Figures and data in High-throughput quantification of population dynamics using luminescence

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Tables
Additional files

10 figures, 7 tables and 1 additional file

Figures

Figure 1 with 19 supplements

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Comparison of colony-forming unit (CFU)-based and luminescence-based rates of change.

For each drug, we generated 2000 bootstrapped datasets by resampling time-series CFU and light-intensity data with replacement and fitted an exponential function to each bootstrap replicate to obtain distributions of rates. Panel (a) shows these distributions for 20 drug-concentration assays across 19 antibiotics; panel (b) shows the antimicrobial peptide pexiganan at 8 μg/mL and 16 μg/mL. The distribution of $ψ_{C F U}$ is shown in blue (lower half of each violin, diamond). The distribution of $ψ_{I}$ is shown in orange (upper half, triangle). Green distributions ( $ψ_{I}^{*}$ ; triangle) represent luminescence-based rates calculated from data starting at the first peak onward. Red distributions show volume-adjusted luminescence rates ( $ψ_{J}$ ; pentagon). Vertical lines mark the 95% confidence intervals. Asterisk (*) or letters (n.s.) indicate whether CFU-based rates differ significantly from the corresponding luminescence-based rate or not (see Materials and methods), with color coding matching the respective luminescence-based distribution. Wide confidence intervals for pexiganan reflect biphasic killing, steep curves, and noisy CFU data.

Figure 1—figure supplement 1

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Ampicillin.

The colony-forming unit (CFU) signal is shown as blue diamonds and the light intensity as orange triangles for data points above their respective detection limits ( $10^{4} C F U / m L$ for CFU and 20 rlu for luminescence). Lines represent log-linear fits to the corresponding signals. The morphology-corrected luminescence signal is shown as orange pentagons with red frames, and the corresponding rate fit indicated by a red line. ‘+’ indicates data points below detection limit or otherwise excluded (for CFU, symbolically plotted at $10^{4} C F U / m L$ to visualize missing data), and if a signal ends early (due to dropping below its detection limit), the corresponding data point of the other signal was cut to the same endpoint for a consistent comparison. These excluded data points are marked as ‘X’.

Figure 1—figure supplement 2

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Cefepime.

The colony-forming unit (CFU) signal is shown as blue diamonds and the light intensity as orange triangles for data points above their respective detection limits ( $10^{4} C F U / m L$ for CFU and 20 rlu for luminescence). Lines represent log-linear fits to the corresponding signals. Green-framed data points and corresponding green fit lines indicate analyses excluding early data points until the first peak.

Figure 1—figure supplement 3

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Ceftazidime.

The colony-forming unit (CFU) signal is shown as blue diamonds and the light intensity as orange triangles for data points above their respective detection limits ( $10^{4} C F U / m L$ for CFU and 20 rlu for luminescence). Lines represent log-linear fits to the corresponding signals. The morphology-corrected luminescence signal is shown as orange pentagons with red frames, and the corresponding rate fit indicated by a red line.

Figure 1—figure supplement 4

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Cefuroxime.

The colony-forming unit (CFU) signal is shown as blue diamonds and the light intensity as orange triangles for data points above their respective detection limits ( $10^{4} C F U / m L$ for CFU and 20 rlu for luminescence). Lines represent log-linear fits to the corresponding signals. ‘+’ indicates data points below detection limit or otherwise excluded (for CFU, symbolically plotted at $10^{4} C F U / m L$ to visualize missing data).

Figure 1—figure supplement 5

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Chloramphenicol.

The colony-forming unit (CFU) signal is shown as blue diamonds and the light intensity as orange triangles for data points above their respective detection limits ( $10^{4} C F U / m L$ for CFU and 20 rlu for luminescence). Lines represent log-linear fits to the corresponding signals.

Figure 1—figure supplement 6

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Ciprofloxacin.

The colony-forming unit (CFU) signal is shown as blue diamonds and the light intensity as orange triangles for data points above their respective detection limits ( $10^{4} C F U / m L$ for CFU and 20 rlu for luminescence). Lines represent log-linear fits to the corresponding signals. The morphology-corrected luminescence signal is shown as orange pentagons with red frames, and the corresponding rate fit indicated by a red line.

Figure 1—figure supplement 7

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Colistin.

The colony-forming unit (CFU) signal is shown as blue diamonds and the light intensity as orange triangles for data points above their respective detection limits ( $10^{4} C F U / m L$ for CFU and 20 rlu for luminescence). Lines represent log-linear fits to the corresponding signals. ‘+’ indicates data points below detection limit or otherwise excluded (for CFU, symbolically plotted at $10^{4} C F U / m L$ to visualize missing data).

Figure 1—figure supplement 8

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Doripenem.

The colony-forming unit (CFU) signal is shown as blue diamonds and the light intensity as orange triangles for data points above their respective detection limits ( $10^{4} C F U / m L$ for CFU and 20 rlu for luminescence). Lines represent log-linear fits to the corresponding signals. Green-framed data points and corresponding green fit lines indicate analyses excluding early data points until the first peak.

Figure 1—figure supplement 9

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Fosfomycin.

The colony-forming unit (CFU) signal is shown as blue diamonds and the light intensity as orange triangles for data points above their respective detection limits ( $10^{4} C F U / m L$ for CFU and 20 rlu for luminescence). Lines represent log-linear fits to the corresponding signals.

Figure 1—figure supplement 10

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Imipenem.

The colony-forming unit (CFU) signal is shown as blue diamonds and the light intensity as orange triangles for data points above their respective detection limits ( $10^{4} C F U / m L$ for CFU and 20 rlu for luminescence). Lines represent log-linear fits to the corresponding signals. ‘+’ indicates data points below detection limit or otherwise excluded (for CFU, symbolically plotted at $10^{4} C F U / m L$ to visualize missing data).

Figure 1—figure supplement 11

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Mecillinam.

The colony-forming unit (CFU) signal is shown as blue diamonds and the light intensity as orange triangles for data points above their respective detection limits ( $10^{4} C F U / m L$ for CFU and 20 rlu for luminescence). Lines represent log-linear fits to the corresponding signals.

Figure 1—figure supplement 12

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Meropenem.

The colony-forming unit (CFU) signal is shown as blue diamonds and the light intensity as orange triangles for data points above their respective detection limits ( $10^{4} C F U / m L$ for CFU and 20 rlu for luminescence). Lines represent log-linear fits to the corresponding signals. The morphology-corrected luminescence signal is shown as orange pentagons with red frames, and the corresponding rate fit indicated by a red line. ‘+’ indicates data points below detection limit or otherwise excluded (for CFU, symbolically plotted at $10^{4} C F U / m L$ to visualize missing data).

Figure 1—figure supplement 13

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Penicillin.

The colony-forming unit (CFU) signal is shown as blue diamonds and the light intensity as orange triangles for data points above their respective detection limits ( $10^{4} C F U / m L$ for CFU and 20 rlu for luminescence). Lines represent log-linear fits to the corresponding signals. ‘+’ indicates data points below detection limit or otherwise excluded (for CFU, symbolically plotted at $10^{4} C F U / m L$ to visualize missing data), and if a signal ends early (due to dropping below its detection limit), the corresponding data point of the other signal was cut to the same endpoint for a consistent comparison. These excluded data points are marked as ‘X’.

Figure 1—figure supplement 14

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Piperacillin.

The colony-forming unit (CFU) signal is shown as blue diamonds and the light intensity as orange triangles for data points above their respective detection limits ( $10^{4} C F U / m L$ for CFU and 20 rlu for luminescence). Lines represent log-linear fits to the corresponding signals.

Figure 1—figure supplement 15

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Polymyxin B.

The colony-forming unit (CFU) signal is shown as blue diamonds and the light intensity as orange triangles for data points above their respective detection limits ( $10^{4} C F U / m L$ for CFU and 20 rlu for luminescence). Lines represent log-linear fits to the corresponding signals. ‘+’ indicates data points below detection limit or otherwise excluded (for CFU, symbolically plotted at $10^{4} C F U / m L$ to visualize missing data).

Figure 1—figure supplement 16

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Rifampicin (25μg/mL).

The colony-forming unit (CFU) signal is shown as blue diamonds and the light intensity as orange triangles for data points above their respective detection limits ( $10^{4} C F U / m L$ for CFU and 20 rlu for luminescence). Lines represent log-linear fits to the corresponding signals.

Figure 1—figure supplement 17

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Tetracycline.

The colony-forming unit (CFU) signal is shown as blue diamonds and the light intensity as orange triangles for data points above their respective detection limits ( $10^{4} C F U / m L$ for CFU and 20 rlu for luminescence). Lines represent log-linear fits to the corresponding signals.

Figure 1—figure supplement 18

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Trimethoprim.

The colony-forming unit (CFU) signal is shown as blue diamonds and the light intensity as orange triangles for data points above their respective detection limits ( $10^{4} C F U / m L$ for CFU and 20 rlu for luminescence). Lines represent log-linear fits to the corresponding signals. The morphology-corrected luminescence signal is shown as orange pentagons with red frames, and the corresponding rate fit indicated by a red line.

Figure 1—figure supplement 19

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Comparison of colony-forming unit (CFU) and luminescence kill curve signals for pexiganan at (a) 8 μg/mL and (b) 16 μg/mL.

The first data points at $t_{0}$ represent the pretreatment CFU and light intensity values.

Figure 2 with 14 supplements

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Density distributions (2.5–97.5% percentile range) of pooled cell volumes acquired by microscopy imaging, shown (a) before and (b) after 2 hr of antibiotic treatment.

Boxes indicate the 25–75% interquartile range, and vertical bars mark the mean. Significance (*) was assessed by bootstrapping cell volumes 200 times with replacement for each replicate (image) and treatment, pooling the bootstrapped volumes by treatment, and comparing the resulting 95% confidence interval of the mean to that of the untreated control (control_2h). ‡ Carbapenems tend to deform cells into a lemon-like shape (Figure 2—figure supplement 10), resulting in poor fitting quality since our algorithm assumes a cylindrical geometry.

Figure 2—figure supplement 1

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Pooled density distributions (95% prediction intervals) of cell widths (red) and lengths (blue), obtained from microscopy images (a) before and (b) after 2 hr of antibiotic treatment.

Boxes indicate the interquartile range (Q1–Q3), and the mean is marked by (|). ‡ Poor fit quality for meropenem-treated cells, which adopt a lemon-like shape (Figure 2—figure supplement 10), due to our algorithm assuming cylindrical geometry.

Figure 2—figure supplement 2

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Microscopy images of control (before treatment) samples.

Each image represents a different replicate. We plotted the green channel of the recorded images in grayscale. The red channel, capturing propidium iodide activity, is overlaid in red. Bacterial shapes detected by the algorithm are outlined in cyan.

Figure 2—figure supplement 3

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Microscopy images of control (after 2 hr) samples.

Each image represents a different replicate. We plotted the green channel of the recorded images in grayscale. The red channel, capturing propidium iodide activity, is overlaid in red. Bacterial shapes detected by the algorithm are outlined in cyan.

Figure 2—figure supplement 4

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Microscopy images of cells treated with ampicillin.

Each image represents a different replicate. We plotted the green channel of the recorded images in grayscale. The red channel, capturing propidium iodide activity, is overlaid in red. Bacterial shapes detected by the algorithm are outlined in cyan.

Figure 2—figure supplement 5

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Microscopy images of cells treated with amoxicillin.

Each image represents a different replicate. We plotted the green channel of the recorded images in grayscale. The red channel, capturing propidium iodide activity, is overlaid in red. Bacterial shapes detected by the algorithm are outlined in cyan.

Figure 2—figure supplement 6

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Microscopy images of cells treated with ceftazidime.

Each image represents a different replicate. We plotted the green channel of the recorded images in grayscale. The red channel, capturing propidium iodide activity, is overlaid in red. Bacterial shapes detected by the algorithm are outlined in cyan.

Figure 2—figure supplement 7

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Microscopy images of cells treated with ciprofloxacin.

Each image represents a different replicate. The white gaps can indicate the start of cell division (cells are not fixed). We plotted the green channel of the recorded images in grayscale. The red channel, capturing propidium iodide activity, is overlaid in red. Bacterial shapes detected by the algorithm are outlined in cyan.

Figure 2—figure supplement 8

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Microscopy images of cells treated with colistin.

Each image represents a different replicate. We plotted the green channel of the recorded images in grayscale. The red channel, capturing propidium iodide activity, is overlaid in red. Bacterial shapes detected by the algorithm are outlined in cyan.

Figure 2—figure supplement 9

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Microscopy images of cells treated with fosfomycin.

Each image represents a different replicate. We plotted the green channel of the recorded images in grayscale. The red channel, capturing propidium iodide activity, is overlaid in red. Bacterial shapes detected by the algorithm are outlined in cyan.

Figure 2—figure supplement 10

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Microscopy images of cells treated with meropenem.

Each image represents a different replicate. We plotted the green channel of the recorded images in grayscale. The red channel, capturing propidium iodide activity, is overlaid in red. Bacterial shapes detected by the algorithm are outlined in cyan.

Figure 2—figure supplement 11

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Microscopy images of cells treated with pexiganan.

Each image represents a different replicate. We plotted the green channel of the recorded images in grayscale. The red channel, capturing propidium iodide activity, is overlaid in red. Bacterial shapes detected by the algorithm are outlined in cyan.

Figure 2—figure supplement 12

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Microscopy images of cells treated with rifampicin.

Each image represents a different replicate. We plotted the green channel of the recorded images in grayscale. The red channel, capturing propidium iodide activity, is overlaid in red. Bacterial shapes detected by the algorithm are outlined in cyan.

Figure 2—figure supplement 13

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Microscopy images of cells treated with tetracycline.

Each image represents a different replicate. We plotted the green channel of the recorded images in grayscale. The red channel, capturing propidium iodide activity, is overlaid in red. Bacterial shapes detected by the algorithm are outlined in cyan.

Figure 2—figure supplement 14

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Microscopy images of cells treated with trimethoprim.

Each image represents a different replicate. We plotted the green channel of the recorded images in grayscale. The red channel, capturing propidium iodide activity, is overlaid in red. Bacterial shapes detected by the algorithm are outlined in cyan.

Figure 3

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Simulations based on the filamentation model quantifying how changes in division rate due to treatment ( $Δ λ$ ) and death rate ( $δ$ ) influence the rate of change of population size ( $ψ_{B}$ , blue), the rate of change of light intensity ( $ψ_{I}$ , orange), and the rate of change of light intensity when the first 2 hr of data are excluded ( $ψ_{I}^{*}$ , green).

These illustrative simulations were conducted using an initial division rate $λ_{0} = 1.5 {h r}^{- 1}$ . More details and all parameter values can be found in Appendix 1.

Appendix 1—figure 1

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Light intensity scales linearly with bacterial density.

Serial tenfold dilutions of bacterial cultures were prepared in a 384-well white microplate, and luminescence was measured immediately. Linear regression of the luminescence signal against bacterial density (CFU) yielded a conversion factor of $m_{f i t} = 0.006 {r l u \cdot m L \cdot C F U}^{- 1}$ . The high correlation ( $R^{2} = 0.987$ in log-log space) confirms a linear relationship between luminescence and bacterial density.

Appendix 1—figure 2

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Panel plot showing the effects of UV treatment on bacterial density (approximated by optical density [OD]) and light intensity (I) over time by comparing treated (UV) and untreated (ctrl) cultures.

(a) OD, (b) light intensity, (c) OD-specific light intensity $ω = I (t) / O D (t)$ , and (d) the difference in OD-specific light intensity between UV-treated and control.

Appendix 1—figure 3

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Example of the probability of colony formation $p_{C} = 1 - p_{E}$ (see Appendix 1) for a single plated bacterium.

Blue shows $p_{C}$ for a purely bactericidal drug ( $λ_{T} = 0$ ) and red for a purely bacteriostatic drug ( $δ_{T} = 0$ ), plotted over the treatment effect $τ = δ_{T} + λ_{T}$ . In this illustrative example, we use $λ_{0} = 1.35 h^{- 1}$ and $δ_{0} = 0.1 h^{- 1}$ .

Appendix 1—figure 4

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Colony-forming unit (CFU) measured over time in supplemented dilution media.

Bacterial cultures treated for 1 min with 16 μg/mL pexiganan were diluted (1:100) in phosphate-buffered saline (PBS) supplemented with varying concentrations of (a) CaCl₂ and (b) MgCl₂. Diluted samples were repeatedly plated over time to test whether supplementation prevents further bacterial killing.

Appendix 1—figure 5

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Colony-forming unit (CFU) time-kill curves for pexiganan, performed manually, comparing dilution in (a) unsupplemented phosphate-buffered saline (PBS) and (b) PBS supplemented with 100 mM MgCl₂.

Time points correspond to: $t_{0} \approx 0 s$ , $t_{1} \approx 20 s$ , $t_{2} \approx 2 m i n$ , $t_{3} \approx 3 m i n 20 s$ , and $t_{4} \approx 5 m i n$ . The black boxplot ( $t_{0}$ ) indicates the pretreatment bacterial density.

Appendix 1—figure 6

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Ampicillin kill curve versus supernatant kill curve.

We show the original ampicillin kill curve in blue and the change of colony-forming unit (CFU) over time in the supernatant. The error bars show the min/max interval of the three replicates. Appendix 1—table 5 lists the confidence interval and mean for the bootstrapped rates. According to the significance criterion (defined in Materials and methods), these two rates are significantly different. The confidence interval of the rate of change of CFU in the supernatant includes zero.

Appendix 1—figure 7

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Illustrative simulations using the filamentation model relating (a–d) bacterial population size (blue) and light intensity (orange) under different combinations of treatment-induced changes in division rate ( $Δ λ$ ) and death rate ( $δ$ ).

Panel (e) shows the distributions of converged cell volumes for $λ = 0.3$ and $λ = 1.5$ . Panel (f) shows the shift of mean cell volumes over time for $Δ λ = - 1.2$ and $Δ λ = 0$ . For all simulations, we used $λ_{0} = 1.5$ , $γ = 150$ , $ϕ = 0.015$ , and $ϵ = 0.04$ . As shown in panel (b), treatment-induced filamentation can lead to a temporary discrepancy between luminescence- and CFU-based rates.

Tables

Key resources table

Reagent type (species) or resource	Designation	Source or reference	Identifiers	Additional information
Strain	K-12 substr. MG1655	Lab collection	GenBank: U00096	Parent strain
Strain	MG1655 galK::luxCDABE-kan	This paper	Available on request	Bioluminescent reporter; λ-Red integration replacing galK
Recombinant DNA reagent	pCS-λ	Kishony and Leibler, 2003		Source of luxCDABE, λ-Pr promoter, and kanamycin cassette
Recombinant DNA reagent	pSIM5	Datta et al., 2006		λ-Red integration helper plasmid
Sequence-based reagent	Integration primers	This paper	Appendix1—table 6	For λ-Red integration
Drug	Antimicrobials		Appendix1—table 1	Suppliers, catalog numbers, and MICs
Software	Analysis scripts, work list generation, colony recognition, and model code	Zenodo	10.5281/zenodo.21321595
Other	Experimental datasets	Zenodo	10.5281/zenodo.21321592
Other	Evo 200 liquid-handling platform	Tecan		Automated CFU plating
Other	STX100 incubator	Liconic		Automated incubator
Other	Infinite F200 plate reader	Tecan		Luminescence reads
Other	Pickolo camera	SciRobotics		Colony imaging
Other	Eclipse Ti2 microscope + DS-Qi2 camera	Nikon		Single-cell imaging

Appendix 1—table 1

Drugs used in this study, their minimum inhibitory concentrations (MICs), working concentrations, and stock solvents.

In the MIC column, we report the highest concentration of the dilution series (numerator) and the maximum inhibiting dilution (denominator). Kanamycin (50 μg/mL) was used as the selection marker for the lux operon.

Drug	MIC[μg/mL]	cwork 10 μg/mL	cwork [MIC]	Solvent	Supplier
Amoxicillin	$\frac{10}{4} = 2.50$	25	10	DMSO	Sigma, A8523
Ampicillin	$\frac{100}{128} = 0.78$	10, 2	12.8, 2.56	Water	Sigma, A9518
Cefepime	$\frac{4}{256} = 0.02$	0.15	9.6	DMSO	Thermo Fisher, J66237
Ceftazidime	$\frac{4}{64} = 0.06$	0.62	10	DMSO	Sigma, PHR1847
Cefuroxime	$\frac{4}{2} = 2.00$	16	8	Water	Sigma, C4417
Chloramphenicol	$\frac{128}{64} = 2.00$	20	10	DMSO	Sigma, C0378
Ciprofloxacin	$\frac{1}{128} = 0.01$	0.08	10	Water	Sigma, 17850
Colistin	$\frac{50}{64} = 0.78$	1.6	2.05	Water	Sigma, C4461
Doripenem	$\frac{4}{256} = 0.02$	0.16	10.24	Water	VWR, ACRO463870010
Fosfomycin	$\frac{4}{4} = 1.00$	8	8	Water	VWR, APOSBIM0107
Imipenem	$\frac{5}{64} = 0.08$	1	12.8	Water	Sigma, PHR1796
Mecilinam	$\frac{10}{128} = 0.08$	0.78	10	DMSO	Sigma, 33447
Meropenem	$\frac{5}{512} = 0.01$	0.1	10.24	Water	Sigma, PHR1772
Penicillin	$\frac{500}{32} = 15.62$	100	6.4	Water	Roth, HP48.2
Pexiganan	$\frac{64}{32} = 2.00$	8, 16	4, 8	Water	Sigma, SML3787
Piperacillin	$\frac{10}{16} = 0.62$	6.25	10	DMSO	Sigma, J66419
Polymyxin B	$\frac{50}{32} = 1.56$	2.5	1.6	Water	Roth, 0235.1
Rifampicin	$\frac{40}{16} = 2.50$	25	10	DMSO	Sigma, R3501
Tetracycline	$\frac{10}{32} = 0.31$	3.12	10	DMSO	Sigma, T3383
Trimethoprim	$\frac{10}{128} = 0.08$	0.78	10	DMSO	Sigma, T7883

Appendix 1—table 2

Point estimates and 95% percentile intervals of $ψ_{C F U}$ , $ψ_{I}$ , $ψ_{I}^{*}$ , and $ψ_{J}$ for different treatments.

Sig_x indicates whether the rate of change of signal $X \in {I, I^{*}, J}$ differs significantly (*) from the distribution of $ψ_{C F U}$ , or not (n.s.), based on the significance criterion defined in the Materials and methods section. Estimates are based on data from the colony-forming unit (CFU)−luminescence assays (see Materials and methods).

	$ψ_{C F U} [\frac{1}{h}]$	$ψ_{I} [\frac{1}{h}]$	sig_I	$ψ_{I}^{*} [\frac{1}{h}]$	sig_I*	$ψ_{J} [\frac{1}{h}]$	sig_J
Ampicillin 10 μg/mL	−3.23 (−4.11, −2.54)	−2.41 (−2.91, −2.11)	*			−2.63 (−3.31, −2.29)	n.s.
Ampicillin 2 μg/mL	−0.81 (−1.19, −0.37)	−0.30 (−0.41, −0.22)	*
Amoxicillin 25 μg/mL	−2.15 (−2.42, −1.88)	−2.04 (−2.21, −1.86)	n.s.
Cefepime 0.15 μg/mL	−1.17 (−1.50, −0.85)	−0.47 (−0.74, −0.24)	*	−0.88 (−1.13, −0.65)	*
Ceftazidime 0.62 μg/mL	−0.88 (−1.06, −0.71)	−0.09 (−0.24, 0.04)	*			−0.38 (−0.52, −0.15)	*
Cefuroxime 16 μg/mL	−1.59 (−2.41, −0.99)	−1.41 (−1.64, −1.25)	n.s.	−1.62 (−1.92, −1.34)	n.s.
Rifampicin 25 μg/mL	0.05 (−0.11, 0.20)	0.04 (−0.03, 0.10)	n.s.
Ciprofloxacin 0.078 μg/mL	−1.96 (−2.29, −1.64)	0.83 (0.44, 1.17)	*			0.48 (0.09, 0.90)	*
Colistin 1.6 μg/mL	−1.00 (−1.45, −0.52)	−0.93 (−1.26, −0.66)	n.s.
Doripenem 0.16 μg/mL	−0.54 (−0.83, −0.25)	−0.24 (−0.37, −0.15)	*	−0.37 (−0.53, −0.26)	*
Fosfomycin 8 μg/mL	−1.22 (−1.65, −0.83)	−1.12 (−1.37, −0.93)	n.s.
Imipenem 1 μg/mL	−1.42 (−2.06, −0.72)	0.00 (−0.16, 0.14)	*	−0.32 (−0.45, −0.22)	*
Mecillinam 0.78 μg/mL	−0.32 (−0.57, −0.10)	0.02 (−0.17, 0.17)	*	−0.34 (−0.47, −0.20)	n.s.
Meropenem 0.1 μg/mL	−1.85 (−2.36, −1.32)	−0.16 (−0.47, 0.10)	*			−0.55 (−0.80, −0.21)	*
Penicillin 100 μg/mL	−2.17 (−2.78, −1.66)	−2.20 (−2.53, −1.94)	n.s.
Rifampicin 25 μg/mL	−0.26 (−0.51, 0.02)	0.09 (−0.01, 0.19)	*
Polymyxin B 2.5 μg/mL	−1.45 (−1.73, −1.22)	−1.41 (−1.87, −1.07)	n.s.
Rifampicin 25 μg/mL	−0.23 (−0.40,0.04)	−0.15 (−0.23,−0.07)	n.s.
Tetracycline 3.125 μg/mL	−0.06 (−0.12, 0.00)	−0.01 (−0.07, 0.05)	n.s.
Trimethoprim 0.78 μg/mL	−0.61 (−0.76, −0.47)	0.48 (0.38, 0.59)	*			0.34 (0.18, 0.51)	*
Pexiganan 8 μg/mL	−44.59 (−66.19, −20.08)	−45.95 (−48.21, −43.34)	n.s.
Pexiganan 16 μg/mL	−61.63 (−93.40,1.26)	−60.27 (−63.06, −56.42)	n.s.

Appendix 1—table 3

Bootstrapped 95% confidence intervals and point estimates for the length, width, and volume of cells after 2 hr of treatment, estimated from microscopy images.

Significance was assessed by comparing the confidence intervals of cell volumes for each antibiotic treatment to the untreated control (control_2 hr), as described in the Materials and methods section of the main paper.

Treatment	Length (μm) mean (95% CI)	Width (μm) mean (95% CI)	Volume (μm³) mean (95% CI)	V-sig.
Control_2h	3.41 (2.28, 4.92)	1.09 (0.77, 1.40)	3.89 (2.00, 6.51)	Ref.
Amoxicillin	5.09 (2.64, 10.77)	1.17 (0.47, 1.37)	6.21 (1.93, 9.67)	n.s.
Ampicillin	12.36 (3.87, 27.14)	1.27 (0.93, 1.67)	16.67 (5.70, 30.39)	Significant
Ceftazidime	54.54 (32.05, 68.69)	0.95 (0.67, 1.22)	40.42 (11.52, 80.86)	Significant
Ciprofloxacin	21.77 (10.88, 36.82)	1.03 (0.70, 1.27)	19.55 (6.35, 45.70)	Significant
Colistin	3.14 (2.19, 4.53)	1.07 (0.60, 1.59)	3.71 (1.00, 8.32)	n.s.
Fosfomycin	3.18 (1.77, 5.32)	0.93 (0.69, 1.15)	2.65 (1.09, 4.75)	n.s.
Meropenem	5.90 (3.44, 10.61)	2.26 (1.13, 3.71)	31.11 (5.33, 71.01)	Significant
Rifampicin	4.77 (2.41, 8.38)	0.98 (0.59, 1.40)	4.32 (1.15, 9.44)	n.s.
Tetracycline	4.71 (2.43, 7.70)	1.08 (0.65, 1.71)	5.25 (1.29, 13.40)	n.s.
Trimethoprim	12.19 (4.67, 30.74)	0.93 (0.65, 1.29)	9.05 (2.64, 19.79)	Significant

Appendix 1—table 4

Estimated group means and 95% confidence intervals from an ordinary least squares (OLS) model fitted to log-transformed colony-forming unit (CFU) data, collected from the first sampled time point after diluting pexiganan-treated strains in supplemented phosphate-buffered saline (PBS).

Grouping is based on the supplement (CaCl₂ or MgCl₂) and concentration (0 mM to 100 mM). Confidence intervals were computed using heteroscedasticity-consistent standard errors (HC3). The compact letter display (cld) indicates groups that are not significantly different by sharing a common letter, based on mutual inclusion of their 95% confidence intervals.

Group	Mean	Low	Up	cld
CaCl₂(0 mM)	−0.00	−0.00	0.00	a
CaCl₂ (1 mM)	2.77	−0.56	6.09	b
CaCl₂ (10 mM)	5.06	4.92	5.20	c
CaCl₂ (100 mM)	5.85	5.38	6.31	d
MgCl₂ (0 mM)	−0.00	−0.00	0.00	a
MgCl₂ (1 mM)	2.77	−0.56	6.09	b
MgCl₂ (10 mM)	5.31	5.20	5.42	e
MgCl₂ (100 mM)	6.36	6.27	6.46	f

Appendix 1—table 5

Comparison of kill rates [ $h^{- 1}$ ] between cultures treated with pexiganan (16 μg/mL) for 5 min and cultures exposed to the supernatant collected after the kill assay.

The two rates differ significantly; the confidence interval of the rate of change of colony-forming unit (CFU) in the supernatant includes zero.

Experiment	Mean	Low	Up
Pexiganan	−46.98	−63.15	−35.80
Supernatant	1.28	−2.19	5.11

Appendix 1—table 6

Primer sequences used for $λ$ -Red-mediated integration of the luxCDABE operon into E. coli.

Lowercase letters indicate homology regions binding to the lux operon on the plasmid; uppercase letters indicate chromosomal homology regions at the integration site.

Primer	Sequence (5’ → 3’)
Forward	CGGTACGGCTGACCATCGGGTGCCAGTGCGGGAGTTTCGTacccagtaaggcagcggtatc
Reverse	AGTCAGCGATATCCATTTTCGCGAATCCGGAGTGTAAGAAtaggtctagggcggcgga

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Malte Muetter
Daniel C Angst
Roland Regoes
Sebastian Bonhoeffer

(2026)

High-throughput quantification of population dynamics using luminescence

eLife 15:RP109213.

https://doi.org/10.7554/eLife.109213.3

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Comparison of colony-forming unit (CFU)-based and luminescence-based rates of change.

Ampicillin.

Cefepime.

Ceftazidime.

Cefuroxime.

Chloramphenicol.

Ciprofloxacin.

Colistin.

Doripenem.

Fosfomycin.

Imipenem.

Mecillinam.

Meropenem.

Penicillin.

Piperacillin.

Polymyxin B.

Rifampicin (25μg/mL).

Tetracycline.

Trimethoprim.

Comparison of colony-forming unit (CFU) and luminescence kill curve signals for pexiganan at (a) 8 μg/mL and (b) 16 μg/mL.

Density distributions (2.5–97.5% percentile range) of pooled cell volumes acquired by microscopy imaging, shown (a) before and (b) after 2 hr of antibiotic treatment.

Pooled density distributions (95% prediction intervals) of cell widths (red) and lengths (blue), obtained from microscopy images (a) before and (b) after 2 hr of antibiotic treatment.

Microscopy images of control (before treatment) samples.

Microscopy images of control (after 2 hr) samples.

Microscopy images of cells treated with ampicillin.

Microscopy images of cells treated with amoxicillin.

Microscopy images of cells treated with ceftazidime.

Microscopy images of cells treated with ciprofloxacin.

Microscopy images of cells treated with colistin.

Microscopy images of cells treated with fosfomycin.

Microscopy images of cells treated with meropenem.

Microscopy images of cells treated with pexiganan.

Microscopy images of cells treated with rifampicin.

Microscopy images of cells treated with tetracycline.

Microscopy images of cells treated with trimethoprim.

Light intensity scales linearly with bacterial density.

Panel plot showing the effects of UV treatment on bacterial density (approximated by optical density [OD]) and light intensity (I) over time by comparing treated (UV) and untreated (ctrl) cultures.

Example of the probability of colony formation pC=1−pE (see Appendix 1) for a single plated bacterium.

Colony-forming unit (CFU) measured over time in supplemented dilution media.

Colony-forming unit (CFU) time-kill curves for pexiganan, performed manually, comparing dilution in (a) unsupplemented phosphate-buffered saline (PBS) and (b) PBS supplemented with 100 mM MgCl2.

Ampicillin kill curve versus supernatant kill curve.

Illustrative simulations using the filamentation model relating (a–d) bacterial population size (blue) and light intensity (orange) under different combinations of treatment-induced changes in division rate (Δλ) and death rate (δ).

Drugs used in this study, their minimum inhibitory concentrations (MICs), working concentrations, and stock solvents.

Point estimates and 95% percentile intervals of ψCFU, ψI, ψI∗, and ψJ for different treatments.

Bootstrapped 95% confidence intervals and point estimates for the length, width, and volume of cells after 2 hr of treatment, estimated from microscopy images.

Estimated group means and 95% confidence intervals from an ordinary least squares (OLS) model fitted to log-transformed colony-forming unit (CFU) data, collected from the first sampled time point after diluting pexiganan-treated strains in supplemented phosphate-buffered saline (PBS).

Comparison of kill rates [h−1] between cultures treated with pexiganan (16 μg/mL) for 5 min and cultures exposed to the supernatant collected after the kill assay.

Primer sequences used for λ-Red-mediated integration of the luxCDABE operon into E. coli.

MDAR checklist

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Example of the probability of colony formation $p_{C} = 1 - p_{E}$ (see Appendix 1) for a single plated bacterium.

Colony-forming unit (CFU) time-kill curves for pexiganan, performed manually, comparing dilution in (a) unsupplemented phosphate-buffered saline (PBS) and (b) PBS supplemented with 100 mM MgCl₂.

Illustrative simulations using the filamentation model relating (a–d) bacterial population size (blue) and light intensity (orange) under different combinations of treatment-induced changes in division rate ( $Δ λ$ ) and death rate ( $δ$ ).

Point estimates and 95% percentile intervals of $ψ_{C F U}$ , $ψ_{I}$ , $ψ_{I}^{*}$ , and $ψ_{J}$ for different treatments.

Comparison of kill rates [ $h^{- 1}$ ] between cultures treated with pexiganan (16 μg/mL) for 5 min and cultures exposed to the supernatant collected after the kill assay.

Primer sequences used for $λ$ -Red-mediated integration of the luxCDABE operon into E. coli.