Design of a large-scale femtoliter droplet array for single-cell analysis of drug-tolerant and drug-resistant bacteria

Iino, Ryota; Matsumoto, Yoshimi; Nishino, Kunihiko; Yamaguchi, Akihito; Noji, Hiroyuki

doi:10.3389/fmicb.2013.00300

Cited by 40 publications

(36 citation statements)

References 46 publications

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“…Other examples of fluorescent molecules used for determination of EPI are the hydrophobic dye Hoechst H33342, which emits intense fluorescence inside the cell upon binding to DNA, 1,2′‐dinaphthylamine, which becomes fluorescent when partitioned into a phospholipid bilayer, and others, such as 1‐anilinonaphthalene‐8‐sulfonate and acriflavine . Efflux inhibition can also be studied at the level of a single cell by direct evaluation of bacterial efflux activity . In this case, individual bacterial cells are isolated in a femtoliter droplet and analyzed by the fluorescence measurement.…”

Section: Methods For Biological Evaluation Of Efflux Pump Inhibitorsmentioning

confidence: 99%

“…In this case, individual bacterial cells are isolated in a femtoliter droplet and analyzed by the fluorescence measurement. Fluorescein‐di‐β‐ d ‐galactopyranoside is used for this purpose, being hydrolyzed by β‐galactosidase in the cytoplasm of a bacterial cell to produce fluorescein …”

Section: Methods For Biological Evaluation Of Efflux Pump Inhibitorsmentioning

confidence: 99%

“…83 Efflux inhibition can also be studied at the level of a single cell by direct evaluation of bacterial efflux activity. [87][88][89][90] In this case, individual bacterial cells are isolated in a femtoliter droplet and analyzed by the fluorescence measurement. Fluorescein-di-β-D-galactopyranoside is used for this purpose, being hydrolyzed by β-galactosidase in the cytoplasm of a bacterial cell to produce fluorescein.…”

Section: Methods For Biological Evaluation Of Efflux Pump Inhibitorsmentioning

confidence: 99%

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Efflux pump inhibitors of clinically relevant multidrug resistant bacteria

Lamut

Mašič

Kikelj

et al. 2019

Medicinal Research Reviews

145

116

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Bacterial infections are an increasingly serious issue worldwide. The inability of existing therapies to treat multidrug‐resistant pathogens has been recognized as an important challenge of the 21st century. Efflux pumps are important in both intrinsic and acquired bacterial resistance and identification of small molecule efflux pump inhibitors (EPIs), capable of restoring the effectiveness of available antibiotics, is an active research field. In the last two decades, much effort has been made to identify novel EPIs. However, none of them has so far been approved for therapeutic use. In this article, we explore different structural families of currently known EPIs for multidrug resistance efflux systems in the most extensively studied pathogens (NorA in Staphylococcus aureus, AcrAB‐TolC in Escherichia coli, and MexAB‐OprM in Pseudomonas aeruginosa). Both synthetic and natural compounds are described, with structure‐activity relationship studies and optimization processes presented systematically for each family individually. In vitro activities against selected test strains are presented in a unifying manner for all the EPIs described, together with the most important toxicity, pharmacokinetic and in vivo efficacy data. A critical evaluation of lead‐likeness characteristics and the potential for clinical development of the most promising inhibitors of the three efflux systems is described. This overview of EPIs is a good starting point for the identification of novel effective antibacterial drugs.

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Section: Methods For Biological Evaluation Of Efflux Pump Inhibitorsmentioning

confidence: 99%

Section: Methods For Biological Evaluation Of Efflux Pump Inhibitorsmentioning

confidence: 99%

Section: Methods For Biological Evaluation Of Efflux Pump Inhibitorsmentioning

confidence: 99%

See 1 more Smart Citation

Efflux pump inhibitors of clinically relevant multidrug resistant bacteria

Lamut

Mašič

Kikelj

et al. 2019

Medicinal Research Reviews

145

116

View full text Add to dashboard Cite

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“…However, upon their adjustment to prolonged mammalian assays, they require multilayer fabrication, either involving high-pressure cell loading or not allowing for different substrates to be used (26,27). Several single-cell platforms that encapsulate cells in stationary droplet arrays are available (5,(28)(29)(30)(31)(32)(33)(34)(35); however, the ability to culture adherent cells for long periods remains difficult (36).…”

mentioning

confidence: 99%

Stationary nanoliter droplet array with a substrate of choice for single adherent/nonadherent cell incubation and analysis

Shemesh

Ben-Arye

Avesar³

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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Microfluidic water-in-oil droplets that serve as separate, chemically isolated compartments can be applied for single-cell analysis; however, to investigate encapsulated cells effectively over prolonged time periods, an array of droplets must remain stationary on a versatile substrate for optimal cell compatibility. We present here a platform of unique geometry and substrate versatility that generates a stationary nanodroplet array by using wells branching off a main microfluidic channel. These droplets are confined by multiple sides of a nanowell and are in direct contact with a biocompatible substrate of choice. The device is operated by a unique and reversed loading procedure that eliminates the need for fine pressure control or external tubing. Fluorocarbon oil isolates the droplets and provides soluble oxygen for the cells. By using this approach, the metabolic activity of single adherent cells was monitored continuously over time, and the concentration of viable pathogens in blood-derived samples was determined directly by measuring the number of colony-formed droplets. The method is simple to operate, requires a few microliters of reagent volume, is portable, is reusable, and allows for cell retrieval. This technology may be particularly useful for multiplexed assays for which prolonged and simultaneous visual inspection of many isolated single adherent or nonadherent cells is required.single cell | nanoliter array | diagnostics C ommon single-cell analysis methods, such as flow cytometry and mass cytometry (1), offer high throughput and accurate single-cell marker quantification, yet they lack the ability to monitor large numbers of single cells continuously and simultaneously in performance-based assays (2, 3). Conventional microscopy may be used for these assays; however, in the case of single cells, they cannot analyze extracellular events, such as secretion. To achieve this, cells must be isolated in compartments that can sustain cell viability and growth while permitting conventional optical analysis over many hours to days. Dropletbased microfluidics, which enables single-cell encapsulation in nano-and subnanoliter droplets by surrounding microscopic aqueous medium with an immiscible carrier fluid (4-8), recently gained interest with the appearance of digital PCR (9-11). Much of the work thus far has been directed toward improving droplet manipulation capabilities (12-16). With these methods, droplets are mobile, and thus cytometry is performed under flow conditions (17), making continuous monitoring of single cells difficult. Continuous monitoring may be achieved by using stationary indexed droplets, but many current droplet immobilization techniques are limited by pressure coupling between droplet generation and capture events, as well as the requirement to adjust droplet volume to nanowell size (6,18,19). The vast majority of methods used to generate water-in-oil droplets begin by priming a continuous oil phase in a microfluidic channel followed by an injection of a dispersed (aqueous) medium (2...

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“…The feasibility of using intrinsic resistome inhibitors for potentiating the effects of clinical drugs is also discussed in this review. Iino et al (2013) used a largescale femtoliter droplet array for single-cell analysis to assess the heterogeneity among the individual cells, enabling the identification of the novel or unconventional mechanisms of antibiotic resistance or resistance against novel antibiotics.…”

mentioning

confidence: 99%

Mechanisms of antibiotic resistance

Cattoir

2015

Frontiers Research Topics

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Design of a large-scale femtoliter droplet array for single-cell analysis of drug-tolerant and drug-resistant bacteria

Cited by 40 publications

References 46 publications

Efflux pump inhibitors of clinically relevant multidrug resistant bacteria

Efflux pump inhibitors of clinically relevant multidrug resistant bacteria

Stationary nanoliter droplet array with a substrate of choice for single adherent/nonadherent cell incubation and analysis

Mechanisms of antibiotic resistance

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