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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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Multiple EF-G mutations confer kanamycin resistance which is increased by second site mutations. (A) Growth curves of the EF-G mutants in 8-kan and (B) plain LB (error bars represent standard deviation from eight replicates; legend from B is applicable throughout the figure). The stationary-phase OD600 for the isolates containing the FusAA608E alone, and in combination with gatC .-CC (cytosine insertions) mutation, is lower in 8-kan than for any of the other mutants. This suggests that the second site mutations in RpoD, CpxA, TopA and CyaA may improve FusAA608E strain's ability to grow at low kanamycin concentrations. Of note, the FusAP610T mutation isolated from 4-kan populations does not grow as well as the other fusA mutant strains in 8-kan. (C) Kanamycin MICs (n = 8; box plots as defined in description of Figure 2C) for the isolates with the mutations in EF-G (fusA). The FusAP610L mutation might provide higher resistance to kanamycin than the FusAP610T mutation (P < 10−5; two-sample independent t-test). However, the FusAA608E mutation alone or in combination with gatC .-CC (cytosine insertions) or TopAS180L confers resistance levels similar to the FusAP610T mutation (P = 0.289, 0.817 and 0.151, respectively). In combination with CyaAN600Y, CpxAF218Y,and RpoDL261Q, the FusAA608E mutation does better and has higher resistance than either FusAP610T or FusAA608E alone (P = 0.006, 0.0003 and <10−5, respectively). The emergence of some of these mutations in P1, although a resistant mutation is already present, may be explained by this observation.
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DSU032F5: Multiple EF-G mutations confer kanamycin resistance which is increased by second site mutations. (A) Growth curves of the EF-G mutants in 8-kan and (B) plain LB (error bars represent standard deviation from eight replicates; legend from B is applicable throughout the figure). The stationary-phase OD600 for the isolates containing the FusAA608E alone, and in combination with gatC .-CC (cytosine insertions) mutation, is lower in 8-kan than for any of the other mutants. This suggests that the second site mutations in RpoD, CpxA, TopA and CyaA may improve FusAA608E strain's ability to grow at low kanamycin concentrations. Of note, the FusAP610T mutation isolated from 4-kan populations does not grow as well as the other fusA mutant strains in 8-kan. (C) Kanamycin MICs (n = 8; box plots as defined in description of Figure 2C) for the isolates with the mutations in EF-G (fusA). The FusAP610L mutation might provide higher resistance to kanamycin than the FusAP610T mutation (P < 10−5; two-sample independent t-test). However, the FusAA608E mutation alone or in combination with gatC .-CC (cytosine insertions) or TopAS180L confers resistance levels similar to the FusAP610T mutation (P = 0.289, 0.817 and 0.151, respectively). In combination with CyaAN600Y, CpxAF218Y,and RpoDL261Q, the FusAA608E mutation does better and has higher resistance than either FusAP610T or FusAA608E alone (P = 0.006, 0.0003 and <10−5, respectively). The emergence of some of these mutations in P1, although a resistant mutation is already present, may be explained by this observation.

Mentions: The FusAP610L mutation grew almost as well in 8-kan (Fig. 5A) as did the wild type in 0-kan (Fig. 5B) and FusAP610T in 4-kan (Fig. 3C). However, the FusAP610T mutation performed relatively poorly in 8-kan (Fig. 5A), suggesting that its effect is more restricted to lower kanamycin concentrations. The difference between the two mutations at the same site may also be reflected in the higher MIC of the FusAP610L variant (Fig. 5C). This isolate had also an intergenic mutation at another locus (ybfQ -/- ybfL); at this point, we do not know whether this adds to the resistance phenotype of FusAP610L. We also noticed that FusAP610L grew much better than FusAA608E in 8-kan (Fig. 5A), despite the two being obtained from the same culture conditions and carrying mutations in adjacent residues. Consistent with this observation, FusAA608E-containing isolates were embellished with additional mutations in P1, but not in P0 (Fig. 4—P1). These, admittedly curious, mutations include RpoDL261Q, CpxAF218Y, TopAS180L and CyaAN600Y. It is notable that the FusAA608E + CpxAF218Y/TopAS180L/CyaAN600Y/RpoDL261Q double mutants showed higher growth than the FusAA608E single mutation alone in 8-kan (Fig. 5A), though the single and the double mutations did not differ from each other in growth in the absence of the antibiotic (Fig. 5B). And the MIC of the double mutants was higher when FusAA608E was present in combination with RpoDL261Q and CyaAN600Y; with the others, the increase was not so evident (Fig. 5C). These might suggest genotype-dependent fine tuning of adaptation to the selected concentration of the antibiotic.Figure 5.


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)

Multiple EF-G mutations confer kanamycin resistance which is increased by second site mutations. (A) Growth curves of the EF-G mutants in 8-kan and (B) plain LB (error bars represent standard deviation from eight replicates; legend from B is applicable throughout the figure). The stationary-phase OD600 for the isolates containing the FusAA608E alone, and in combination with gatC .-CC (cytosine insertions) mutation, is lower in 8-kan than for any of the other mutants. This suggests that the second site mutations in RpoD, CpxA, TopA and CyaA may improve FusAA608E strain's ability to grow at low kanamycin concentrations. Of note, the FusAP610T mutation isolated from 4-kan populations does not grow as well as the other fusA mutant strains in 8-kan. (C) Kanamycin MICs (n = 8; box plots as defined in description of Figure 2C) for the isolates with the mutations in EF-G (fusA). The FusAP610L mutation might provide higher resistance to kanamycin than the FusAP610T mutation (P < 10−5; two-sample independent t-test). However, the FusAA608E mutation alone or in combination with gatC .-CC (cytosine insertions) or TopAS180L confers resistance levels similar to the FusAP610T mutation (P = 0.289, 0.817 and 0.151, respectively). In combination with CyaAN600Y, CpxAF218Y,and RpoDL261Q, the FusAA608E mutation does better and has higher resistance than either FusAP610T or FusAA608E alone (P = 0.006, 0.0003 and <10−5, respectively). The emergence of some of these mutations in P1, although a resistant mutation is already present, may be explained by this observation.
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Related In: Results  -  Collection

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DSU032F5: Multiple EF-G mutations confer kanamycin resistance which is increased by second site mutations. (A) Growth curves of the EF-G mutants in 8-kan and (B) plain LB (error bars represent standard deviation from eight replicates; legend from B is applicable throughout the figure). The stationary-phase OD600 for the isolates containing the FusAA608E alone, and in combination with gatC .-CC (cytosine insertions) mutation, is lower in 8-kan than for any of the other mutants. This suggests that the second site mutations in RpoD, CpxA, TopA and CyaA may improve FusAA608E strain's ability to grow at low kanamycin concentrations. Of note, the FusAP610T mutation isolated from 4-kan populations does not grow as well as the other fusA mutant strains in 8-kan. (C) Kanamycin MICs (n = 8; box plots as defined in description of Figure 2C) for the isolates with the mutations in EF-G (fusA). The FusAP610L mutation might provide higher resistance to kanamycin than the FusAP610T mutation (P < 10−5; two-sample independent t-test). However, the FusAA608E mutation alone or in combination with gatC .-CC (cytosine insertions) or TopAS180L confers resistance levels similar to the FusAP610T mutation (P = 0.289, 0.817 and 0.151, respectively). In combination with CyaAN600Y, CpxAF218Y,and RpoDL261Q, the FusAA608E mutation does better and has higher resistance than either FusAP610T or FusAA608E alone (P = 0.006, 0.0003 and <10−5, respectively). The emergence of some of these mutations in P1, although a resistant mutation is already present, may be explained by this observation.
Mentions: The FusAP610L mutation grew almost as well in 8-kan (Fig. 5A) as did the wild type in 0-kan (Fig. 5B) and FusAP610T in 4-kan (Fig. 3C). However, the FusAP610T mutation performed relatively poorly in 8-kan (Fig. 5A), suggesting that its effect is more restricted to lower kanamycin concentrations. The difference between the two mutations at the same site may also be reflected in the higher MIC of the FusAP610L variant (Fig. 5C). This isolate had also an intergenic mutation at another locus (ybfQ -/- ybfL); at this point, we do not know whether this adds to the resistance phenotype of FusAP610L. We also noticed that FusAP610L grew much better than FusAA608E in 8-kan (Fig. 5A), despite the two being obtained from the same culture conditions and carrying mutations in adjacent residues. Consistent with this observation, FusAA608E-containing isolates were embellished with additional mutations in P1, but not in P0 (Fig. 4—P1). These, admittedly curious, mutations include RpoDL261Q, CpxAF218Y, TopAS180L and CyaAN600Y. It is notable that the FusAA608E + CpxAF218Y/TopAS180L/CyaAN600Y/RpoDL261Q double mutants showed higher growth than the FusAA608E single mutation alone in 8-kan (Fig. 5A), though the single and the double mutations did not differ from each other in growth in the absence of the antibiotic (Fig. 5B). And the MIC of the double mutants was higher when FusAA608E was present in combination with RpoDL261Q and CyaAN600Y; with the others, the increase was not so evident (Fig. 5C). These might suggest genotype-dependent fine tuning of adaptation to the selected concentration of the antibiotic.Figure 5.

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