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. 2021 Nov 17;65(12):e0146821.
doi: 10.1128/AAC.01468-21. Epub 2021 Sep 27.

Uropathogenic Escherichia coli Shows Antibiotic Tolerance and Growth Heterogeneity in an In Vitro Model of Intracellular Infection

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Uropathogenic Escherichia coli Shows Antibiotic Tolerance and Growth Heterogeneity in an In Vitro Model of Intracellular Infection

Ivana Kerkez et al. Antimicrob Agents Chemother. .

Abstract

Uropathogenic Escherichia coli (UPEC), the major causative agent of urinary tract infections, can invade different types of host cells. To compare the pharmacodynamic properties of antibiotics against intra- and extracellular UPEC, an in vitro model of intracellular infection was established in J774 mouse macrophages infected by the UPEC strain CFT073. We tested antibiotics commonly prescribed against urinary tract infections (gentamicin, ampicillin, nitrofurantoin, trimethoprim, sulfamethoxazole, and ciprofloxacin) and the investigational fluoroquinolone finafloxacin. The metabolic activity of individual bacteria was assessed by expressing the fluorescent reporter protein TIMERbac within CFT073. Concentration-response experiments revealed that all tested antibiotics were much less effective against intracellular bacteria than extracellular ones. Most antibiotics, except fluoroquinolones, were unable to reach a bactericidal effect intracellularly at clinically achievable concentrations. Ciprofloxacin and finafloxacin killed 99.9% of extracellular bacteria at concentrations around the MIC, while for intracellular bacteria, concentrations more than ×ばつ over the MIC were required to achieve a bactericidal effect. Time-kill curves showed that finafloxacin was more rapidly bactericidal in acidic medium than at neutral pH, while the reverse observation was made for ciprofloxacin. Intracellularly, kill curves showed biphasic kinetics for both fluoroquinolones, suggesting the presence of drug-tolerant subpopulations. Flow cytometry analysis of TIMERbac fluorescence revealed a marked heterogeneity in intracellular growth of individual bacteria, suggesting that the presence of subpopulations reaching a state of metabolic dormancy was the main reason for increased antibiotic tolerance of intracellular UPEC.

Keywords: TIMERbac; UPEC; UTI; flow cytometry; fluorescent reporter; fluoroquinolones; intracellular infection; macrophages; persisters; pharmacodynamics.

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Figures

FIG 1
FIG 1
J774 macrophage infection model for pharmacodynamic evaluation of antibiotics against intracellular E. coli CFT073. (A) Schematic representation of infection protocol. Gentamicin (Gen) treatment (100 mg/liter) was used to remove of the extracellular bacteria. (B) Number of intracellular bacteria at time zero for different infection loads indicated as multiplicity of infection (MOI). Results are mean values ± standard deviations from at least 3 experiments (each experiment was done in triplicate). (C) Representative flow cytometry analysis for one experiment of red and green fluorescence in J774 macrophages after infection by CFT073 expressing TIMERbac and elimination of extracellular gentamicin (time zero in panel A). Overlay of control cells (noninfected; blue color) and macrophages that were exposed to bacteria (time zero in panel A; red color) gives two distinct subpopulations; one with high red fluorescence (infected cells) and another that overlaps with the control (noninfected cells).
FIG 2
FIG 2
Fluorescent microscopy and flow cytometry analyses on bacterial growth and activity with TIMERbac reporter-protein. (A) Confocal microscopy image of infected macrophages at time zero with overlay of three pseudocolored channels. Blue color (Ex, 458 nm; Em, 474 to 554 nm) indicates signal from Lyso Sensor dye DND-189; green (Ex, 488 nm; Em, 493 to 533nm) and red color (Ex, 561 nm; Em, 566 to 616 nm) indicate signal from reporter protein TIMERbac. Intracellular bacteria are indicated with white arrows. (B) Fluorescent image is overlaid with transmitted light image from 488-nm laser, and the dotted line indicates the section from which quantitative fluorescence intensities are measured and presented in panel C. (C) Bacteria expressing TIMERbac protein (green and red fluorescence) are localized within acidic intracellular compartments (blue fluorescence). (D) Three bacterial subpopulations Q1, Q2, and Q3 detected by FACS by analyzing intracellular (IC) and extracellular (EC) control bacteria. Abundances of respective bacterial subpopulations (shown in percentages) are presented in panel E. Results are averages ± standard deviations from three experiments with at least two parallels.
FIG 3
FIG 3
Antibiotic concentration-response curves for planktonic (extracellular) versus internalized (intracellular) bacteria after 24 h of incubation. (A) The effect of three different gentamicin concentrations on extracellular contamination (bacterial CFU/ml, red triangles); total number of macrophages per ml of DMEM after trypsinization (black dots) and LDH release (gray bars). All three parameters were acquired from the same well. (B to H) Activity of antibiotics against extracellular (open symbols, black line) and intracellular bacteria (closed symbols, red solid line; apparent intracellular bacteria counts at sub-MIC concentrations are presented with a dotted red line) were expressed in changes log10 CFU units from time zero to 24 h, and the concentrations tested (mg/liter) are presented in log10 scale. The first vertical black-dotted line corresponds to the MIC of the antibiotic as measured in DMEM at pH 7.4, and the second vertical blue-dotted line corresponds to the Cmax of the drug in patient urine. Results are mean values ± standard errors of the means (SEM) from at least three experiments (each experiment was done in triplicate). The horizontal dotted lines correspond to the initial inoculum and to the limit of detection, respectively.
FIG 4
FIG 4
Effect of pH on ciprofloxacin and finafloxacin pharmacodynamics. (A and B) Concentration-response curves for extracellular bacteria in media at pH 7.4 or 5.5 or intracellularly exposed during 24 h to ciprofloxacin or finafloxacin at the indicated concentrations (expressed as multiples of the MIC at pH 7.4). (C) Evaluation of the number of bacteria over time of incubation under control conditions or with fluoroquinolones at the indicated concentrations in media at pH 7.4 or 5.5. Results on the graphs are mean values ± SEM from at least three different experiments (each experiment performed in triplicate). CIP, ciprofloxacin; FIN, finafloxacin; Ctr, control condition without antibiotics.
FIG 5
FIG 5
Intracellular time-kill curves comparing ciprofloxacin and finafloxacin. (A) Time-dependent killing of intracellular bacteria (presented as log10 CFU per ml of J744 lysate from one well) by ciprofloxacin and finafloxacin at an extracellular concentration of 150 mg/liter or in the presence of gentamicin (at ×ばつ MIC to avoid extracellular contamination). (B to D) In parallel with CFU determination, the same samples were analyzed by flow cytometry (FC) for TIMERbac fluorescence in the green and red channels as described in Fig. 2. Results on graphs are mean values ± standard deviations from at least three different experiments (each experiment performed in triplicate).

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