiotics. The following sensitivity was determined experimentally as an example for the determination of inhibitory substances using the Streptococcus thermophilus B19 test culture and rezazurin indicatorin the analysis of 14 drugs: aminoglycosides 1 mg/dm3, lincosamides 0,1 mg/dm3.

Background Colistin resistance is considered a serious problem due to a lack of alternative antibiotics. The Rapid ResaPolymyxin Acinetobacter/Pseudomonas NP test is a resazurin reduction-based technique that relies on the visual detection of bacterial growth in the presence of a defined concentration of colistin. The aim of this study was to evaluate the performance of the Rapid ResaPolymyxin Acinetobacter/Pseudomonas NP test in the detection of colistin susceptibility in common clinical Gram-negative bacteria. Results A total of 253 clinical isolates from a teaching hospital, including Acinetobacter baumanii (n = 58, 8 colistin-resistant), Pseudomonas aeruginosa (n = 61, 11 colistin-resistant), Klebsiella pneumoniae (n = 70, 20 colistin-resistant) and Escherichia coli (n = 64, 14 colistin-resistant) were tested in this study. The sensitivity and specificity of the Rapid ResaPolymyxin Acinetobacter/Pseudomonas NP test compared to Broth microdilution method was 100 and 99%, respectively. Conclusions Our results suggest that Rapid ResaPolymyxin Acinetobacter/Pseudomonas NP test could be used as an accurate detection method for colistin resistance.

Aim: To evaluate an assay to detect minimum bactericidal concentration (MBC) in Mycobacterium tuberculosis, using as single model rifampicin, isoniazid, levofloxacin (LVX) and linezolid (LNZ) and in combination. Material & methods: MBCs were carried out directly from resazurin microtiter assay plate and 3D checkerboard in M. tuberculosis H37Rv and five resistant clinical isolates. Results: The proposed MBC assay showed similar values to those determined by MGIT™, used as control. LVX and LNZ's MBC values were close to their MIC values. LNZ or LVX combined with isoniazid and rifampicin showed MBC value reduced in 63.7% of the assays. Conclusion: The proposed assay to determine MBCs of drugs can be applied to the study of new compounds with anti- M. tuberculosis activity to detect their bactericidal effect and also in laboratory routine for clinical dose adjustment of drugs according to the patient's profile.

Objective: To evaluate the anti-bacteria! and anti-biofilm activity of ethyl acetate fraction of Rotula aquatica Lour. (EFRA) against clinically isolated uropathogenic Escherichia coli. Methods: In vitro antibacterial and anti-biofilm studies were employed. The antimicrobial activity of EFRA was assayed by the well diffusion method. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the active fraction were determined by Resazurin method. The time-kill kinetic assay, acridine orange-ethidium bromide staining, propidium iodide uptake assay, and scanning electron microscopic (SEM) analysis were done to evaluate the efficacy of EFRA in killing uropathogenic Escherichia coli. The anti-biofilm activity was determined by 3-[4,5- dimethyl-2-thiazolyl]-2, 5-diphenyl-2H-tetrazolium-bromide (MTT) assay and specific biofilm formation assay. Results: The well diffusion assay of EFRA showed a very clear zone of inhibition against Escherichia coli BRL-17. The MIC and MBC of EFRA were 2.5 mg/mL and 5 mg/mL, respectively. The time-kill kinetic assay, fluorescence microscopic analysis, propidium iodide uptake assay, and SEM analysis displayed the effect of EFRA in killing the bacteria. The MTT assay and specific biofilm formation assay showed that EFRA prevented the formation of biofilms. Conclusions: The results of the present study confirm that EFRA could prevent bacterial growth and inhibit its biofilm formation.

Modern industrial milk processing, based on high-tech processes, places high demands on its quality. The article presents the results of laboratory experiments on the sensitivity of the method of GOST 23454-2016 to antibiotics from the group of fluoroquinolones. The method itself states that chloramphenicol-related levomycetin is determined at a concentration of 5 mg / dm3. But there are no references at all to the group of fluoroquinolones. The following sensitivity for quinolones was determined experimentally using the Streptococcus thermophilus B19 test culture method and the resazurin indicator in an analysis of ten drugs as an example: enrofloxacin from 0,01 to 1 mg / dm3, levofloxacin, marbofloxacin and moxifloxacin 0,1 mg / dm3. We determined the same sensitivity for several chloramphenicol – 0,1 mg / dm3.

Objectives: Diagnosis of tuberculosis (TB) is challenging, especially in a resource-limited setting. Non-commercial alternatives such as colorimetric assays must be used for effective diagnosis and prompt treatment. In this study we aimed to determine the diagnostic performance, time to detection, cost, contamination rate, and ease of performance of the oxidation-reduction assays; Resazurin tube assay (RETA) and Malachite Green decolorization assay (MGDA) and their modifications using para-nitro benzoic acid (PNB) for detection of TB against the results on Lowenstein-Jensen medium (LJ) medium. Methods: Two-hundred-seventeen samples were subjected to colorimetric assays by incubating the inoculated media at 370C. Dyes were added on days 10, 14, 18, 28, and 42 days of incubation, and color change was noted after 24 hours and was compared with results on the LJ medium.Results: Diagnostic performance of colorimetric assays increased from day 10 to day 28 (maximum at day 28), while it was the same on days 28 and 42. The sensitivity, specificity, and diagnostic accuracy of RETA were 87.6%, 95.7%, and 88.6%, while that of MGDA were 81.7%, 95.5%, and 86.5%, respectively. On day 18, more than 51% of the samples were positive by colorimetric assays, whereas only 20.4% were positive by LJ culture. In addition, the colorimetric assays were as economical as LJ culture. Conclusion: Colorimetric methods can potentially become non-commercial alternatives for rapidly detecting Mycobacterium. tuberculosis, especially in TB laboratories with limited resources. The ease of interpreting the results and its cost-effectiveness is an additional advantage. J Microbiol Infect Dis 2022; 12(3):108-115.

Aims Caries and periodontal disease are associated with inadequate control of oral bacteria. Since conventional microbiological evaluations are impractical in dental clinics or public engagement activities, a rapid test for the quantification of oral bacteria represents a useful tool. We describe the development of a colour change test to rapidly estimate bacterial colonisation density in the mouth. Methods and results Volunteers rinsed with milk or milkshake. Viability indicators were added and colour changes quantified during incubation. Using milkshake and the resazurin-based solution PrestoBlue (9% v/v), the method distinguished between samples before and after brushing within 5 min. Colour changes were quantified and viable counts were obtained using oral rinses. Measured colour changes strongly correlated with total counts of both anaerobes and streptococci (Spearman's correlation coefficient of 0·782 and 0·769, respectively, P ≤ 0·001) and with perceived changes, as determined by volunteers (n = 10) visually ranking images. Conclusions The resazurin milkshake test can rapidly and visually quantify viable bacteria in oral samples. Significance and impact of the study The resazurin milkshake test could serve as a sensitive semi-quantitative method for measuring oral bacteria in human oral rinse samples.