11 research outputs found
Droplet-based digital antibiotic susceptibility screen reveals single-cell clonal heteroresistance in an isogenic bacterial population
Since antibiotic resistance is a major threat to global health, recent observations that the traditional test of minimum inhibitory concentration (MIC) is not informative enough to guide effective antibiotic treatment are alarming. Bacterial heteroresistance, in which seemingly susceptible isogenic bacterial populations contain resistant sub-populations, underlies much of this challenge. To close this gap, here we developed a droplet-based digital MIC screen that constitutes a practical analytical platform for quantifying the single-cell distribution of phenotypic responses to antibiotics, as well as for measuring inoculum effect with high accuracy. We found that antibiotic efficacy is determined by the amount of antibiotic used per bacterial colony forming unit (CFU), not by the absolute antibiotic concentration, as shown by the treatment of beta-lactamase-carrying Escherichia coli with cefotaxime. We also noted that cells exhibited a pronounced clustering phenotype when exposed to near-inhibitory amounts of cefotaxime. Overall, our method facilitates research into the interplay between heteroresistance and antibiotic efficacy, as well as research into the origin and stimulation of heterogeneity by exposure to antibiotics. Due to the absolute bacteria quantification in this digital assay, our method provides a platform for developing reference MIC assays that are robust against inoculum-density variations
Droplet-based digital antibiotic susceptibility screen reveals single-cell clonal heteroresistance in an isogenic bacterial population
Since antibiotic resistance is a major threat to global health, recent observations that the traditional test of minimum inhibitory concentration (MIC) is not informative enough to guide effective antibiotic treatment are alarming. Bacterial heteroresistance, in which seemingly susceptible isogenic bacterial populations contain resistant sub-populations, underlies much of this challenge. To close this gap, here we developed a droplet-based digital MIC screen that constitutes a practical analytical platform for quantifying the single-cell distribution of phenotypic responses to antibiotics, as well as for measuring inoculum effect with high accuracy. We found that antibiotic efficacy is determined by the amount of antibiotic used per bacterial colony forming unit (CFU), not by the absolute antibiotic concentration, as shown by the treatment of beta-lactamase-carrying Escherichia coli with cefotaxime. We also noted that cells exhibited a pronounced clustering phenotype when exposed to near-inhibitory amounts of cefotaxime. Overall, our method facilitates research into the interplay between heteroresistance and antibiotic efficacy, as well as research into the origin and stimulation of heterogeneity by exposure to antibiotics. Due to the absolute bacteria quantification in this digital assay, our method provides a platform for developing reference MIC assays that are robust against inoculum-density variations
Wound dressings for a proteolytic-rich environment
Wound dressings have experienced continuous and significant changes over the years based on the knowledge of the biochemical events associated with chronic wounds. The development goes from natural
materials used to just cover and conceal the wound to interactive materials that can facilitate the healing process, addressing specific issues in non-healing wounds. These
new types of dressings often relate with the proteolytic wound environment and the bacteria load to enhance the healing. Recently, the wound dressing research is focusing on the replacement of synthetic polymers by natural protein materials to delivery bioactive agents to the wounds. This
article provides an overview on the novel protein-based wound dressings such as silk fibroin keratin and elastin.
The improved properties of these dressings, like the release of antibiotics and growth factors, are discussed. The different types of wounds and the effective parameters of
healing process will be reviewed
Droplet Microfluidic Technique for the Study of Fermentation
We demonstrate a technique that uses microdroplets for culturing and selecting bacterial cultures in a model biotechnological application. We propose an assay for determination of ethanol concentration that provides increased dynamic range and is compatible with droplet microfluidic screening technologies. The assay comprises two enzymes—alcohol oxidase (AOX) and horseradish peroxidase (HRP)—and a colorimetric readout system of phenol-4-sulfonic acid (PSA) and 4-aminoantipyrine (4-AAP). The microdroplet method provides high repeatability (a relative error of measured ethanol concentration < 5%), high specificity for ethanol, low consumption of reagents and wide dynamic range (1–70 g·L-1) compared to existing assays. We report the use of this method in a screen of ethanol generation efficiency of Zymomonas mobilis (strain 3881) against the concentration of glucose in the culture media
Combinatorial Antimicrobial Susceptibility Testing Enabled by Non-Contact Printing
We demonstrate the utility of non-contact printing to fabricate the mAST—an easy-to-operate, microwell-based microfluidic device for combinatorial antibiotic susceptibility testing (AST) in a point-of-care format. The wells are prefilled with antibiotics in any desired concentration and combination by non-contact printing (spotting). For the execution of the AST, the only requirements are the mAST device, the sample, and the incubation chamber. Bacteria proliferation can be continuously monitored by using an absorbance reader. We investigate the profile of resistance of two reference Escherichia coli strains, report the minimum inhibitory concentration (MIC) for single antibiotics, and assess drug–drug interactions in cocktails by using the Bliss independence model
Lifetime of Phosphorescence from Nanoparticles Yields Accurate Measurement of Concentration of Oxygen in Microdroplets, Allowing One To Monitor the Metabolism of Bacteria
A method
to monitor the level of oxygen in microdroplets is presented.
Optical sensor nanoparticles are dispersed in the aqueous phase of
the microfluidic droplets for culturing bacteria. The oxygen sensor
nanoparticles consist of phosphorescent indicator dye embedded in
poly(styrene-<i>block</i>-vinylpyrrolidone) nanobeads. The
nanoparticles are excitable by red light and emit in the near-infrared
spectra region which minimizes background fluorescence from biological
matter. The biocompatibility of the nanoparticles was proven. Nanoparticles
sensors were read out by adapted miniaturized oxygen meters. The instruments
can be easily integrated into the microfluidic system by placing it
next to the tubing and measuring through the tubing wall. The phosphorescence
lifetime-based measurement circumvents the drawbacks of intensity-based
measurements and enables the determination of the absolute oxygen
concentration in individual moving droplets. The technique can also
be used for monitoring the growth of bacteria in microdroplets. We
demonstrate simultaneous measurement of concentration of oxygen and
optical density (OD) from micro cultures of E. coli and M. smegmatis
Physicochemical Properties Predict Retention of Antibiotics in Water-in-Oil Droplets
Water-in-oil droplet microfluidics promises capacity
for high-throughput
single-cell antimicrobial susceptibility assays and investigation
of drug resistance mechanisms. Every droplet must serve as an isolated
environment with a controlled antibiotic concentration in such assays.
While technologies for generation, incubation, screening, and sorting
droplets mature, predictable retention of active molecules inside
droplets remains a major outstanding
challenge. Here, we analyzed 36 descriptors of the antibiotic molecules
against experimental results on the cross-talk of antibiotics in droplets.
We show that partition coefficient and fractional polar surface area
are the key physicochemical properties that predict antibiotic retention.
We verified the prediction by monitoring growth inhibition by antibiotic-loaded
neighboring droplets. Our experiments also demonstrate that transfer
of antibiotics between droplets is concentration- and distance-dependent.
Our findings immediately apply to designing droplet antibiotic assays
and give deeper insight into the retention of small molecules in water-in-oil
emulsions
Droplet-based digital antibiotic susceptibility screen reveals single-cell clonal heteroresistance in an isogenic bacterial population
Abstract Since antibiotic resistance is a major threat to global health, recent observations that the traditional test of minimum inhibitory concentration (MIC) is not informative enough to guide effective antibiotic treatment are alarming. Bacterial heteroresistance, in which seemingly susceptible isogenic bacterial populations contain resistant sub-populations, underlies much of this challenge. To close this gap, here we developed a droplet-based digital MIC screen that constitutes a practical analytical platform for quantifying the single-cell distribution of phenotypic responses to antibiotics, as well as for measuring inoculum effect with high accuracy. We found that antibiotic efficacy is determined by the amount of antibiotic used per bacterial colony forming unit (CFU), not by the absolute antibiotic concentration, as shown by the treatment of beta-lactamase-carrying Escherichia coli with cefotaxime. We also noted that cells exhibited a pronounced clustering phenotype when exposed to near-inhibitory amounts of cefotaxime. Overall, our method facilitates research into the interplay between heteroresistance and antibiotic efficacy, as well as research into the origin and stimulation of heterogeneity by exposure to antibiotics. Due to the absolute bacteria quantification in this digital assay, our method provides a platform for developing reference MIC assays that are robust against inoculum-density variations
Valorizing the Unexplored Filtration Waste of Brewing Industry for Green Silver Nanocomposite Synthesis
The brewing industry generates a substantial amount of by-products rich in polyphenols, carbohydrates, sugars, sulfates, nitrogen compounds, organic carbon, and several elements, including chlorine, magnesium, and phosphorus. Although limited quantities of these by-products are used in fertilizers and composts, a large amount is discarded as waste. Therefore, it is crucial to identify different ways of valorizing the by-products. Research regarding the valorization of the brewery by-products is still in its nascent stage; therefore, it still has high potential. Herein, we report the valorization of the brewery by-product from the filtration stage of the brewing process (BW9) to synthesize silver nanocomposites as this waste has remained largely unexplored. The BW9 nanocomposites have been compared to those obtained from the brewery product B. The chemical composition analysis of BW9 and B revealed several organic moieties capable of reducing metal salts and capping the formed nanoparticles. Therefore, the brewery waste from stage 9 was valorized as a precursor and added to silver-based precursor at various temperatures (25, 50, and 80 °C) and for various time periods (10, 30, and 120 min) to synthesize silver nanocomposites. The nanocomposites obtained using BW9 were compared to those obtained using the main product of the brewing industry, beer (B). Synthesized nanocomposites composed of AgCl as a major phase and silver metal (Agmet) was incorporated in minor quantities. In addition, Ag3PO4 was also found in B nanocomposites in minor quantities (up to 34 wt.%). The surface morphology depicted globular nanoparticles with layered structures. Small ball-like aggregates on the layer representative of Ag3PO4 were observed in B nanocomposites. The surface of nanocomposites was capped with organic content and functional groups present in the brewery products. The nanocomposites demonstrated high antibacterial activity against Escherichia coli (E. coli), with BW9 nanocomposites exhibiting a higher activity than B nanocomposites