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Antimicrobial Agents and Chemotherapy, October 2006, p. 3454-3456, Vol. 50, No. 10
0066-4804/06/$08.00+0     doi:10.1128/AAC.00783-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Weak Mutators Can Drive the Evolution of Fluoroquinolone Resistance in Escherichia coli

Hanna Örlén and Diarmaid Hughes^*

Department of Cell and Molecular Biology, Box 596, Biomedical Center, Uppsala University, S-751 24 Uppsala, Sweden

Received 29 June 2006/ Returned for modification 12 July 2006/ Accepted 14 July 2006

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Weak mutators are common among clinical isolates of Escherichia coli. We show that the relative mutation rate and the "evolvability of fluoroquinolone resistance" are related by a power law slope of 1.2 over 3 orders of magnitude. Thus, even weak mutators can drive the evolution of fluoroquinolone resistance under selection pressure.

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Fluoroquinolones are a widely used group of antimicrobial agents. The development of resistance to clinically achievable levels of fluoroquinolones in most organisms, including Escherichia coli, is a multistep mutational process. Although several plasmid-borne resistance determinants have been described (10, 13, 14, 20), the great majority of the genetic alterations associated with fluoroquinolone resistance are mutations of chromosomal genes, including gyrA, gyrB, parC, parE, marR, and acrR (11, 12). E. coli isolates with MICs above the CLSI (formerly NCCLS)-defined breakpoint for ciprofloxacin (4 µg/ml) usually have four or more mutations distributed among these genes (17). This implies that resistant lineages have gone through several cycles of mutation and selection. The relative mutation rate might be a significant factor that determines the probability that resistance will evolve in a lineage by increasing the rate of supply of rare new mutations.

The focus of interest regarding the influence of mutators on evolution has been on strong mutators such as mutS mutants (3, 7, 21, 24, 25). These increase mutation rates 500-fold and are found in up to 1% of natural E. coli isolates (2, 9, 17, 18, 21). They have strong selective benefits in experimental models (8, 19, 24, 27) but in vivo also lead to the accumulation of detrimental mutations that severely reduce fitness, measured as reduced competitiveness during transmission to and recolonization of eukaryotic hosts (8). In contrast, studies of hundreds of clinical E. coli isolates, also from a variety of sources, have found that about 25% are weakly hypermutable (up to 13-fold increase in the mutation rate) (2, 21). The relative preponderance of weak and moderate hypermutators among clinical isolates, including multiply resistant isolates (5), may reflect their better ability to evolve resistance, be transmitted to new hosts, and persist longer than strong mutators without incurring major fitness costs (4, 28).

We have previously noted an increased mutation rate correlated with fluoroquinolone resistance in clinical isolates of E. coli (17). The increases are modest, with the mutation rate in 80% of resistant isolates increasing by less than 20-fold the rate for the wild type. The influence of small increases in the mutation rate on the evolution of antibiotic resistance has not previously been tested experimentally. This raises the question of whether the link between fluoroquinolone resistance and a moderately increased mutation rate (17) is fortuitous or causal. We tested whether small and moderate increases in the mutation rate were sufficient to proportionately increase the ability of E. coli to evolve fluoroquinolone resistance.

Seven different mutator alleles were introduced into E. coli K12 MG1655 by P1 transduction (Table 1). These mutator alleles have been described in the literature, and their reported mutator phenotypes differ widely in magnitude (1, 15, 16, 22, 26, 29). Fifty independent lineages of each strain were evolved by serial passage in Luria-Bertani medium with successively higher concentrations of ciprofloxacin (Bayer AG, Wuppertal, Germany) in a BioscreenC machine (Oy Growth Curves Ab Ltd., Helsinki, Finland). The ciprofloxacin concentration steps were 0, 0.01, 0.02, 0.04, 0.08, 0.16, 0.32, 0.64, 1.25, 2.5, 5, 10, 20, 40, 80, 160, 320, 640, 1,280, and 2,560 µg/ml. Each cycle was 23 to 24 h of growth in a 200 µl, with the transfer of 5 µl at each step (initially 10⁶ to 10⁷ CFU). During the course of the evolution experiments, different lineages grew to different cell concentrations, reflecting their independent evolutionary trajectories; and thus, biological bottleneck sizes were lineage and step specific. The optical density at 600 nm (OD₆₀₀) of each well was measured at 10-min intervals throughout the culture period. Extinction of a lineage was defined as the failure to obtain growth (OD₆₀₀ < 0.03) after overnight incubation. The extinction coefficient (K_E) is defined as the lowest drug concentration step at which 50% of the lineages were extinct.

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TABLE 1. Bacterial strains and mutator genotypes

The MIC of ciprofloxacin was determined by Etest, according to the instructions of the manufacturer (AB BIODISK, Solna, Sweden). The mutator mutations do not themselves significantly affect the MICs (Table 2).

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TABLE 2. Relationship between mutation rate and KE

Mutation rates were measured by fluctuation tests with 40 independent cultures of each strain, assaying for rifampin resistance as described previously (17). This is a standard assay and measures the occurrence of at least 69 different base substitutions in rpoB (6). The results (Table 2) are close to those expected on the basis of the measurements for the parental strains. The range of mutation rates covered by these strains is 500-fold, with an emphasis on the lower end of the range up to 10-fold the rate for the wild type.

All lineages of each strain, with the exception of a few mutS lineages, went to extinction within the course of the experiment. At 1,280 and 2,560 µg/ml ciprofloxacin, 20% and 13% of the mutS lineages were still living, respectively. The K_E values for each strain (Table 2) ranged over 3 orders of magnitude. When the K_E values were plotted against the relative mutation rate, the data fit a straight line (linear regression R² value, 0.77; P value, <0.001 at the 95% confidence level) to a power law with a slope of 1.2 (Fig. 1). Thus, "evolvability" (K_E) increases as the 1.2 power of the mutation rate. The results for one miaA strain deviated notably from this regression line, possibly because it failed to generate an increased rate of some mutation required for the evolution of fluoroquinolone resistance, although pleiotropic side effects due to the absence of this tRNA modification cannot be ruled out. Removal of the data for the miaA strain from the analysis gives a slope of 1.2 for the remaining seven strains (linear regression R² value, 0.95; P value, < 0.001). This particular power law relationship may reflect the average value of synergistic epistasis between different mutations and their effects on the resistance level. For example, a parC mutation does not show a resistance phenotype except in the presence of an appropriate gyrA mutation (30).

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FIG. 1. KE values for wild-type and mutator strains plotted as a function of relative mutation rate.

In conclusion, we found a good correlation between the mutation rate and K_E that extended over a wide range of mutation rates. This shows that even small increases in the mutation rate have a positive and measurable effect on the evolution of fluoroquinolone resistance by mutation and that the effect is in proportion to the magnitude of the mutation rate increase. There is no minimum threshold of mutability for increasing the rate of evolution to drug resistance.

 
This work was supported by grants from the Swedish Research Council (Vetenskapsrådet) and the European Union Sixth Framework Programme (EAR project, contract LSHM-CT-2005-518152) to D. Hughes.

We thank the scientists listed in Table 1, Santanu Dasgupta (Uppsala University), and Monica Rydén Aulin (Stockholm University) for kindly sending us mutator strains.

 
* Corresponding author. Mailing address: Department of Cell and Molecular Biology, Box 596, Biomedical Center, Uppsala University, S-751 24 Uppsala, Sweden. Phone: 46-18-471 4354. Fax: 46-18-530396. E-mail: diarmaid.hughes{at}icm.uu.se.

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1
1. Balashov, S., and M. Z. Humayun. 2004. Specificity of spontaneous mutations induced in mutA mutator cells. Mutat. Res. 548:9-18.[Medline]
2
2. Baquero, M. R., A. I. Nilsson, C. Turrientes Mdel, D. Sandvang, J. C. Galan, J. L. Martinez, N. Frimodt-Moller, F. Baquero, and D. I. Andersson. 2004. Polymorphic mutation frequencies in Escherichia coli: emergence of weak mutators in clinical isolates. J. Bacteriol. 186:5538-5542.[Abstract/Free Full Text]
3
3. Boe, L., M. Danielsen, S. Knudsen, J. B. Petersen, J. Maymann, and P. R. Jensen. 2000. The frequency of mutators in populations of Escherichia coli. Mutat. Res. 448:47-55.[Medline]
4
4. Denamur, E., and I. Matic. 2006. Evolution of mutation rates in bacteria. Mol. Microbiol. 60:820-827.[CrossRef][Medline]
5
5. Denamur, E., O. Tenaillon, C. Deschamps, D. Skurnik, E. Ronco, J. L. Gaillard, B. Picard, C. Branger, and I. Matic. 2005. Intermediate mutation frequencies favor evolution of multidrug resistance in Escherichia coli. Genetics 171:825-827.[Abstract/Free Full Text]
6
6. Garibyan, L., T. Huang, M. Kim, E. Wolff, A. Nguyen, T. Nguyen, A. Diep, K. Hu, A. Iverson, H. Yang, and J. H. Miller. 2003. Use of the rpoB gene to determine the specificity of base substitution mutations on the Escherichia coli chromosome. DNA Repair (Amsterdam) 2:593-608.
7
7. Giraud, A., I. Matic, M. Radman, M. Fons, and F. Taddei. 2002. Mutator bacteria as a risk factor in treatment of infectious diseases. Antimicrob. Agents Chemother. 46:863-865.[Abstract/Free Full Text]
8
8. Giraud, A., I. Matic, O. Tenaillon, A. Clara, M. Radman, M. Fons, and F. Taddei. 2001. Costs and benefits of high mutation rates: adaptive evolution of bacteria in the mouse gut. Science 291:2606-2608.[Abstract/Free Full Text]
9
9. Giraud, A., M. Radman, I. Matic, and F. Taddei. 2001. The rise and fall of mutator bacteria. Curr. Opin. Microbiol. 4:582-585.[CrossRef][Medline]
10
10. Hata, M., M. Suzuki, M. Matsumoto, M. Takahashi, K. Sato, S. Ibe, and K. Sakae. 2005. Cloning of a novel gene for quinolone resistance from a transferable plasmid in Shigella flexneri 2b. Antimicrob. Agents Chemother. 49:801-803.[Abstract/Free Full Text]
11
11. Hooper, D. C. 2001. Emerging mechanisms of fluoroquinolone resistance. Emerg. Infect. Dis. 7:337-341.[Medline]
12
12. Hopkins, K. L., R. H. Davies, and E. J. Threlfall. 2005. Mechanisms of quinolone resistance in Escherichia coli and Salmonella: recent developments. Int. J. Antimicrob. Agents 25:358-373.[CrossRef][Medline]
13
13. Jacoby, G. A., N. Chow, and K. B. Waites. 2003. Prevalence of plasmid-mediated quinolone resistance. Antimicrob. Agents Chemother. 47:559-562.[Abstract/Free Full Text]
14
14. Jacoby, G. A., K. E. Walsh, D. M. Mills, V. J. Walker, H. Oh, A. Robicsek, and D. C. Hooper. 2006. qnrB, another plasmid-mediated gene for quinolone resistance. Antimicrob. Agents Chemother. 50:1178-1182.[Abstract/Free Full Text]
15
15. Jurado, J., A. Maciejewska, J. Krwawicz, J. Laval, and M. K. Saparbaev. 2004. Role of mismatch-specific uracil-DNA glycosylase in repair of 3,N4-ethenocytosine in vivo. DNA Repair (Amsterdam) 3:1579-1590.[CrossRef]
16
16. Klapacz, J., and A. S. Bhagwat. 2005. Transcription promotes guanine to thymine mutations in the non-transcribed strand of an Escherichia coli gene. DNA Repair (Amsterdam) 4:806-813.[CrossRef]
17
17. Komp Lindgren, P., A. Karlsson, and D. Hughes. 2003. Mutation rate and evolution of fluoroquinolone resistance in Escherichia coli isolates from patients with urinary tract infections. Antimicrob. Agents Chemother. 47:3222-3232.[Abstract/Free Full Text]
18
18. LeClerc, J. E., B. Li, W. L. Payne, and T. A. Cebula. 1996. High mutation frequencies among Escherichia coli and Salmonella pathogens. Science 274:1208-1211.[Abstract/Free Full Text]
19
19. Mao, E. F., L. Lane, J. Lee, and J. H. Miller. 1997. Proliferation of mutators in a cell population. J. Bacteriol. 179:417-422.[Abstract/Free Full Text]
20
20. Martinez-Martinez, L., A. Pascual, and G. A. Jacoby. 1998. Quinolone resistance from a transferable plasmid. Lancet 351:797-799.[CrossRef][Medline]
21
21. Matic, I., M. Radman, F. Taddei, B. Picard, C. Doit, E. Bingen, E. Denamur, and J. Elion. 1997. Highly variable mutation rates in commensal and pathogenic Escherichia coli. Science 277:1833-1834.[Free Full Text]
22
22. Miller, J. H. 1998. Mutators in Escherichia coli. Mutat. Res. 409:99-106.[Medline]
23
23. Miller, J. H. 1992. A short course in bacterial genetics: a laboratory manual and handbook for Escherichia coli and related bacteria. Cold Spring Harbor Laboratory Press, Plainview, N.Y.
24
24. Oliver, A., R. Canton, P. Campo, F. Baquero, and J. Blazquez. 2000. High frequency of hypermutable Pseudomonas aeruginosa in cystic fibrosis lung infection. Science 288:1251-1254.[Abstract/Free Full Text]
25
25. Orencia, M. C., J. S. Yoon, J. E. Ness, W. P. Stemmer, and R. C. Stevens. 2001. Predicting the emergence of antibiotic resistance by directed evolution and structural analysis. Nat. Struct. Biol. 8:238-242.[CrossRef][Medline]
26
26. Parker, B., and M. G. Marinus. 1988. A simple and rapid method to obtain substitution mutations in Escherichia coli: isolation of a dam deletion/insertion mutation. Gene 73:531-535.[CrossRef][Medline]
27
27. Sniegowski, P. D., P. J. Gerrish, and R. E. Lenski. 1997. Evolution of high mutation rates in experimental populations of E. coli. Nature 387:703-705.[CrossRef][Medline]
28
28. Taddei, F., M. Radman, J. Maynard-Smith, B. Toupance, P. H. Gouyon, and B. Godelle. 1997. Role of mutator alleles in adaptive evolution. Nature 387:700-702.[CrossRef][Medline]
29
29. Zhao, J., H. E. Leung, and M. E. Winkler. 2001. The miaA mutator phenotype of Escherichia coli K-12 requires recombination functions. J. Bacteriol. 183:1796-1800.[Abstract/Free Full Text]
30
30. Zhao, X., C. Xu, J. Domagala, and K. Drlica. 1997. DNA topoisomerase targets of the fluoroquinolones: a strategy for avoiding bacterial resistance. Proc. Natl. Acad. Sci. USA 94:13991-13996.[Abstract/Free Full Text]

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Antimicrobial Agents and Chemotherapy, October 2006, p. 3454-3456, Vol. 50, No. 10
0066-4804/06/$08.00+0     doi:10.1128/AAC.00783-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

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