Microtiter Assay Research Articles

Fabrication of a Crystalline Nanocellulose Embedded Agarose Biomaterial Ink for Bone Marrow-Derived Mast Cell Culture.

Three-dimensional (3D) bioprinting utilizes hydrogel-based composites (or biomaterial inks) that are deposited in a pattern, forming a substrate onto which cells are deposited. Because many biomaterial inks can be potentially cytotoxic to primary cells, it is necessary to determine the biocompatibility of these hydrogel composites prior to their utilization in costly 3D tissue engineering processes. Some 3D culture methods, including bioprinting, require that cells be embedded into a 3D matrix, making it difficult to extract and analyze the cells for changes in viability and biomarker expression without eliciting mechanical damage. This protocol describes as proof of concept, a method to assess the biocompatibility of a crystalline nanocellulose (CNC) embedded agarose composite, fabricated into a 24-well culture system, with mouse bone marrow-derived mast cells (BMMCs) using flow cytometric assays for cell viability and biomarker expression. After 18 h of exposure to the CNC/agarose/D-mannitol matrix, BMMC viability was unaltered as measured by propidium iodide (PI) permeability. However, BMMCs cultured on the CNC/agarose/D-mannitol substrate appeared to slightly increase their expression of the high-affinity IgE receptor (FcεRI) and the stem cell factor receptor (Kit; CD117), although this does not appear to be dependent on the amount of CNC in the bioink composite. The viability of BMMCs was also assessed following a time course exposure to hydrogel scaffolds that were fabricated from a commercial biomaterial ink composed of fibrillar nanocellulose (FNC) and sodium alginate using a 3D extrusion bioprinter. Over a period of 6-48 h, the FNC/alginate substrates did not adversely affect the viability of the BMMCs as determined by flow cytometry and microtiter assays (XTT and lactate dehydrogenase). This protocol describes an efficient method to rapidly screen the biochemical compatibility of candidate biomaterial inks for their utility as 3D scaffolds for post-print seeding with mast cells.

Will Triazoles Still Be of Importance in Disease Control of Zymoseptoria tritici in the Future?

Septoria tritici blotch (STB), caused by Zymoseptoria tritici, is one of the most important foliar wheat diseases worldwide. Current control strategies of STB rely mainly on fungicides, whereby triazoles (demethylation inhibitors; DMIs) have been the backbone in the control of Z. tritici in the last decades. However, in recent years a gradual loss of sensitivity of Z. tritici to several active ingredients of the triazole group has been reported in several European wheat-growing areas. Nevertheless, a new triazole fungicide, namely, mefentrifluconazole, has recently become available in disease management of STB, which belongs to a completely new triazole subclass, the so-called isopropanol triazoles. In this study, the trend in sensitivity development of Z. tritici towards older triazoles (tebuconazole, prothioconazole, and propiconazole) and the new isopropanol triazole mefentrifluconazole was determined in microtiter assays using Z. tritici field populations isolated in 1999, 2009, 2014, and 2020 in a high-disease-pressure and high-fungicide-input area in northern Germany in order to investigate whether the loss of sensitivity of Z. tritici to older triazoles also applies to mefentrifluconazole. For the three triazole fungicides tebuconazole, prothioconazole and propiconazole, a significant shift towards decreasing sensitivity of Z. tritici field populations was observed from 1999 to 2020, whereas the efficacy of mefentrifluconazole in reducing the in vitro fungal growth by 50% (EC50) remained unchanged over the investigated period, demonstrating a stable sensitivity of Z. tritici towards mefentrifluconazole. Although older triazoles are suffering from a loss of sensitivity of Z. tritici field populations due to the selection and spread of less triazole sensitive strains within the Z. tritici population, the efficacy of the new triazole mefentrifluconazole with its unique isopropanol unit was not affected by these changes within the Z. tritici population. Thus, the introduction of such new molecular units could also represent an important contribution for older groups of active ingredients, which previously suffered from a loss of sensitivity.

Fungicide Management of Pasmo Disease of Flax and Sensitivity of Septoria linicola to Pyraclostrobin and Fluxapyroxad.

Among the diseases that have the potential to cause damage to flax (Linum usitatissimum L.) every year, the fungal disease pasmo, caused by Septoria linicola, is the most important. Fungicide application and a diverse crop rotation are the most important strategies to control this disease because there is little variation in resistance among flax cultivars. However, few fungicide products are available to flax growers. Field studies were conducted at four locations in Western Canada in 2014, 2015, and 2016 to determine the effect of two fungicide active ingredients applied singly and in combination: pyraclostrobin, fluxapyroxad, and fluxapyroxad + pyraclostrobin; and two application timings (early-flower, mid-flower, and at both stages) on pasmo disease severity, seed yield, and quality of flaxseed. The results indicated that among the three fungicide treatments, both pyraclostrobin and fluxapyroxad + pyraclostrobin controlled pasmo effectively; however, fluxapyroxad + pyraclostrobin was the most beneficial to improve the quality and quantity of the seed for most of the site-years. Disease severity in the fungicide-free control was 70%, and application of fluxapyroxad + pyraclostrobin decreased disease severity to 18%, followed by pyraclostrobin (23%) and fluxapyroxad (48%). Application of fluxapyroxad + pyraclostrobin also improved seed yield to 2,562 kg ha-1 compared with 1,874 kg ha-1 for the fungicide-free control, followed by pyraclostrobin (2,391 kg ha-1) and fluxapyroxad (2,340 kg ha-1). Fungicide application at early and mid-flowering stage had the same effects on disease severity and seed yield; however, seed quality was improved more when fungicide was applied at mid-flowering stage. Continuous use of the same fungicide may result in the development of fungicide insensitivity in the pathogen population. Thus, sensitivity of S. linicola isolates to pyraclostrobin and fluxapyroxad fungicides was determined by the spore germination and microtiter assay methods. Fungicide insensitivity was not detected among the 73 isolates of S. linicola tested against either of these fungicides.

Drug susceptibiity testing of nontuberculous mycobacteria by broth microdilution method

Introduction-Incidence of Nontuberculous mycobacteria (NTM) has been increasing in past few years. Treatment of NTM differs from Mycobacterium tuberculosis. For proper treatment, it's important to carry out Drug Susceptibility Testing of NTM. Method of DST for NTM is different from MTB and is not available in most laboratories. Method-We performed DST on 122 isolates of NTM. Amikacin, Ciprofloxacin, Trimethoprim, Doxycycline, Moxifloxacin, Clarithromycin, Minocycline and Cefoxitin were used for Rapid Growing Mycobacteria (RGM) and Rifampicin, Clarithromycin, Ethambutol, Isoniazid and Moxifloxacin for Slow Growing Mycobacteria (SGM). M. avium Complex (MAC) was tested against Clarithromycin. Minimum inhibitor concentration was calculated as recommended by standard Clinical and Laboratory Standards Institute (CLSI) and Resazurin Microtitre Assay (REMA). Result-Most of Rapid Growing Mycobacteria were sensitive to Amikacin (76.1%) and Moxifloxacin (46.47%) while Slow Growing Mycobacteria showed only 33.3% sensitivity to Rifampicin and Moxifloxacin and 42% to Clarithromycin. M. avium-intracellulare complex showed 45–50% sensitivity to Clarithromycin. Overall, 98% concordance (Kappa = 0.98; almost perfect; 95% CI = 0.966 to 0.996) was seen between standard and REMA method of DST of NTM. Conclusion-Rapid growers showed good sensitivity to Amikacin and Moxifloxacin, while only one third SGM showed sensitivity to Rifampicin, Moxifloxacin and Clarithromycin. For proper management of NTM of eastern Rajasthan its important to know the DST profile in our area to initiate empirical therapy till the results of specific patient are available. REMA was found to give excellent concordance with standard method.

Outlining In Vitro and In Silico Cholinesterase Inhibitory Activity of Twenty-Four Natural Products of Various Chemical Classes: Smilagenin, Kokusaginine, and Methyl Rosmarinate as Emboldening Inhibitors.

Cholinesterase (ChE) inhibition is an important treatment strategy for Alzheimer’s disease (AD) as acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) are involved in the pathology of AD. In the current work, ChE inhibitory potential of twenty-four natural products from different chemical classes (i.e., diosgenin, hecogenin, rockogenin, smilagenin, tigogenin, astrasieversianins II and X, astragalosides I, IV, and VI, cyclocanthosides E and G, macrophyllosaponins A-D, kokusaginin, lamiide, forsythoside B, verbascoside, alyssonoside, ipolamide, methyl rosmarinate, and luteolin-7-O-glucuronide) was examined using ELISA microtiter assay. Among them, only smilagenin and kokusaginine displayed inhibitory action against AChE (IC50 = 43.29 ± 1.38 and 70.24 ± 2.87 µg/mL, respectively). BChE was inhibited by only methyl rosmarinate and kokusaginine (IC50 = 41.46 ± 2.83 and 61.40 ± 3.67 µg/mL, respectively). IC50 values for galantamine as the reference drug were 1.33 ± 0.11 µg/mL for AChE and 52.31 ± 3.04 µg/mL for BChE. Molecular docking experiments showed that the orientation of smilagenin and kokusaginine was mainly driven by the interactions with the peripheral anionic site (PAS) comprising residues of hAChE, while kokusaginine and methyl rosmarinate were able to access deeper into the active gorge in hBChE. Our data indicate that similagenin, kokusaginine, and methyl rosmarinate could be hit compounds for designing novel anti-Alzheimer agents.

Temporal Changes in Sensitivity of Zymoseptoria tritici Field Populations to Different Fungicidal Modes of Action

Septoria tritici blotch (STB; Zymoseptoria tritici), one of the most important foliar diseases in wheat, is mainly controlled by the intensive use of fungicides during crop growth. Unfortunately, Z. tritici field populations have developed various extents of resistance to different groups of fungicides. Due to the complete resistance to quinone outside inhibitors (QoIs), fungicidal control of STB relies mainly on demethylation inhibitors (DMIs) and succinate dehydrogenase inhibitors (SDHIs) as well as multi-site inhibitors. In this study, temporal changes in the sensitivity of Z. tritici to selected DMIs (tebuconazole, propiconazole, prothioconazole, prochloraz), SDHIs (boscalid, bixafen), and multi-site inhibitors (chlorothalonil, folpet) were determined in microtiter assays using Z. tritici field populations isolated in 1999, 2009, 2014, and 2020 in a high-disease-pressure and high-fungicide-input area in Northern Germany. For the four tested DMI fungicides, a significant shift towards decreasing sensitivity of Z. tritici field populations was observed between 1999 and 2009, whereby concentrations inhibiting fungal growth by 50% (EC50) increased differentially between the four DMIs. Since 2009, EC50 values of tebuconazole, propiconazole, and prochloraz remain stable, whereas for prothioconazole a slightly increased sensitivity shift was found. A shift in sensitivity of Z. tritici was also determined for both tested SDHI fungicides. In contrast to DMIs, EC50 values of boscalid and bixafen increased continuously between 1999 and 2020, but the increasing EC50 values were much smaller compared to those of the four tested DMIs. No changes in sensitivity of Z. tritici were observed for the multi-site inhibitors chlorothalonil and folpet over the last 21 years. The sensitivity adaptation of Z. tritici to both groups of single-site inhibitors (DMIs, SDHIs) mainly used for STB control represents a major challenge for future wheat cultivation.

Phytochemical Profiling of Methanolic Fruit Extract of Gardenia latifolia Ait. by LC-MS/MS Analysis and Evaluation of Its Antioxidant and Antimicrobial Activity.

Gardenia latifolia Ait. (Rubiaceae) is also known as Indian Boxwood is a small deciduous tree often growing in southern states of India. In the present study, phytochemical profiling of methanolic extract of G. latifolia fruits were carried out using FTIR and LC-MS/MS analysis. Besides, its antioxidant and antimicrobial potential have been analysed using DPPH activity, differential pulse voltammetry and resazurin microtiter assay, respectively. Phytochemical profiling revealed the presence of 22 major diversified compounds and main were 3-caffeoyl quinic acid (chlorogenic acid), 3,4-Di-O-caffeoyl quinic acid, 6-O-trans-feruloylgenipin gentiobioside, 10-(6-O-trans sinapoyl glucopyranosyl) gardendiol, isoquercitrin, scortechinones, secaubryenol, iridoids and quercetin 3-rutinoside (rutin). The extract showed antioxidant activity (IC50 = 65.82) and powerful antibacterial activity with lowest minimum inhibitory concentration against Gram-positive Staphylococcus aureus (15.62 µg/µL), Bacillus subtilis (31.25 µg/µL) than gram negative Escherichia coli (62.5 µg/µL), Klebsiella pneumoniae (62.5 µg/µL), Pseudomonas aeruginosa (31.25 µg/µL). This study shows that the fruits of G. latifolia have tremendous potential to be used in food industries, phyto-therapeutics and cosmetic industries.

Biosurfactant-mediated biosynthesis of CuO nanoparticles and their antimicrobial activity

In the design and synthesis of novel functional materials, a few important challenges that have to be addressed by the material research community are (1) developing robust, reproducible, and environmentally friendly green methods for the synthesis of various nanomaterials and (2) exploring new functional nanomaterials to combat with multi-drug-resistant pathogens. This paper addresses the above-mentioned challenges by (1) demonstrating a green synthesis for the CuO nanoparticles using environmentally benign biosurfactant rhamnolipid (RL) and (2) exploring the antimicrobial activity of RL functionalized/capped CuO nanoparticles (RL-CuO NPs) against various bacterial and fungal species. CuO nanoparticles (CuO NPs) were synthesized using RL by hydrothermal method at pH 9–10. Capping of RL on CuO NPs was probed by characterizing the sample with Fourier-Transform Infrared Spectroscopy (FT-IR) and Thermo Gravimetric Analysis (TGA). Whereas X-Ray Diffraction (XRD), Scanning Electron Microscope (SEM), Transmission Electron Microscope (TEM) were used to understand the phase, crystallinity, and morphology of CuO NPs. The antimicrobial activity of RL-CuO NPs was examined using resazurin microtitre assay, against Gram-positive bacteria, such as Streptococcus mutans, Staphylococcus aureus, Enterococcus faecalis, for which the minimum inhibition concentration (MIC) values were observed at low concentrations, i.e., 7.8 µg/L of RL-CuO NPs. For Gram-negative bacteria viz. Shigella dysentriae and Salmonella typhi (except Pseudomonas aeruginosa) MIC values were observed at little higher concentrations of RL-CuO NPs, i.e., 250 µg/L. Anti-fungal activity of RL-CuO NPs was examined against Candida albicans, Aspergillus niger and MIC values for these fungi are observed at 125 µg/L and 62.5 µg/L, respectively.

The use of an Ebola virus reporter cell line in a semi-automated microtitration assay

A variety of methods have been developed for quantification of infectious Ebola virus in clinical or laboratory samples, but existing methods often require extensive operator involvement, manual assay scoring, or the use of custom reagents. In this study, we utilize a recently developed Ebola-specific reporter cell line that expresses ZsGreen in response to Ebola virus infection, in conjunction with semi-automated processing and quantification techniques, to develop an unbiased, high-throughput microtitration assay for quantification of infectious Ebola virus in vitro. This assay was found to have equivalent sensitivity to a standardized plaque assay for quantifying viral titers. However, the new assay could be implemented with fewer reagents and processing steps, reduced subjectivity, and higher throughput. This assay may be useful for a variety of applications, particularly studies that require the detection or quantification of infectious Ebola virus in large numbers of samples.

Study of quorum-sensing LasR and RhlR genes and their dependent virulence factors in Pseudomonas aeruginosa isolates from infected burn wounds

BackgroundPseudomonas aeruginosa (P. aeruginosa) is an opportunistic pathogen responsible for burn-wound infection. High incidence, infection severity and increasing resistance characterize P. aeruginosa -induced burn infection.PurposeTo estimate quorum-sensing (QS)-dependent virulence factors of P. aeruginosa isolates from burn wounds and correlate it to the presence of QS genes.MethodsA cross-sectional descriptive study included 50 P . aeruginosa isolates from burn patients in Mansoura University Plastic and Burn Hospital, Egypt. Antibiotic sensitivity tests were done. All isolates were tested for their ability to produce biofilm using a micro-titration assay method. Protease, pyocyanin and rhamnolipid virulence factors were determined using skimmed milk agar, King’s A medium and CTAB agar test, respectively. The identity of QS lasR and rhlR genes was confirmed using PCR.Results In total, 86 % of isolates had proteolytic activity. Production of pyocyanin pigment was manifested in 66 % of isolates. Altogether, 76 % of isolates were rhamnolipid producers. Biofilm formation was detected in 96 % of isolates. QS lasR and rhlR genes were harboured by nearly all isolates except three isolates were negative for both lasR and rhlR genes and two isolates were positive for lasR gene and negative for rhlR gene. Forty-nine isolates were considered as extremely QS-proficient strains as they produced QS-dependent virulence factors. In contrast, one isolate was a QS deficient strain.ConclusionsQS affects P. aeruginosa virulence-factor production and biofilm in burn wounds. Isolates containing lasR and rhlR seem to be a crucial regulator of virulence factors and biofilm formation in P. aeruginosa whereas the lasR gene positively regulates biofilm formation, proteolytic activity, pyocyanin production and rhamnolipid biosurfactant synthesis. The QS regulatory RhlR gene affects protease and rhamnolipid production positively.

Anti-mycobacterial Activity of 22 Iranian Endemic or Rare Plant Extracts Against Multi-drug and Extensively Drug-resistant Mycobacterium tuberculosis.

Due to side-effects and low efficacy of common drugs on new resistant strains of Mycobacterium tuberculosis (Mtb), investigation on novel drugs and natural compounds from rich sources of endemic plants is required. Thus, in the present study, the anti-mycobacterial effects of 22 Iranian endemic or rare plant extracts on multi-drug resistance (MDR) and extensively-drug resistance (XDR) Mtb isolates were evaluated. Twenty-two Iranian endemic and rare plant species from 9 families were collected and extracted by methanol. Their inhibitory-effects were then evaluated against Mtb H37Rv strain, seven clinical MDR-TB, and two XDR-TB isolates using the resazurin microtiter assay (REMA) method. The best of them were then fractionalized by five different polar solvents (Petroleum- Ether, Dichloromethane, Ethyl-Acetate, n-butanol, and water). To find anti-mycobacterial fractions, the inhibitory effect of isolated fractions was tested on Mtb H37Rv. Out of the 22 plants, 14 plant extracts demonstrated anti-mycobacterial activity with minimum inhibitory concentration (MIC) ranging from 4 to 30μg/mL against Mtb H37Rv. Eight plant extracts also exhibited anti-mycobacterial activity against MDR and XDR clinical strains of Mtb by MICs, i.e., 15-60μg/ml. Crinitaria grimmii and Linum album were the best antimycobacterial plants. Among fractions of Crinitaria grimmii, dichloromethane and n-butanol, and for Linum album, dichloromethane and Ethyl-Acetate fractions displayed more anti-mycobacterial effect as compared to crude extract on Mtb. The present study confirms the potential role of some plants to treat respiratory diseases as our results have demonstrated that these plants exhibit anti-mycobacterial activity in the acceptable range against Mtb. Thus, these plants could be good sources and alternatives of plant metabolites for anti-TB-drug development.

Tuberculosis cases in a prison in the extreme south of Brazil.

Introduction. Tuberculosis (TB) control is a challenge, especially in vulnerable populations, such as prisoners.Hypothesis. In prison houses, the transmission of micro-organisms that cause infectious diseases can occur due to the susceptibility and immune compromise of prisoners, and due to the precarious physical conditions of the prison houses. However, strategies such as monitoring by health professionals, can mitigate the transmission of these micro-organisms, as well as, reduce the number of coinfections and antimicrobials resistance.Aim. This study attempted to analyse the dynamics of transmission and the antimicrobial resistance profile of Mycobacterium tuberculosis strains obtained from prisoners and to characterize the epidemiological, clinical and laboratory profiles of prisoners diagnosed with TB.Methodology. A cross-sectional and retrospective study was conducted with sputum samples collected from 228 distinct prisoners who were treated at the Health Unit located in the Regional Penitentiary of Rio Grande, Rio Grande do Sul, Brazil. The antimicrobial resistance profile of the strains was evaluated using the Resazurin Microtiter Assay and the transmission dynamics was investigated using 15-loci MIRU-VNTR.Results. Thirty-five patients (15.4 %) were diagnosed with TB, and when a TB/HIV coinfection was assessed, 8.6 % (3/35) of the patients were positive. In addition, all patients with results available for HBV, HCV, syphilis and diabetes mellitus were negative. Based on the genotypic profile, 55.9 % of the clinical isolates were grouped into five groups. One isolate with mono-resistance to isoniazid and two with mono-resistance to streptomycin were found.Conclusion. The presence of a Health Unit may have influenced the low numbers of TB/HIV, TB/HBV, TB/HCV, TB/syphilis coinfections and TB cases resistant to antimicrobials. Recent M. tuberculosis transmission can be inferred based on the high percentage of formatting of clusters. This situation stresses the need to improve active and passive detection, the screening of individuals for TB upon entrance into prison for early detection, and the implementation of prophylactic measures to reduce M. tuberculosis transmission.

Synthesis of ZnO nanoparticles using Theobroma cacao L. pod husks, and their antibacterial activities against foodborne pathogens

Zinc oxide nanoparticles (ZnONPs) with antimicrobial properties have potential applications in the food industry. In the present work, the biosynthesis of ZnONPs was carried out using Theobroma cacao L. pod husk extract. UV-Vis spectroscopy confirmed NP formation by the presence of a peak in the range of 390 - 360 nm. Different parameters affecting the biosynthesis were assessed. A temperature of 28°C and pH of 7 were the best conditions for the formation of small-sized NPs. The antibacterial activity of ZnONPs was evaluated using the resazurin microtiter assay. The minimum inhibitory concentrations of ZnONPs for Escherichia coli and Staphylococcus aureus were 6.25 and 12.5 μg/mL, respectively. ZnONPs were more potent than chloramphenicol, suggesting that they are effective against chloramphenicol-resistant bacteria. Based on scanning electron microscopy and transmission electron microscopy, the ZnONPs were irregular in shape, with an average size of 81 nm. The results of the present work provide a simple, cost-effective, and eco-friendly method for the large-scale production of ZnONPs. Moreover, the study highlights the potential of ZnONPs as an antimicrobial agent that can be applied to food packaging systems, textiles, and medical devices.

Design and fabrication of hydrogel incorporated with copper nanoparticles loaded microsponge for antimicrobial and antioxidant activity

A hydrogel has been designed as an antimicrobial agent with low toxicity and reduced side effects. The hydrogel has been fabricated with green synthesised copper nanoparticles loaded microsponges. Here, the copper nanoparticles have been synthesised from the leaf extract of Ocimum Sanctum. This fabricated hydrogel is checked for its pH and particle size by particle size analyser (PSA). The best hydrogel is assessed through resazurin microtitre assay for its antimicrobial activity. Using DPPH assay, it is also checked for its antioxidant activity. The results show that the fabricated hydrogel incorporated with copper nanoparticles loaded microsponge act as an excellent antimicrobial gel and has shown good antioxidant potential.

Bacterial Adhesion Kinetics in a High Throughput Setting in Seconds-minutes Time Resolution.

Bacterial surface adhesion, the first step in many important processes including biofilm formation and tissue invasion, is a fast process that occurs on a time scale of seconds. Adhesion patterns tend to be stochastic and spatially heterogeneous, especially when bacteria are present in low population densities and at early stages of adhesion to the surface. Thus, in order to observe this process, a high degree of temporal resolution is needed across a large surface area in a way that allows several replicates to be monitored. Some of the current methods used to measure bacterial adhesion include microscopy, staining-based microtiter assays, spectroscopy, and PCR. Each of these methods has advantages in assaying aspects of bacterial surface adhesion, but none can capture all features of the process. In the protocol presented here, adapted from Shteindel et al., 2019 , fluorescently-labeled bacteria are monitored in a multi-titer setting using a standard plate fluorimeter and a dye that absorbs light in the fluorophore excitation and emission wavelengths. The advantage of using this dye is that it restricts the depth of the optic layer to the few microns adjacent to the bottom of the microtiter well, eliminating fluorescence originating from unattached bacteria. Another advantage of this method is that this setting does not require any preparatory steps, which enables reading of the sample to be repeated or continuous. The use of a standard multi-titer well allows easy manipulation and provides flexibility in experimental design.

In vitro Anti-Mycobacterium ulcerans and cytotoxic activities of some selected medicinal plants and an indoloquinoline alkaloid.

Buruli ulcer (BU) is a neglected tropical disease caused by the Mycobacterium ulcerans. BU is an endemic disease in many communities in sub-Saharan Africa where population have long history of using medicinal plants for treatment. Indeed, several medicinal plants have been documented against BU and related conditions. The present study was undertaken to prove the efficacy of seven medicinal plants documented for the treatment of mycobacterial infections and related symptoms in Ghana. Antimycobacterial activity of the stem bark extracts and reference control drugs were conducted using the resazurin microtiter assay (REMA) assay method in clear round bottom 96-well microtiter plates. The extracts that showed anti-mycobacterium ulcerans activity were assessed for cytotoxicity using the Alamar blue assay. Overall, The Cryptolepis sanguinolenta root aqueous extract exhibited the highest antimycobacterial activity (MIC=64 μg/mL) followed by Cleistopholis patens (MIC=256 μg/mL). Based on the marked activity of the Cryptolepis sanguinolenta extracts, pure cryptolepine, its major metabolite recorded a MIC value of 32 μg/mL. These extracts with considerable antimycobacterial activity showed 50% cytotoxic concentration (CC50) ranging from 94 to 384 μg/mL. Thus, Cleistopholis patens and Cryptolepis sanguinolenta are primed for further studies and could afford novel drugs for the mitigation of buruli ulcer disease.

Antimicrobial activity of Streptomyces spp. isolated from Apis dorsata combs against some phytopathogenic bacteria.

The aim of this study was to investigate the antimicrobial potential of actinomycetes isolated from combs of the giant honey bee, Apis dorsata. In total, 25 isolates were obtained from three different media and were screened for antimicrobial activity against four plant pathogenic bacteria (Ralstonia solanacearum, Xanthomonas campestris pv. campestris, Xanthomonas oryzae pv. oryzae and Pectobacterium carotovorum). Following screening using a cross-streaking method, three isolates showed the potential to inhibit the growth of plant pathogenic bacteria. Based on a 96-well microtiter assay, the crude extract of DSC3-6 had minimum inhibitory concentration (MIC) values against X. oryzae pv. oryzae, X. campestris pv. campestris, R. solanacearum and P. carotovorum of 16, 32, 32 and 64 mg L−1, respectively. The crude extract of DGA3-20 had MIC values against X. oryzae pv. oryzae, X. campestris pv. campestris, R. solanacearum and P. carotovorum of 32, 32, 32 and 64 mg L−1, respectively. The crude extract of DGA8-3 at 32 mgL−1 inhibited the growth of X. oryzae pv. oryzae, X. campestris pv. campestris, R. solanacearum and P. carotovorum. Based on their 16S rRNA gene sequences, all isolates were identified as members of the genus Streptomyces. The analysis of 16S rRNA gene sequence similarity and of the phylogenetic tree based on the maximum likelihood algorithm showed that isolates DSC3-6, DGA3-20 and DGA8-3 were closely related to Streptomyces ramulosus (99.42%), Streptomyces axinellae (99.70%) and Streptomyces drozdowiczii (99.71%), respectively. This was the first report on antibacterial activity against phytopathogenic bacteria from actinomycetes isolated from the giant honey bee.

Distribution of Alternaria alternata isolates with resistance to quinone outside inhibitor (QoI) fungicides in Brazilian orchards of tangerines and their hybrids

Alternaria brown spot (ABS), caused by Alternaria alternata, has been controlled with sprays of quinone outside inhibitors (QoI), demethylation inhibitors (DMI) and copper-based fungicides. QoI fungicides have been intensively used for citrus disease control in São Paulo (SP) since 2003, when ABS was first found in the state. In 2017, citrus growers from several regions in SP stated experiencing failures in ABS control with the use of QoI fungicides. This study was then proposed to assess the sensitivity of A. alternata isolates to QoI fungicides by conidial germination and resazurin microtiter assay, and also to determine the geographic distribution of sensitive and resistant isolates in areas cultivated with tangerines and their hybrids in SP. One hundred and twenty-eight A. alternata isolates were sampled in nine municipalities in different SP regions in 2017 and 2018. Isolates collected in 2003/2004, immediately after the first report of ABS in SP, were used as baseline. Effective concentrations of azoxystrobin and pyraclostrobin to reduce conidial germination by 50% (EC50) were estimated and A. alternata isolates were classified as resistant when the conidial germination rate was ≥50% at a discriminatory rate of 1 μg mL−1. Isolates resistant to QoI fungicides were widely found throughout SP regions, and no pattern in resistance distribution was observed. In five out of the nine municipalities, 100% of isolates were resistant to both fungicides, while in other four municipalities the percentage of resistant isolates to azoxystrobin (46%–100%) was higher than that of pyraclostrobin (15%–93%). The G143A mutation positively correlated with the resistance phenotype to azoxystrobin for all A. alternata isolates and to pyraclostrobin for most of them. The resazurin microtiter assay proved useful for rapid qualitative classification of sensitive and resistant A. alternata isolates. Our results suggest the need for anti-resistance strategies in the chemical management of the disease in the state.

Endogenous antisense walR RNA modulates biofilm organization and pathogenicity of Enterococcus faecalis.

Enterococcus faecalis (E. faecalis) is regarded as the major pathogen for persistent periapical periodontitis. The aim of the present study was to investigate the role of antisense walR RNA in the regulation of adjacent downstream genes. Reverse transcription-PCR assays were performed to validate walR. Adjacent downstream genes walK, EF1195, EF1196, and EF1197 were co-transcribed and detect antisense walR RNA. Northern blotting and 5'-rapid amplification of cDNA ends (5'-RACE) assays were conducted to detect and confirm a novel walR antisense (ASwalR) RNA. ASwalR overexpression mutants were constructed, and the biofilm biomass was determined using a crystal violet microtiter assay. The present study detected and confirmed a 550-bp noncoding antisense RNA with the potential to attenuate the activities of the essential response regulator WalR. The levels of antisense walR RNA transcripts were inversely associated with the production of WalR protein. It was showed that overexpression of ASwalR leads to reduced biofilm formation and exopolysaccharide synthesis. Furthermore, the pathogenicity of E. faecalis was markedly decreased by ASwalR overexpression in an in vivo periapical periodontitis model. In summary, the present study detected a novel antisense walR RNA that leads to a reduction in biofilm formation and the pathogenicity of E. faecalis. Collectively, the data suggest a role for ASwalR as a post-transcriptional modulator of the WalR regulator in E. faecalis.

Advantages and limitations of microtiter biofilm assays in the model of antibiofilm activity of Klebsiella phage KP34 and its depolymerase

One of the potential antibiofilm strategies is to use lytic phages and phage-derived polysaccharide depolymerases. The idea is to uncover bacteria embedded in the biofilm matrix making them accessible and vulnerable to antibacterials and the immune system. Here we present the antibiofilm efficiency of lytic phage KP34 equipped with virion-associated capsule degrading enzyme (depolymerase) and its recombinant depolymerase KP34p57, depolymerase-non-bearing phage KP15, and ciprofloxacin, separately and in combination, using a multidrug-resistant K. pneumoniae biofilm model. The most effective antibiofilm agents were (1) phage KP34 alone or in combination with ciprofloxacin/phage KP15, and (2) depolymerase KP34p57 with phage KP15 and ciprofloxacin. Secondly, applying the commonly used biofilm microtiter assays: (1) colony count, (2) LIVE/DEAD BacLight Bacterial Viability Kit, and (3) crystal violet (CV) biofilm staining, we unravelled the main advantages and limitations of the above methods in antibiofilm testing. The diverse mode of action of selected antimicrobials strongly influenced obtained results, including a false positive enlargement of biofilm mass (CV staining) while applying polysaccharide degrading agents. We suggest that to get a proper picture of antimicrobials’ effectiveness, multiple examination methods should be used and the results must be read considering the principle of each technique and the antibacterial mechanism.