Bacterial charity work leads to population-wide resistance

Journal name:
Nature
Volume:
467,
Pages:
82–85
Date published:
DOI:
doi:10.1038/nature09354
Received
Accepted

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 isolates1, 2, 3, 4, 5, 6, 7. 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 cells8. 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 selection9 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.

At a glance

Figures

  1. Tracking a population of E. coli developing antibiotic resistance.
    Figure 1: Tracking a population of E. coli developing antibiotic resistance.

    a, A clonal wild-type E. coli MG1655 population was continuously cultured in a bioreactor for ten 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 wild-type cells. Every 24h thereafter, the population MIC (red lines) was measured. Following increases in group MIC, the bioreactor concentration (dashed green lines) was adjusted accordingly at the next sampling interval. Twelve individual isolates were selected from plating daily populations on non-selective plates and their MICs (grey bars) were determined. 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 ten days of continuous culture. Daily populations were serially diluted and spotted on plates with a range of norfloxacin concentrations. The percentage resistance (circles coloured 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.

  2. Indole production by isolates and the protective effect of extracellular indole.
    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 (1,500ngml−1). 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, Quantification by high-pressure liquid chromatography of extracellular indole production by isolates with varying norfloxacin resistance: wild type (white bars); c10,6 (striped bars); c10,12 (green bars); and c10,12ΔtnaA (ND, not detected). With the exception of c10,12ΔtnaA, all isolates produce approximately 300μM of indole in the absence of antibiotic stress. Under norfloxacin stress (1,500ngml−1), c10,12 continued to produce up to 300μM of indole whereas wild type and c10,6 produced <50μM of indole. No indole was detected for c10,12ΔtnaA. Data shown, mean±s.e.m. (n3). c, MBC, 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 concentrations for day nine (1,000ngml−1) and for day ten (1,500ngml−1) are also shown. d, Total growth of mutants under norfloxacin stress (1,500ngml−1) 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 an HRI-to-LRI ratio of 1:100 (1 in 100). Results shown are representative of biological replicates and are expressed as mean±s.e.m.

  3. Whole-genome sequencing of various mutants.
    Figure 3: Whole-genome sequencing of various mutants.

    a, Five total genomes were sequenced using the Solexa GA2: wild type, three HRIs from days eight to ten, and an LRI from day ten. Sequencing coverage for each isolate is plotted, according to colour, on concentric tracks with the wild-type genome (orange) in the centre. Intervals within each track represent ×25 coverage per 1,000 bases (Mb) of the genome. Each SNP, represented by circles coloured according to isolate, is marked at the appropriate genomic position on the genome(s) in which it was found. PmdtK denotes the promoter of the gene mdtK. b, Allelic frequency of each SNP over the course of the ten-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).

  4. A population-based antibiotic-resistance mechanism.
    Figure 4: A population-based antibiotic-resistance mechanism.

    A bacterial population is shown. 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.

Accession codes

Primary accessions

Gene Expression Omnibus

References

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Affiliations

  1. Howard Hughes Medical Institute, Center for BioDynamics, Boston, Massachusetts 02115, USA

    • Henry H. Lee,
    • Michael N. Molla &
    • James J. Collins
  2. Center for Advanced Biotechnology, Department of Biomedical Engineering, Boston University, Boston, Massachusetts 02215, USA

    • Henry H. Lee,
    • Michael N. Molla,
    • Charles R. Cantor &
    • James J. Collins
  3. Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, Massachusetts 02215, USA

    • James J. Collins

Contributions

All authors designed the study. H.H.L. and M.N.M. performed and analysed the experiments with input from C.R.C. and J.J.C. All authors prepared and commented on the manuscript.

Competing financial interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to:

The microarray data have been deposited in the NCBI Gene Expression Omnibus under GEO Series accession number GSE22833.

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