Genomic analysis reveals distinct concentration-dependent evolutionary trajectories for antibiotic resistance in Escherichia coli.
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.
Affiliation: National Centre for Biological Sciences, Tata Institute of Fundamental Research, GKVK, Bellary Road, Bangalore, Karnataka 560065, India email@example.com firstname.lastname@example.org.Show MeSH
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Mentions: What are the characteristics of the bacterial population resulting from the above-described growth in the antibiotic, in terms of its ability to grow in the presence or absence of kanamycin? For this, we first grew cells—produced by P0 cultures in antibiotic-free, 4-kan or 8-kan media—on LB agar plates. Individual colonies so obtained were then checked for their ability to grow in the absence of kanamycin (plain LB, 0-kan), moderately sublethal levels of kanamycin (4-kan) and lethal levels of kanamycin (20-kan). We note here that this experimental strategy is limited in the sense that the growth experiments performed to assess fitness also contribute to further evolution and selection. However, these steps were performed in the same manner for 4-kan and 8-kan populations, and therefore, the results thus obtained enable a direct comparison between the two antibiotic concentrations. Control colonies derived from all the populations immediately after inoculation displayed wild-type characteristics: good growth in 0-kan, poor growth at 4-kan and no growth at 20-kan (Supplementary Fig. S5A–F, 0 h.). Similarly, colonies derived from populations not treated with kanamycin retained this response throughout the course of the experiment (Supplementary Fig. S5A and S5D). Most colonies derived from 4-kan populations grew well in LB and in 4-kan; in addition, they were more resistant to 20-kan than the wild type (Fig. 2A, and Supplementary Fig. S5B and S5E). In contrast, a proportion of colonies derived from 8-kan populations, though more resistant to the antibiotic than the wild type, showed severe growth defects (Fig. 2B, and Supplementary Fig. S5C and S5F). The proportion of colonies with a growth defect varied between trials (Fig. 2B, compare group-1 and -2 colonies in 8-kan 1 and 2 at 24 h, between the two replicates). After another passage in the same concentration of the antibiotic (P1), both 4-kan and 8-kan cultures produced antibiotic-resistant colonies with little growth defect (Fig. 2A and 2B, group-3 colonies increase at P1).Figure 2.
Affiliation: National Centre for Biological Sciences, Tata Institute of Fundamental Research, GKVK, Bellary Road, Bangalore, Karnataka 560065, India email@example.com firstname.lastname@example.org.