Very rapid flow cytometric assessment of antimicrobial susceptibility during the apparent lag phase of microbial (re)growth

Jindal, Srijan; Thampy, Harish; Day, Philip J.; Kell, Douglas B.

doi:10.1099/mic.0.000777

Cited by 16 publications

(23 citation statements)

References 142 publications

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“…Microbial resistance to antibiotics (AMR) remains a huge problem (e.g. 67–72 ). To this end, a major cause is the ability of efflux pumps to create resistance to antibiotics by pumping them out from the cytoplasm of cells (e.g.…”

Section: Discussionmentioning

confidence: 99%

Why most transporter mutations that cause antibiotic resistance are to efflux pumps rather than to import transporters

Mendes

Gottardi

Superti‐Furga

et al. 2020

Preprint

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Genotypic microbial resistance to antibiotics with intracellular targets commonly arises from mutations that increase the activities of transporters (pumps) that cause the efflux of intracellular antibiotics. A priori it is not obvious why this is so much more common than are mutations that simply inhibit the activity of uptake transporters for the antibiotics. We analyse quantitatively a mathematical model consisting of one generic equilibrative transporter and one generic concentrative uptake transporter (representing any number of each), together with one generic efflux transporter. The initial conditions are designed to give an internal concentration of the antibiotic that is three times the minimum inhibitory concentration (MIC). The effect of varying the activity of each transporter type 100-fold is dramatically asymmetric, in that lowering the activities of individual uptake transporters has comparatively little effect on internal concentrations of the antibiotic. By contrast, increasing the activity of the efflux transporter lowers the internal antibiotic concentration to levels far below the MIC. Essentially, these phenomena occur because inhibiting individual influx transporters allows others to ‘take up the slack’, whereas increasing the activity of the generic efflux transporter cannot easily be compensated. The findings imply strongly that inhibiting efflux transporters is a much better approach for fighting antimicrobial resistance than is stimulating import transporters. This has obvious implications for the development of strategies to combat the development of microbial resistance to antibiotics and possibly also cancer therapeutics in human.

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Section: Discussionmentioning

confidence: 99%

Why most transporter mutations that cause antibiotic resistance are to efflux pumps rather than to import transporters

Mendes

Gottardi

Superti‐Furga

et al. 2020

Preprint

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“…As previously [17,18], we used a high-throughput flow cytometer, the Intellicyt® iQue Screener Plus (Sartorius, Göttingen, Germany), with the following protocol: buffer equilibration (QSol, Sartorius) and plate shaking at 2000 rpm for 50 sec, sampling for 2 sec with 1 sec upload time, 5 sec wash in Qsol buffer every 3 wells, and further probe wash for 10 sec every 12 wells. The instrument has three LED lasers (405nm, 488nm, 640nm) and collects data for two light scattering and 13 fluorescence channels for all samples.…”

Section: Methodsmentioning

confidence: 99%

“…Fluorescence flow cytometry provides a convenient and high-throughput means for assessing the extent of uptake and accumulation of a given fluorophore (e.g. [6][7][8][9][10][11][12][13][14][15][16]), and in recent work [17,18] we have found this to be true in E. coli in vivo. In particular, the availability of the 'Keio' collection of single-gene knockouts in 'non-essential' genes [19,20] allowed up to assess the contribution of many of these genes to uptake.…”

Section: Introductionmentioning

confidence: 99%

A palette of fluorophores that are differentially accumulated by wild-type and mutant strains ofEscherichia coli: surrogate ligands for bacterial membrane transporters

Salcedo-Sora

Jindal

O’Hagan

et al. 2020

Preprint

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Our previous work had demonstrated that two commonly used fluorescent dyes that were accumulated by wild-type E. coli MG1655 were accumulated differentially in single-gene knockout strains, and also that they might be used as surrogates in flow cytometric transporter assays. We summarise the desirable properties of such stains, and here survey 143 candidate dyes. We triage them eventually (on the basis of signal, accumulation levels, and cost) to a palette of 39 commercially available and affordable fluorophores that are accumulated significantly by wild-type cells of the ‘Keio’ strain BW25113, as measured flow cytometrically. Cheminformatic analyses indicate both their similarities and their (much more considerable) structural differences. We describe the effects of pH and of the efflux pump inhibitor chlorpromazine on the accumulation. Even the ‘wild-type’ MG1655 and BW25113 strains can differ significantly in their ability to take up such dyes. We illustrate the highly differential uptake of our dyes into strains with particular lesions in, or overexpressed levels of, three particular transporters or transporter components (yhjV, yihN, and tolC). The relatively small collection of dyes described offers a rapid, inexpensive, convenient and valuable approach to the assessment of microbial physiology and transporter function.

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“…This instrument does not have user-adjustable photomultiplier tubes so the numbers simply reflect the extent of fluorescence it registers. The following settings [107] were used for the flow cytometry: automatic prime -60 s (in Qsol buffer); pre-plate shake -15 s and 900 r.p.m. ; sip time -2 s (actual sample uptake); additional sip time -0.5 s (the gap between sips); pump speed -29 r.p.m.…”

Section: Methodsmentioning

confidence: 99%

“…The strategy used to assess diS-C3(5) uptake, gating on light scattering in 'forward' and 'side' directions by the bacteria and assessing uptake via red fluorescence (which is not interfered with by any autofluorescence) is precisely as was described previously [124]. It is obvious ( Fig 2) that even in the wild-type strain there is considerable heterogeneity in the distribution of uptake between individual cells; this becomes even greater for the cells (∆atpB) lacking the β subunit of the membrane ATP synthase.…”

Section: Variation Of the Uptake Of A Lipophilic Carbocyanine Cationmentioning

confidence: 99%

Involvement of multiple influx and efflux transporters in the accumulation of cationic fluorescent dyes byEscherichia coli

Jindal¹,

Yang

Day

et al. 2019

Preprint

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We used high-throughput flow cytometry to assess the ability of individual gene knockout strains ofE colito take up two membrane-permeable, cationic fluorescent dyes, viz the carbocyanine diS-C3(5) and the DNA dye SYBR Green. Individual strains showed a large range of distributions of uptake. The range of modal steady-state uptakes for the carbocyanine between the different strains was 36-fold. Knockouts of the ATP synthase α- and β-subunits greatly inhibited uptake, implying that most uptake was ATP-driven rather than being driven by say a membrane potential. Dozens of transporters changed the steady-state uptake of the dye by more than 50% with respect to that of the wild type, in both directions (increased or decreased); knockouts in known influx and efflux transporters behaved as expected, giving confidence in the general strategy. Many of the knockouts with the most reduced uptake were transporter genes of unknown function (‘y-genes’). Similarly, several overexpression variants in the ‘ASKA’ collection had the anticipated, opposite effects. Similar findings were made with SYBR Green (the range being some 69-fold), though despite it too containing a benzimidazole motif there was negligible correlation between its uptake and that of the carbocyanine when compared across the various strains. Overall, we conclude that the uptake of these dyes may be catalysed by a great many transporters of possibly broad and presently unknown specificity. This casts serious doubt upon the use of such dyes as quantitative stains for representing either bioenergetic parameters or the amount of cellular DNA in unfixed cells (in vivo). By contrast, it opens up their potential use as transporter assay substrates in high-throughput screening.

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Very rapid flow cytometric assessment of antimicrobial susceptibility during the apparent lag phase of microbial (re)growth

Abstract: Rapid changes in the number and flow cytometric behaviour of cells of E. coli taken from a stationary phase and inoculated into rich medium.

Cited by 16 publications

References 142 publications

Why most transporter mutations that cause antibiotic resistance are to efflux pumps rather than to import transporters

Why most transporter mutations that cause antibiotic resistance are to efflux pumps rather than to import transporters

A palette of fluorophores that are differentially accumulated by wild-type and mutant strains ofEscherichia coli: surrogate ligands for bacterial membrane transporters

Involvement of multiple influx and efflux transporters in the accumulation of cationic fluorescent dyes byEscherichia coli

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