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Coupling Microplate-Based Antibacterial Assay with Liquid Chromatography for High-Resolution Growth Inhibition Profiling of Crude Extracts: Validation and Proof-of-Concept Study with Staphylococcus aureus.

ABSTRACT: With the identification of novel antibiotics from nature being pivotal in the fight against human pathogenic bacteria, there is an urgent need for effective methodologies for expedited screening of crude extracts. Here we report the development and validation of a simple and dye-free antimicrobial assay in 96-well microplate format, for both determination of IC50 values and high-resolution inhibition profiling to allow pin-pointing of bioactive constituents directly from crude extracts. While commonly used antimicrobial assays visualize cell viability using dyes, the developed and validated assay conveniently uses OD600 measurements directly on the fermentation broth. The assay was validated with an investigation of the inhibitory activity of DMSO against Staphylococcus aureus, temperature robustness, interference by coloured crude extracts as well as inter-day reproducibility. The potential for high-resolution S. aureus growth inhibition profiling was evaluated on a crude extract of an inactive Alternaria sp., spiked with ciprofloxacin.

SUBMITTER: Ardalani H

PROVIDER: S-EPMC8001363 | biostudies-literature | 2021 Mar

REPOSITORIES: biostudies-literature

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Coupling Microplate-Based Antibacterial Assay with Liquid Chromatography for High-Resolution Growth Inhibition Profiling of Crude Extracts: Validation and Proof-of-Concept Study with <i>Staphylococcus aureus</i>.

Ardalani Hamidreza H Anam Syariful S Kromphardt Kresten J K KJK Staerk Dan D Kongstad Kenneth T KT

Molecules (Basel, Switzerland) 20210311 6

With the identification of novel antibiotics from nature being pivotal in the fight against human pathogenic bacteria, there is an urgent need for effective methodologies for expedited screening of crude extracts. Here we report the development and validation of a simple and dye-free antimicrobial assay in 96-well microplate format, for both determination of IC<sub>50</sub> values and high-resolution inhibition profiling to allow pin-pointing of bioactive constituents directly from crude extract ...[more]

PMID: 33799878

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Project description:Whether Candida within the patient microbiome drives the pathogenesis of Staphylococcus aureus bacteremia, described as microbial hitchhiking, cannot be directly studied. Group-level observations from studies of various decontamination and non-decontamination-based ICU infection prevention interventions and studies without study interventions (observational groups) collectively enable tests of this interaction within causal models. Candidate models of the propensity for Staphylococcus aureus bacteremia to arise with versus without various antibiotic, anti-septic, and antifungal exposures, each identified as singleton exposures, were tested using generalized structural equation modelling (GSEM) techniques with Candida and Staphylococcus aureus colonization appearing as latent variables within the models. Each model was tested by confrontation against blood and respiratory isolate data, obtained from 467 groups within 284 infection prevention studies. Introducing an interaction term between Candida colonization and Staphylococcus aureus colonization substantially improved GSEM model fit. Model-derived coefficients for singular exposure to anti-septic agents (- 1.28; 95% confidence interval; - 2.05 to - 0.5), amphotericin (- 1.49; - 2.3 to - 0.67), and topical antibiotic prophylaxis (TAP; + 0.93; + 0.15 to + 1.71) as direct effects versus Candida colonization were similar in magnitude but contrary in direction. By contrast, the coefficients for singleton exposure to TAP, as with anti-septic agents, versus Staphylococcus colonization were weaker or non-significant. Topical amphotericin would be predicted to halve both candidemia and Staphylococcus aureus bacteremia incidences versus literature derived benchmarks for absolute differences of < 1 percentage point. Using ICU infection prevention data, GSEM modelling validates the postulated interaction between Candida and Staphylococcus colonization facilitating bacteremia.

Project description:Antibiotic resistance has become a serious global concern, and the discovery of antimicrobial herbal constituents may provide valuable solutions to overcome the problem. In this study, the effects of therapies combining antibiotics and four medicinal herbs on methicillin-resistant Staphylococcus aureus (MRSA) were investigated. Specifically, the synergistic effects of Magnolia officinalis, Verbena officinalis, Momordica charantia, and Daphne genkwa in combination with oxacillin or gentamicin against methicillin-resistant (ATCC43300) and methicillin-susceptible (ATCC25923) S. aureus were examined. In vitro susceptibility and synergistic testing were performed to measure the minimum inhibitory concentration and fractional inhibitory concentration (FIC) index of the antibiotics and medicinal herbs against MRSA and methicillin-susceptible S. aureus. To identify the active constituents in producing these synergistic effects, in silico molecular docking was used to investigate the binding affinities of 139 constituents of the four herbs to the two common MRSA inhibitory targets, penicillin binding proteins 2a (PBP2a) and 4 (PBP4). The physicochemical and absorption, distribution, metabolism, and excretion properties and drug safety profiles of these compounds were also analyzed. D. genkwa extract potentiated the antibacterial effects of oxacillin against MRSA, as indicated by an FIC index value of 0.375. M. officinalis and V. officinalis produced partial synergistic effects when combined with oxacillin, whereas M. charantia was found to have no beneficial effects in inhibiting MRSA. Overall, tiliroside, pinoresinol, magnatriol B, and momorcharaside B were predicted to be PBP2a or PBP4 inhibitors with good drug-like properties. This study identifies compounds that deserve further investigation with the aim of developing therapeutic agents to modulate the effect of antibiotics on MRSA. Impact statement Antibiotic resistant is a well-known threat to global health and methicillin-resistant Staphylococcus aureus is one of the most significant ones. These resistant bacteria kill thousands of people every year and therefore a new effective antimicrobial treatment is necessary. This study identified the herbs and their associated bioactive ingredients that can potential the effects of current antibiotics. These herbs have long history of human usage in China and have well-defined monograph in the Chinese Pharmacopeia. These indicate their relatively high clinical safety and may have a quicker drug development process than that of a new novel antibiotic. Based on the results of this study, the authors will perform further in vitro and animal studies, aiming to accumulate significant data for the application of clinical trial.

Project description:BackgroundThe antibacterial activities of aqueous leaf extracts of Moringa oleifera, Vernonia amygdalina, Azadirachta indica and Acalypha wilkesiana against multidrug resistance (MDR) Staphylococcus aureus associated with skin and soft tissue infections were investigated.MethodsStaphylococcus aureus (n = 183) from the skin and soft tissue infections with evidence of purulent pus, effusions from aspirates, wounds, and otorrhea were biotyped, and evaluated for biofilm production. The phenotypic antibiotic resistance and MDR strains susceptibility to plant leaves extract were determined using disc diffusion and micro-broth dilution assays respectively. The correlation of plant extract bioactive components with inhibitory activities was determined.ResultsHigh occurrence rate of S. aureus were recorded among infant and adult age groups and 13.2% mild biofilm producers from the wound (p < 0.05). Of 60.2% MDR strains with overall significant MARI of more than 0.85 (p < 0.05), high resistant rates to linozidine (92.7%; 95% CI:7.27-10.52), ofloxacin (94.2%; 95% CI:6.09-8.15), chloramphenicol (91.2%; 95% CI:6.11-8.32), gentamicin (97.3%; 95% CI:6.20-8.22), ciprofloxacin (92.7%; 95% CI: 5.28-7.99) and vancomycin (86.6%; 95% CI:6.81-9.59) were observed. Vernonia amygdalina and Azadirachta indica showed significant antimicrobial activity at 100 mg/ml and 75 mg/ml, with low susceptibility of less than 10% to 25 mg/ml, 50 mg/ml, and 75 mg/ml Moringa oleifera. Alkaloids, saponin and terpenoids were significant in Moringa oleifera, Acalypha wilkesiana, Azadirachta indica and Vernonia amygdalina leaves extracts (p < 0.05). High inhibitory concentrations at IC50; 3.23, 3.75 and 4.80 mg/ml (p = 0.02, CI: - 0.08 - 11.52) and IC90; 12.9, 7.5, and 9.6 mg/ml (p = 0.028, CI: 2.72-23.38) were shown by Acalypha wilkesiana, Vernonia amygdalina and Moringa oleifera respectively. Comparative outcome of the plant extracts showed Acalypha wilkesiana, Vernonia amygdalina and Moringa oleifera to exhibit significant inhibition activities (p < 0.05) compared to other extracts. Significant median inhibitory concentration (15.3 mg/ml) of Azadirachta indica were observed (p < 0.01) and strong associations of phytochemical compounds of Azadirachta indica (eta = 0.527,p = 0.017), Vernonia amygdalina (eta = 0.123,p = 0.032) and Acalypha wilkesiana (eta = 0.492,p = 0.012) with their respective inhibitory values.ConclusionObserved high occurrence rate of skin and soft tissue infections caused by biofilm-producing MDR S. aureus requires alternative novel herbal formulations with rich bioactive compounds from Moringa oleifera, Acalypha wilkesiana, Azadirachta indica and Vernonia amygdalina as skin therapeutic agents.

Dataset Information

Coupling Microplate-Based Antibacterial Assay with Liquid Chromatography for High-Resolution Growth Inhibition Profiling of Crude Extracts: Validation and Proof-of-Concept Study with Staphylococcus aureus.

Publications

Coupling Microplate-Based Antibacterial Assay with Liquid Chromatography for High-Resolution Growth Inhibition Profiling of Crude Extracts: Validation and Proof-of-Concept Study with <i>Staphylococcus aureus</i>.

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