Epigenetic inheritance based evolution of antibiotic resistance in bacteria

Abstract

Background: The evolution of antibiotic resistance in bacteria is a topic of major medical importance. Evolution is the result of natural selection acting on variant phenotypes. Both the rigid base sequence of DNA and the more plastic expression patterns of the genes present define phenotype.

Results: We investigated the evolution of resistant E. coli when exposed to low concentrations of antibiotic. We show that within an isogenic population there are heritable variations in gene expression patterns, providing phenotypic diversity for antibiotic selection to act on. We studied resistance to three different antibiotics, ampicillin, tetracycline and nalidixic acid, which act by inhibiting cell wall synthesis, protein synthesis and DNA synthesis, respectively. In each case survival rates were too high to be accounted for by spontaneous DNA mutation. In addition, resistance levels could be ramped higher by successive exposures to increasing antibiotic concentrations. Furthermore, reversion rates to antibiotic sensitivity were extremely high, generally over 50%, consistent with an epigenetic inheritance mode of resistance. The gene expression patterns of the antibiotic resistant E. coli were characterized with microarrays. Candidate genes, whose altered expression might confer survival, were tested by driving constitutive overexpression and determining antibiotic resistance. Three categories of resistance genes were identified. The endogenous beta-lactamase gene represented a cryptic gene, normally inactive, but when by chance expressed capable of providing potent ampicillin resistance. The glutamate decarboxylase gene, in contrast, is normally expressed, but when overexpressed has the incidental capacity to give an increase in ampicillin resistance. And the DAM methylase gene is capable of regulating the expression of other genes, including multidrug efflux pumps.

Conclusion: In this report we describe the evolution of antibiotic resistance in bacteria mediated by the epigenetic inheritance of variant gene expression patterns. This provides proof in principle that epigenetic inheritance, as well as DNA mutation, can drive evolution.

Figure 1

E. coli ampicillin survival curves. All E. coli are isogenic, recently derived from a single colony. Triangles show survival rates for cells previously grown in the absence of ampicillin. Resistance rates were ramped higher by prior exposure to ampicillin. Cells surviving selection of 1 μg/ml ampicillin (circles) showed improved survival on both 1 μg/ml and 2.5 μg/ml ampicillin. Cells surviving 2.5 μg/ml ampicillin (squares), showed even more dramatic improvement, with an approximately three log increase in survival on 2.5 μg/ml ampicillin. Nevertheless, reversion rates were extremely high, with for example over 95% of cells from 2.5 μg/ml ampicillin not surviving dispersion and immediate re-plating on another LB agar plate with the same antibiotic concentration, 2.5 μg/ml ampicillin. Survival rates for previously unexposed E. coli at ampicillin concentrations of 5 μg/ml and higher were extremely low, below 1/10⁶.

Figure 2

Heat map showing changing gene expression patterns in ampicillin resistant E. coli. Each column represents one population grown in the absence of amp (C), or in the concentration of amp shown at the bottom. Each row shows the expression level across populations for one gene, with blue for low, red for high and yellow for intermediate expression. The endogenous β-lactamase gene, AmpC, shows elevated expression in most, but not all, ampicillin resistant populations.

Figure 3

Expression levels of endogenous β-lactamase gene in E. coli surviving selection at different ampicillin concentrations. Transcript abundances were determined using a total of 29 Affymetrix E. coli 2.0 oligonucleotide microarrays. Expression levels are in arbitrary Affymetrix expression units. Survivors at higher ampicillin concentrations show significantly increased β-lactamase expression.

Figure 4

Elevated expression of GadA and GadB genes in ampicillin resistant populations. Both genes encoding the two glutamate decarboxylase isozymes show significantly elevated expression in ampicillin resistant E. coli populations.

Figure 5

Structures of glutamate and ampicillin. Similarities between the lactam ring and the peptide backbone have been proposed to be responsible for the antibiotic function of ampicillin.

Figure 6

E. coli nalidixic acid survival curves. As observed for ampicillin, the survival rates at low nalidixic acid concentrations are too high to be accounted for by spontaneous mutation. Cells surviving prior antibiotic exposure show dramatically improved resistance rates, yet reversion to antibiotic sensitivity is very common, with over 95% of cells from 40 μg/ml nalidixic acid not surviving re-exposure to the same antibiotic concentration. No previous nalidixic acid exposure, triangles. From 40 μg/ml nalidixic acid, circles.

Figure 7

E. coli tetracycline survival curves. Survival ratios for E. coli not previously exposed to tetracycline (triangles), and for E. coli previously surviving exposure to 2 μg/ml tetracycline (circles).

Figure 8

Model of evolution of antibiotic resistance through epigenetic inheritance. Within an isogenic population of E. coli there is random variation in expression levels of genes. Antibiotic exposure (horizontal arrows) selects cells with gene expression patterns that allow survival. For example, elevated expression of the GadA and β-lactamase genes promote ampicillin survival. A combination of continued selection, epigenetic inheritance and stochastic variation can evolve populations with gene expression patterns providing increasing antibiotic resistance.