A single-cell drug efflux assay in bacteria by using a directly accessible femtoliter droplet array

Iino, Ryota; Hayama, Kohei; Amezawa, Hiromi; Sakakihara, Shouichi; Kim, Soo Hyeon; Matsumono, Yoshimi; Nishino, Kunihiko; Yamaguchi, Akihito; Noji, Hiroyuki

doi:10.1039/c2lc40394c

Cited by 47 publications

(41 citation statements)

References 33 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%

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%

Efflux pump inhibitors of clinically relevant multidrug resistant bacteria

Lamut

Mašič

Kikelj

et al. 2019

Medicinal Research Reviews

145

116

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“…As a result of heterogeneity, any isolation technique may only capture a fraction of the persisters present, yielding a limited sample of the persister population. Single-cell analysis techniques offer means to interrogate individual cells (Iino et al, 2012, 2013); however, the identification of persisters before they exit their non-replicative state is not presently possible. Perhaps a viable path forward is to study model persisters generated following the over-expression of genes that have been shown to increase persister levels (Korch and Hill, 2006; Vázquez-Laslop et al, 2006).…”

Section: Challenges In the Study Of Persister Metabolismmentioning

confidence: 99%

The role of metabolism in bacterial persistence

et al. 2014

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Bacterial persisters are phenotypic variants with extraordinary tolerances toward antibiotics. Persister survival has been attributed to inhibition of essential cell functions during antibiotic stress, followed by reversal of the process and resumption of growth upon removal of the antibiotic. Metabolism plays a critical role in this process, since it participates in the entry, maintenance, and exit from the persister phenotype. Here, we review the experimental evidence that demonstrates the importance of metabolism to persistence, highlight the successes and potential of targeting metabolism in the search for anti-persister therapies, and discuss the current methods and challenges to understand persister physiology.

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“…In our new microdevice, a large number of dome-shaped femtoliter droplets can be prepared that enclose individual bacterial cells (Iino et al, 2012a). One prominent feature of this array is that the individual droplets containing the enclosed cells can be accessed and collected with a micropipette.…”

Section: Large-scale Femtoliter Droplet Array For Single Bacterial Cementioning

confidence: 99%

“…We have recently developed a single-cell drug efflux assay using the femtoliter droplet array ( Figure 4 ; Iino et al, 2012a). In this assay, E. coli cultured in test tubes was mixed with a fluorogenic substrate, fluorescein-di-β - D -galactopyranoside (FDG), enclosed in a droplet array, and then cultured for 15–20 min at room temperature.…”

Section: A Single-cell Drug Efflux Assay In a Femtoliter Droplet Arraymentioning

confidence: 99%

“…Collected single cells divide multiple times after transfer to growth medium in a test tube. The plasmid in the divided cells can be extracted and used for subsequent gene analysis (Iino et al, 2012a). Culturing after cell collection can be omitted by amplifying the DNA by single-cell PCR (Ottesen et al, 2006).…”

Section: Phenotypic Change After Genetic Transformationmentioning

confidence: 99%

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

Iino¹,

Matsumoto²,

Nishino³

et al. 2013

Front. Microbiol.

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Single-cell analysis is a powerful method to assess the heterogeneity among individual cells, enabling the identification of very rare cells with properties that differ from those of the majority. In this Methods Article, we describe the use of a large-scale femtoliter droplet array to enclose, isolate, and analyze individual bacterial cells. As a first example, we describe the single-cell detection of drug-tolerant persisters of Pseudomonas aeruginosa treated with the antibiotic carbenicillin. As a second example, this method was applied to the single-cell evaluation of drug efflux activity, which causes acquired antibiotic resistance of bacteria. The activity of the MexAB-OprM multidrug efflux pump system from Pseudomonas aeruginosa was expressed in Escherichia coli and the effect of an inhibitor D13-9001 were assessed at the single cell level.

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A single-cell drug efflux assay in bacteria by using a directly accessible femtoliter droplet array

Cited by 47 publications

References 33 publications

Efflux pump inhibitors of clinically relevant multidrug resistant bacteria

Efflux pump inhibitors of clinically relevant multidrug resistant bacteria

The role of metabolism in bacterial persistence

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

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