Emergence of antibiotic resistance from multinucleated bacterial filaments

Abstract

Bacteria can rapidly evolve resistance to antibiotics via the SOS response, a state of high-activity DNA repair and mutagenesis. We explore here the first steps of this evolution in the bacterium Escherichia coli. Induction of the SOS response by the genotoxic antibiotic ciprofloxacin changes the E. coli rod shape into multichromosome-containing filaments. We show that at subminimal inhibitory concentrations of ciprofloxacin the bacterial filament divides asymmetrically repeatedly at the tip. Chromosome-containing buds are made that, if resistant, propagate nonfilamenting progeny with enhanced resistance to ciprofloxacin as the parent filament dies. We propose that the multinucleated filament creates an environmental niche where evolution can proceed via generation of improved mutant chromosomes due to the mutagenic SOS response and possible recombination of the new alleles between chromosomes. Our data provide a better understanding of the processes underlying the origin of resistance at the single-cell level and suggest an analogous role to the eukaryotic aneuploidy condition in cancer.

Keywords: SOS response; antibiotic resistance; evolution; filamentation; mutation.

Fig. 1.

(A) E. coli bacteria form filaments in microhabitats after 3 h of exposure to a ciprofloxacin gradient. (B) Cipro-resistant bacteria that have reverted to a normal-length phenotype (arrows) in one of the microhabitats.

Fig. 2.

(A) Semilog plot of the contour length of a collection of filaments (n = 20) growing on agar containing 0.125× MIC of cipro vs. time. (B) Example of a budding process indicated by the white arrows at either end of a filament. Yellow arrows show budded cells resuming normal division. (Scale bar, 5 μm.) (C) Distribution of budding positions along the filaments (n = 95 cells, average length 42 ± 16 μm, average time at division 332 ± 71 min).

Fig. 3.

(A) Distribution of the different fates of the buds: normal symmetrical division (red), asymmetrical division (yellow), no division and no growth (black), and filamentation (gray). (B) Death of long filaments after budding, imaged after 5 h (Top) and 22 h (Bottom) of low cipro exposure using the membrane-impermeable dye propidium iodide. Bright-field (BF), GFP, and RFP (PI) images are shown. (Scale bar, 2 μm.) (C) Microscopic images of filaments and buds expressing sulA-gfp (Top) and constitutively expressed rfp (Bottom) in the same cells after 4 h of growth in low cipro. (Scale bar, 2 μm.) (D) Histogram of average sulA-gfp expression within filaments (n = 98 cells). The red line represents a GFP mean intensity of 528 ± 122 of the population. (E) Histogram of average sulA-gfp expression within buds (n = 110 cells). The red line represents a GFP mean intensity of 393 ± 107 of the population.

Fig. 4.

(A) Chromosome count as a function of length as measured by DAPI stain (red crosses) and R2-CFP loci (blue crosses). (B) Distribution of individual chromosome (R2-CFP) positions along the filaments after 4 h of growth with 0.125× MIC cipro (n = 50 cells, mean R2 locus counts per cell 4.5 ± 1.9, average filament length 20 ± 4 μm). (C) Time-lapse images of R2-CFP showing complex movement of individual chromosomes along the filament length. (Scale bar, 1 μm.) Each red circle surrounding an R2 locus denotes an individual chromosome and results from automated tracking analysis software (Materials and Methods). White arrowheads indicate events of merging/splitting between two chromosomes. The yellow arrowhead indicates a chromosome duplication/separation event. Dashed lines show the cell contours. (D) Distribution of R2-CFP locus positions in individual cells shown in C. Traces were obtained by using a MATLAB-based particle tracking methodology; automated (filled circle) or manual tracking (open circles). (Materials and Methods). The black arrow indicates a replication/separation event near the tip. Gray arrows show merging and splitting events between two chromosomes.

Fig. 5.

Resistance of the wild-type strain, a QZ030 hyperresistant isolate (1), and three different progeny clones to cipro as measured by growth curves. The cipro concentration at which growth was monitored is indicated.

Fig. 6.

Basic model for the initial stages of resistance in filamentous cells. Light gray cells are initial sensitive cells, dark gray cells have double-strand breaks and stalled replication forks, green cells have evolved moderate antibiotic resistance, and red cells are undergoing death. Chromosomes are shown within the cells.