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Genomic analysis reveals distinct concentration-dependent evolutionary trajectories for antibiotic resistance in Escherichia coli.

Mogre A, Sengupta T, Veetil RT, Ravi P, Seshasayee AS - DNA Res. (2014)

Bottom Line: A second class of mutations, recovered only during evolution in higher sublethal concentrations of the antibiotic, deleted the C-terminal end of the ATP synthase shaft.This mutation confers basal-level resistance to kanamycin while showing a strong growth defect in the absence of the antibiotic.In conclusion, the early dynamics of the development of resistance to an aminoglycoside antibiotic is dependent on the levels of stress (concentration) imposed by the antibiotic, with the evolution of less costly variants only a matter of time.

View Article: PubMed Central - PubMed

Affiliation: National Centre for Biological Sciences, Tata Institute of Fundamental Research, GKVK, Bellary Road, Bangalore, Karnataka 560065, India aswin@ncbs.res.in aalapbm@ncbs.res.in.

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Resistance developed as a result of growth in sublethal kanamycin concentration. (A) Growth of two replicate populations of Escherichia coli MG1655 in ∼25% of the lethal level of kanamycin (4 µg/ml: 4-kan 1 and 2; for additional growth curves, see Supplementary Figure S3A). Control populations in plain LB are 0-kan 1 and 2. Populations at various time points from the 4-kan curves were sequenced while only mid-exponential- and stationary-phase populations from the 0-kan curves were sequenced. (B) MICs of evolving populations. P0 indicates the first round of growth in kanamycin (i.e. the growth curves in A) while P1–P10 indicate serial transfers by 100-fold dilution. Black bars indicate MIC levels of control populations passaged in plain LB (0-kan). Red bars indicate populations evolving in 4-kan, with data available for multiple time points in P0. Green bars indicate populations grown in 4-kan in P0 but subsequently transferred into plain LB for the remaining passages P1–P10 (4-kan → 0-kan). The maintenance of increased MIC levels suggests the presence of resistant mutations and not a temporary stress response. The average MIC level of the two biological replicates (from three technical replicates) is plotted with the total length of the positive and negative error bars representing the difference in MICs between the two replicates (range). (C) Growth of four replicate populations of E. coli MG1655 in ∼50% lethal levels of kanamycin (8 µg/ml: 8-kan 1-4; more growth curves can be found in Supplementary Figure S3). (D) Isolates derived from (four replicates of) the P0 8-kan and the P1 8-kan populations were tested for their MIC (red bars) and growth in plain LB (black bars, taken at 7 h. after inoculation). Many isolates in P0 were sick, i.e. had a lower OD600 in plain LB but could resist kanamycin. The incidence of these sick isolates decreases after P1, and fit resistant forms seem to dominate. For comparison, kanamycin MICs (red bars) and OD600 (black bars) of multiple isolates, from P0 and P1 control wild-type populations grown in plain LB, are shown in the right panels.
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DSU032F1: Resistance developed as a result of growth in sublethal kanamycin concentration. (A) Growth of two replicate populations of Escherichia coli MG1655 in ∼25% of the lethal level of kanamycin (4 µg/ml: 4-kan 1 and 2; for additional growth curves, see Supplementary Figure S3A). Control populations in plain LB are 0-kan 1 and 2. Populations at various time points from the 4-kan curves were sequenced while only mid-exponential- and stationary-phase populations from the 0-kan curves were sequenced. (B) MICs of evolving populations. P0 indicates the first round of growth in kanamycin (i.e. the growth curves in A) while P1–P10 indicate serial transfers by 100-fold dilution. Black bars indicate MIC levels of control populations passaged in plain LB (0-kan). Red bars indicate populations evolving in 4-kan, with data available for multiple time points in P0. Green bars indicate populations grown in 4-kan in P0 but subsequently transferred into plain LB for the remaining passages P1–P10 (4-kan → 0-kan). The maintenance of increased MIC levels suggests the presence of resistant mutations and not a temporary stress response. The average MIC level of the two biological replicates (from three technical replicates) is plotted with the total length of the positive and negative error bars representing the difference in MICs between the two replicates (range). (C) Growth of four replicate populations of E. coli MG1655 in ∼50% lethal levels of kanamycin (8 µg/ml: 8-kan 1-4; more growth curves can be found in Supplementary Figure S3). (D) Isolates derived from (four replicates of) the P0 8-kan and the P1 8-kan populations were tested for their MIC (red bars) and growth in plain LB (black bars, taken at 7 h. after inoculation). Many isolates in P0 were sick, i.e. had a lower OD600 in plain LB but could resist kanamycin. The incidence of these sick isolates decreases after P1, and fit resistant forms seem to dominate. For comparison, kanamycin MICs (red bars) and OD600 (black bars) of multiple isolates, from P0 and P1 control wild-type populations grown in plain LB, are shown in the right panels.

Mentions: The P0 growth curve of E. coli grown in 4 µg/ml kanamycin (4-kan) is characterized by a long lag phase (6–7 h) followed by an exponential phase defined by low growth rate (Fig. 1A and Supplementary Fig. S3 for statistics). The lag phase period increased considerably to at least 11 h in 8 µg/ml kanamycin (8-kan) (Fig. 1C and Supplementary Fig. S3); the cultures typically reached a final OD after 24 h that was less than what was observed for the 4-kan cultures (compare Fig. 1A and C, and Supplementary Fig. S3B).Figure 1.


Genomic analysis reveals distinct concentration-dependent evolutionary trajectories for antibiotic resistance in Escherichia coli.

Mogre A, Sengupta T, Veetil RT, Ravi P, Seshasayee AS - DNA Res. (2014)

Resistance developed as a result of growth in sublethal kanamycin concentration. (A) Growth of two replicate populations of Escherichia coli MG1655 in ∼25% of the lethal level of kanamycin (4 µg/ml: 4-kan 1 and 2; for additional growth curves, see Supplementary Figure S3A). Control populations in plain LB are 0-kan 1 and 2. Populations at various time points from the 4-kan curves were sequenced while only mid-exponential- and stationary-phase populations from the 0-kan curves were sequenced. (B) MICs of evolving populations. P0 indicates the first round of growth in kanamycin (i.e. the growth curves in A) while P1–P10 indicate serial transfers by 100-fold dilution. Black bars indicate MIC levels of control populations passaged in plain LB (0-kan). Red bars indicate populations evolving in 4-kan, with data available for multiple time points in P0. Green bars indicate populations grown in 4-kan in P0 but subsequently transferred into plain LB for the remaining passages P1–P10 (4-kan → 0-kan). The maintenance of increased MIC levels suggests the presence of resistant mutations and not a temporary stress response. The average MIC level of the two biological replicates (from three technical replicates) is plotted with the total length of the positive and negative error bars representing the difference in MICs between the two replicates (range). (C) Growth of four replicate populations of E. coli MG1655 in ∼50% lethal levels of kanamycin (8 µg/ml: 8-kan 1-4; more growth curves can be found in Supplementary Figure S3). (D) Isolates derived from (four replicates of) the P0 8-kan and the P1 8-kan populations were tested for their MIC (red bars) and growth in plain LB (black bars, taken at 7 h. after inoculation). Many isolates in P0 were sick, i.e. had a lower OD600 in plain LB but could resist kanamycin. The incidence of these sick isolates decreases after P1, and fit resistant forms seem to dominate. For comparison, kanamycin MICs (red bars) and OD600 (black bars) of multiple isolates, from P0 and P1 control wild-type populations grown in plain LB, are shown in the right panels.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4263303&req=5

DSU032F1: Resistance developed as a result of growth in sublethal kanamycin concentration. (A) Growth of two replicate populations of Escherichia coli MG1655 in ∼25% of the lethal level of kanamycin (4 µg/ml: 4-kan 1 and 2; for additional growth curves, see Supplementary Figure S3A). Control populations in plain LB are 0-kan 1 and 2. Populations at various time points from the 4-kan curves were sequenced while only mid-exponential- and stationary-phase populations from the 0-kan curves were sequenced. (B) MICs of evolving populations. P0 indicates the first round of growth in kanamycin (i.e. the growth curves in A) while P1–P10 indicate serial transfers by 100-fold dilution. Black bars indicate MIC levels of control populations passaged in plain LB (0-kan). Red bars indicate populations evolving in 4-kan, with data available for multiple time points in P0. Green bars indicate populations grown in 4-kan in P0 but subsequently transferred into plain LB for the remaining passages P1–P10 (4-kan → 0-kan). The maintenance of increased MIC levels suggests the presence of resistant mutations and not a temporary stress response. The average MIC level of the two biological replicates (from three technical replicates) is plotted with the total length of the positive and negative error bars representing the difference in MICs between the two replicates (range). (C) Growth of four replicate populations of E. coli MG1655 in ∼50% lethal levels of kanamycin (8 µg/ml: 8-kan 1-4; more growth curves can be found in Supplementary Figure S3). (D) Isolates derived from (four replicates of) the P0 8-kan and the P1 8-kan populations were tested for their MIC (red bars) and growth in plain LB (black bars, taken at 7 h. after inoculation). Many isolates in P0 were sick, i.e. had a lower OD600 in plain LB but could resist kanamycin. The incidence of these sick isolates decreases after P1, and fit resistant forms seem to dominate. For comparison, kanamycin MICs (red bars) and OD600 (black bars) of multiple isolates, from P0 and P1 control wild-type populations grown in plain LB, are shown in the right panels.
Mentions: The P0 growth curve of E. coli grown in 4 µg/ml kanamycin (4-kan) is characterized by a long lag phase (6–7 h) followed by an exponential phase defined by low growth rate (Fig. 1A and Supplementary Fig. S3 for statistics). The lag phase period increased considerably to at least 11 h in 8 µg/ml kanamycin (8-kan) (Fig. 1C and Supplementary Fig. S3); the cultures typically reached a final OD after 24 h that was less than what was observed for the 4-kan cultures (compare Fig. 1A and C, and Supplementary Fig. S3B).Figure 1.

Bottom Line: A second class of mutations, recovered only during evolution in higher sublethal concentrations of the antibiotic, deleted the C-terminal end of the ATP synthase shaft.This mutation confers basal-level resistance to kanamycin while showing a strong growth defect in the absence of the antibiotic.In conclusion, the early dynamics of the development of resistance to an aminoglycoside antibiotic is dependent on the levels of stress (concentration) imposed by the antibiotic, with the evolution of less costly variants only a matter of time.

View Article: PubMed Central - PubMed

Affiliation: National Centre for Biological Sciences, Tata Institute of Fundamental Research, GKVK, Bellary Road, Bangalore, Karnataka 560065, India aswin@ncbs.res.in aalapbm@ncbs.res.in.

Show MeSH
Related in: MedlinePlus