Phenotypic heterogeneity enables uropathogenic Escherichia coli to evade killing by antibiotics and serum complement

Abstract

Uropathogenic strains of Escherichia coli (UPEC) are the major cause of bacteremic urinary tract infections. Survival in the bloodstream is associated with different mechanisms that help to resist serum complement-mediated killing. While the phenotypic heterogeneity of bacteria has been shown to influence antibiotic tolerance, the possibility that it makes cells refractory to killing by the immune system has not been experimentally tested. In the present study we sought to determine whether the heterogeneity of bacterial cultures is relevant to bacterial targeting by the serum complement system. We monitored cell divisions in the UPEC strain CFT073 with fluorescent reporter protein. Stationary-phase cells were incubated in active or heat-inactivated human serum in the presence or absence of different antibiotics (ampicillin, norfloxacin, and amikacin), and cell division and complement protein C3 binding were measured by flow cytometry and immunofluorescence microscopy. Heterogeneity in the doubling times of CFT073 cells in serum enabled three phenotypically different subpopulations to be distinguished, all of them being recognized by the C3 component of the complement system. The population of rapidly growing cells resists serum complement-mediated lysis. The dominant subpopulation of cells with intermediate growth rate is susceptible to serum. The third population, which does not resume growth upon dilution from stationary phase, is simultaneously protected from serum complement and antibiotics.

FIG 1

Serum killing coincides with the start of bacterial growth. E. coli CFT073 stationary-phase cells were diluted in either fresh LB medium, PBS, 50% HIS in 1× PBS, or 50% serum in 1× PBS and grown statically at 37°C without shaking. The CFU were determined at the indicated time points. The averages and standard deviations of at least three independent experiments are presented.

FIG 2

Subpopulation of cells surviving antibiotics and complement killing. Cells were grown to stationary phase in LB medium and diluted in fresh LB medium or PBS supplemented or not with HIS or serum (final concentration, 50%). Cells were incubated in the presence of ampicillin (A), norfloxacin (B), or amikacin (C). The number of surviving cells (CFU/ml) was determined after 150 min by plating. The averages and standard deviations of at least three independent experiments are presented.

FIG 3

A bacterial population that survives serum treatment is enriched in rapidly dividing and nondividing cells. Stationary-phase CFT073 cells [carrying plasmid pETgfp-mut2AGGAGG(3)] were diluted in PBS, LB medium, 50% HIS, or 50% serum. The cells were incubated at 37°C without shaking for 150 min, and their fluorescence was measured by using flow cytometry. Density blots of GFP fluorescence (GFP-H) and the SSC parameter (SSC-H) are shown for cells in PBS at zero time (A), in PBS after 150 min (B), in LB medium (C), in HIS (D), or in serum (E). Each dot represents the fluorescence of a single event (particle). (F) The distribution of the fluorescence level of events (single cells) analyzed by flow cytometry is presented as a histogram consisting of 376 repartition bins. The number of events with the respective GFP fluorescence levels in 1-ml cell cultures grown under particular conditions is shown. (G and H) Scanning electron micrographs illustrate the morphology of the E. coli cells after 150 min of incubation in 50% HIS (G) and in 50% serum (H). Arrows indicate the different bacterial cells during lysis.

FIG 4

Cell division profile of persister cells indicates survival of nondividing cells. Cells were grown to stationary phase in LB medium and diluted in 50% HIS (A and B) or 50% serum (C and D). Cells were incubated without antibiotics (no AB) and in the presence of ampicillin (Amp; 200 μg/ml), norfloxacin (Nor; 5 μg/ml), or amikacin (Ami; 25 μg/ml).The distributions of the fluorescence levels of events (single cells) analyzed by flow cytometry are presented as histograms consisting of 376 repartition bins. The numbers of events with the respective GFP fluorescence levels in 1-ml cell cultures from HIS (A) or serum (C) are shown. Differently colored histograms represent conditions as follows: filled gray, without antibiotics; yellow, ampicillin; green, norfloxacin; and dashed red, amikacin. The norfloxacin and amikacin lines mostly overlap. (B and D) After 150 min, the CFU were determined by plating, and the number GFP-positive events was measured by flow cytometry (FACS events).

FIG 5

Recognition and opsonization of CFT073 strain bacterial cells by serum complement. C3 deposition was measured by sequential C3 antibody staining and immunofluorescence microscopy. Cells were grown to stationary phase in LB medium and diluted in fresh medium (in PBS supplemented with either 50% HIS or 50% serum or in PBS alone) and incubated for 150 min at 37°C. (A) Immunofluorescence micrographs of GFP and C3-bound Alexa 568 fluorescence. Cells were grown in serum in the presence of ampicillin (200 μg/ml), norfloxacin (5 μg/ml), or amikacin (25 μg/ml), and C3 binding was detected by using immunofluorescence. From left to right is shown the GFP fluorescence inside the cells (visible GFP signal indicates intact cells) and the C3 deposition on cells (together with no GFP signal enables lysed cells to be identified); figure enlargements for GFP and C3 tile figures are also shown. Red dashed boxes show the exact locations of the figure enlargements on the tile figures. (B) Dot plot of C3-bound Alexa 568 and GFP fluorescence of cells incubated in HIS, PBS, or serum. Each dot represents the mean intensity (GFP and C3-bound Alexa 568) per cell measured. The dashed horizontal line indicates threshold value for cells with specifically bound C3. The dashed vertical line separates subpopulations of nondividing (high GFP levels) and dividing (low GFP levels) cells. (C) Relative distribution of cells by mean C3-bound Alexa 568 fluorescence. The objects examined were distinguished by the presence or absence of GFP. Objects with a mean GFP fluorescence higher than 0.01 (includes all cells shown in panel B) were considered live (GFP-positive cells). Objects with a GFP fluorescence ≤0.01 were considered lysed.

FIG 6

Exponentially growing cells are susceptible to serum. (A) Cells were grown to exponential phase in LB medium and diluted in 50% HIS or 50% serum. Cell division profiles of exponential-phase cells were determined at the start and 60 and 120 min after dilution. The relative number of events with particular GFP fluorescence levels in the same culture volume is shown. (B) Cells were grown to stationary (Stats) or exponential (Exp) phase in LB medium and diluted in 50% HIS or serum. To inhibit cell growth, chloramphenicol (Cam; 50 μg/ml) was added to one parallel sample. The CFU were counted after the indicated time by plating, and the percentage of surviving cells compared to the zero time point is shown. The averages and standard deviations of at least three independent experiments are presented.

FIG 7

Model of phenotypic heterogeneity of UPEC cells during infection. Phenotypic heterogeneity leads to the formation of three different subpopulations. Dormant nondividing cells are simultaneously protected from serum and antibiotics. The dominant subpopulation of cells with an intermediate growth rate is susceptible to antibiotics and serum. Rapidly dividing cells are susceptible to antibiotics but can resist serum complement-mediated lysis. Bacteria are depicted as dark gray ovals, and lighter gray ovals indicate dilution of the color by cell division.