Bacterial charity work leads to population-wide resistance

Abstract

Bacteria show remarkable adaptability in the face of antibiotic therapeutics. Resistance alleles in drug target-specific sites and general stress responses have been identified in individual end-point isolates. Less is known, however, about the population dynamics during the development of antibiotic-resistant strains. Here we follow a continuous culture of Escherichia coli facing increasing levels of antibiotic and show that the vast majority of isolates are less resistant than the population as a whole. We find that the few highly resistant mutants improve the survival of the population's less resistant constituents, in part by producing indole, a signalling molecule generated by actively growing, unstressed cells. We show, through transcriptional profiling, that indole serves to turn on drug efflux pumps and oxidative-stress protective mechanisms. The indole production comes at a fitness cost to the highly resistant isolates, and whole-genome sequencing reveals that this bacterial altruism is made possible by drug-resistance mutations unrelated to indole production. This work establishes a population-based resistance mechanism constituting a form of kin selection whereby a small number of resistant mutants can, at some cost to themselves, provide protection to other, more vulnerable, cells, enhancing the survival capacity of the overall population in stressful environments.

Figure 1. Tracking a population of Escherichia coli developing antibiotic resistance

(a)A clonal wildtype Escherichia coli MG1655 population was continuously cultured in a bioreactor for 10 days with increasing concentrations of the quinolone, norfloxacin. MIC is defined as the drug concentration inhibiting no more than 60% of unstressed cell growth. The initial bioreactor concentration was set as the MIC of wildtype cells. Every 24 hours thereafter, the population MIC was measured, red lines. Following increases in group MIC, the bioreactor concentration, dashed green lines, was adjusted at the next sampling interval accordingly. Twelve individual isolates were selected from plating daily populations on non-selective plates and their MICs determined, grey bars. MICs shown are representative of biological duplicates. (b) Daily population analysis profiles, representing the fraction of the population resistant to each drug level, were taken throughout the 10 days of continuous culture. Daily populations were serially diluted and spotted on plates with a range of norfloxacin concentrations. Percent resistance, circles colored according to norfloxacin concentration, was calculated as the number of colonies at specific norfloxacin concentrations relative to the total number of cells (plated on non-restrictive plates). Results shown are representative of biological duplicates.

Figure 2. Indole production by isolates and the protective effect of extracellular indole

(a) Proteins were detected in the supernatant of c10,12 when grown clonally under the bioreactor concentration of norfloxacin (1500ng/mL). These protein bands were subjected to mass spectrometry for protein identification. The top hit for the dominant protein band matched over 75% of residues for tnaA, which encodes the enzyme tryptophanase. The major enzymatic activity of tryptophanase yields indole. This dominant band was absent from the supernatant of c10,12ΔtnaA. No proteins were found in the supernatant of c10,6. (b) HPLC quantification of extracellular indole production by isolates with varying norfloxacin resistance: wildtype, white bars; c10,6, striped bars; c10,12, green bars; and c10,12ΔtnaA, not detected. With the exception of c10,12ΔtnaA, all isolates produce approximately 300µM indole in the absence of antibiotic stress. Under norfloxacin stress (1500ng/mL), c10,12 continued to produce up to 300µM of indole while wildtype and c10,6 produced <50µM of indole. No indole was detected for c10,12ΔtnaA. Results are means ± s.e.m (n≥3). (c) MBC, defined as the minimum concentration of norfloxacin that kills 99.9% of the cells in a culture, is shown for c10,6 with and without the addition of 300µM of indole. The bioreactor concentration for day 9 (1000ng/mL) and for day 10 (1500ng/mL) is also shown. (d) Total growth of mutants under norfloxacin stress (1500ng/mL) in isolation or in co-culture: c10,6, striped bars; c10,12, green bars; and c10,12ΔtnaA, grey bars. Each condition starts with the same total number of cells and co-cultures are mixed in a highly resistant isolate to less resistant isolate ratio of 1:100. Results shown are representative of biological replicates and expressed as means ± s.e.m.

Figure 3. >Whole-genome sequencing of various mutants

(a) Five total genomes were sequenced using the Solexa GA2: wildtype, three highly resistant isolates from days 8 through 10, and a less resistant isolate from day 10. Sequencing coverage for each isolate is plotted, according to color, on concentric tracks with the wildtype genome, orange, in the center. Intervals within each track represent 25x coverage per 1000 bases of the genome. Each SNP, represented by circles colored according to isolate, is marked at the appropriate genomic position on the genome(s) in which it was found. (b) Allelic frequency of each SNP over the course of the 10-day evolution experiment was estimated, using Sequenom’s iPLEX platform, in the total population, black circles, and in an enriched highly resistant population by norfloxacin selection, green triangles.

Figure 4. A population-based antibiotic resistance mechanism

A bacterial population is diagrammed. (a) In the absence of antibiotic stress, wild-type cells naturally produce indole. (b) Under antibiotic stress, wild-type cells stop producing indole and eventually die. (c) When a drug resistant mutant emerges, it is able to produce indole even under antibiotic stress. This indole allows the more vulnerable cells in the population to survive the antibiotic stress by inducing various antibiotic tolerance mechanisms, thereby boosting the survival capacity of the population.