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Antimicrob Agents Chemother. Nov 2007; 51(11): 4205–4208.
Published online Aug 6, 2007. doi:  10.1128/AAC.00647-07
PMCID: PMC2151436

Contributions of the Combined Effects of Topoisomerase Mutations toward Fluoroquinolone Resistance in Escherichia coli[down-pointing small open triangle]

Abstract

In defined, isogenic strains, at least three mutations, two of which must be in gyrA, were required to exceed the CLSI breakpoint for fluoroquinolone resistance. Strains with double mutations in both gyrA and parC had even higher MICs of fluoroquinolones than strains with totals of three mutations.

Fluoroquinolones are widely prescribed antibiotics used to treat a broad range of bacterial infections (13). Fluoroquinolones target the type 2 topoisomerases, gyrase (gyrA and gyrB), and topoisomerase IV (parC and parE) (5, 6). Mutations in the target genes increase the MICs of fluoroquinolones (16). Escherichia coli clinical isolates with high MICs frequently contain double gyrA mutations in the codons for amino acid positions 83 and 87; some also contain double parC mutations in the codons for amino acid positions 80 and 84 (7, 11, 19). The genetic background of clinical isolates is undefined and highly variable; therefore, it is not possible to conclude that the analyzed mutations caused the observed increases in MIC. Indeed, isolates with the same gyrA and parC mutations can have MICs that differ >10-fold (11).

The strains and plasmid used in this study are listed in Table Table1,1, and the oligonucleotides used in this study are listed in Table Table2.2. The effects of double gyrA mutations on MIC in isogenic strains created by drug selection of an E. coli clinical isolate were measured previously (2, 17). Double gyrA mutations combined with a single parC mutation caused 12-fold-increased MICs for ciprofloxacin, the only drug tested (2, 17). However, in defined, isogenic Salmonella strains, the same combinations of mutations caused 30- and 85-fold (depending upon the gyrA mutation)-increased ciprofloxacin MICs (18). Because the genetic background of the E. coli clinical isolate is unknown, it is impossible to conclude whether the differences between the E. coli and Salmonella data are species specific. In addition, no one has measured directly the effect of double parC mutations on the MICs of fluoroquinolones in defined, isogenic strains.

TABLE 1.
Bacterial strains and plasmida
TABLE 2.
Oligonucleotides used in this study

Here, we determined the effects of gyrA and parC double mutations on fluoroquinolone susceptibility in defined, isogenic E. coli strains by Etest (AB Biodisk, Solna, Sweden) per the manufacturer's instructions. MICs exceeding Etest detection limits were determined by broth dilution (macrodilution), following CLSI (formerly NCCLS) guidelines (15). Where indicated, mutants were constructed using the λ Red temperature-sensitive plasmid pSIM5 as described previously (3), except that cells were allowed to recover overnight following electroporation. Strains were cured of the plasmid at the nonpermissive temperature 37°C, and plasmid loss was confirmed by lack of growth on LB agar containing 30 μg/ml chloramphenicol.

The parental strain MICs were the same as those previously reported (Fig. (Fig.1A)1A) (10, 14). The effects of gyrA mutations on the MICs are shown relative to the MIC of the parental strain (1609) to allow direct comparison of the different fluoroquinolones and control for drug-specific and cellular factors (Fig. (Fig.1B).1B). gyrA(L83,Y87) increased the MICs of the fluoroquinolones ~5- to 15-fold, approximately the same result as that for single mutants. Thus, double gyrA mutations, by themselves, do not increase the MICs of the fluoroquinolones in E. coli.

FIG. 1.
Effects of topoisomerase mutations on fluoroquinolone resistance. (A) Fluoroquinolone MICs for the parental C600 strain (1609). The cumulative effects of gyrase and topoisomerase IV mutations were determined by calculating the severalfold increase in ...

We measured the effects of parC mutations on MIC in the gyrA(L83) background (Fig. (Fig.1C).1C). gyrA(L83) parC(I80,G84) showed no additional MIC increases compared to gyrA(L83) with parC single mutations. The norfloxacin MIC increased the most (~5-fold); the moxifloxacin MIC was not significantly increased. Surprisingly, when we analyzed strains carrying only parC mutations, as controls, we found that parC(K84) had significantly (P < 0.01) decreased sparfloxacin, grepafloxacin, gemifloxacin, and moxifloxacin MICs (averages of 0.008, 0.014, 0.007, and 0.028 μg/ml, respectively).

Double gyrA mutations with parC(L80) caused high MIC increases for the fluoroquinolones. Shown (Fig. (Fig.1D)1D) is the severalfold increase for gyrA(L83,Y87) parC(L80) (SKM9) relative to that for gyrA(L83) parC(L80) (1596). gyrA(L83,Y87) parC(L80) MICs increased 9- to 60-fold, depending upon the fluoroquinolone (Fig. (Fig.1D).1D). The ciprofloxacin MIC increased the most (~60-fold), and the moxifloxacin MIC increased the least (~9-fold). This magnitude of increase is the same as that for defined, isogenic Salmonella strains (18); therefore, the previous difference was not species specific but was likely a consequence of the unknown E. coli background (2, 17).

To determine the effects of double parC mutations on MICs, we compared gyrA(L83,Y87) parC(I80,G84) (SKM18) to gyrA(L83,Y87) parC(L80) (SKM9) (Fig. (Fig.1E).1E). The gatifloxacin MIC increased the least (twofold). The moxifloxacin MIC increased the most (~10-fold), which is interesting because the first parC mutation did not significantly increase the moxifloxacin MIC over that for the single gyrA mutation. Double parC mutations increased MICs, but only with double gyrA mutations; otherwise, the second parC mutation was “silent.” Additionally, the magnitudes of increase were significantly lower for parC mutations than for gyrA mutations. Thus, although parC mutations themselves did not greatly increase MICs, they were required for gyrA mutations to cause high MICs.

Plotting the severalfold increases in MIC versus the MICs for the parental strain revealed informative trends with regard to fluoroquinolone- and mutation-specific differences (Fig. (Fig.2).2). In the double gyrA, combined with single or double parC, mutants, the increases in MIC for gemifloxacin, ciprofloxacin, and norfloxacin grouped together at ~10,000-fold. Likewise, the increases for levofloxacin, moxifloxacin, and gatifloxacin grouped together at ~1,000-fold (Fig. (Fig.2).2). Levofloxacin, moxifloxacin, and gatifloxacin all have C-8 substitutions, which may explain their lower MIC increases (22).

FIG. 2.
Comparison of fluoroquinolone resistance in mutants. The x axis shows strains. The y axis denotes the severalfold increases in the MICs of all fluoroquinolones for each strain compared to the MICs for the parental C600 strain (1609). Ofloxacin (OFX) MICs ...

In E. coli, gyrase is the primary target for all fluoroquinolones; topoisomerase IV is a secondary target (reviewed in reference 9). Purified gyrase is more susceptible to fluoroquinolones, which may explain why mutations in topoisomerase IV are not effective until gyrase is mutated (reviewed in reference 8). As the organism acquires mutations, the primary target of the fluoroquinolones switches between gyrase and topoisomerase IV. This idea is supported by previous studies examining stepwise mutants in which the first-, second-, third-, and fourth-step mutations occurred in gyrA, parC, gyrA, and parC, respectively (12). A single mutation in gyrase decreases its susceptibility sufficiently that topoisomerase IV becomes targeted. The silent phenotype of a second gyrase mutation in a parC wild-type background implies that the fluoroquinolones are targeting primarily topoisomerase IV; however, once parC has a single mutation, gyrase once again becomes targeted, explaining why double gyrase mutants have up to 60-fold-increased MICs over those for single gyrA, single parC mutants. An additional parC mutation in the double gyrA mutant strain increased MICs, indicating that topoisomerase IV becomes the target once again.

Acknowledgments

We thank Hana M. El Sahly, Richard J. Hamill, and Sheila I. Hull for critically reading the manuscript.

L.Z. was supported by National Institutes of Health grant R01-AI054830 and The Burroughs Wellcome Fund. S.K.M. was supported by a Houston Area Molecular Biophysics Program predoctoral fellowship, NIH T32-GM008280.

Footnotes

[down-pointing small open triangle]Published ahead of print on 6 August 2007.

REFERENCES

1. Appleyard, R. K. 1954. Segregation of new lysogenic types during growth of a doubly lysogenic strain derived from Escherichia coli K12. Genetics 39:440-452. [PMC free article] [PubMed]
2. Bagel, S., V. Hullen, B. Wiedemann, and P. Heisig. 1999. Impact of gyrA and parC mutations on quinolone resistance, doubling time, and supercoiling degree of Escherichia coli. Antimicrob. Agents Chemother. 43:868-875. [PMC free article] [PubMed]
3. Costantino, N., and D. L. Court. 2003. Enhanced levels of lambda Red-mediated recombinants in mismatch repair mutants. Proc. Natl. Acad. Sci. USA 100:15748-15753. [PMC free article] [PubMed]
4. Datta, S., N. Costantino, and D. L. Court. 2006. A set of recombineering plasmids for gram-negative bacteria. Gene 379:109-115. [PubMed]
5. Drlica, K. 1999. Mechanism of fluoroquinolone action. Curr. Opin. Microbiol. 2:504-508. [PubMed]
6. Froelich-Ammon, S. J., and N. Osheroff. 1995. Topoisomerase poisons: harnessing the dark side of enzyme mechanism. J. Biol. Chem. 270:21429-21432. [PubMed]
7. Heisig, P. 1996. Genetic evidence for a role of parC mutations in development of high-level fluoroquinolone resistance in Escherichia coli. Antimicrob. Agents Chemother. 40:879-885. [PMC free article] [PubMed]
8. Hooper, D. C. 2000. Mechanisms of action and resistance of older and newer fluoroquinolones. Clin. Infect. Dis. 31(Suppl. 2):S24-S28. [PubMed]
9. Hooper, D. C. 2001. Mechanisms of action of antimicrobials: focus on fluoroquinolones. Clin. Infect. Dis. 32(Suppl. 1):S9-S15. [PubMed]
10. Khodursky, A. B., E. L. Zechiedrich, and N. R. Cozzarelli. 1995. Topoisomerase IV is a target of quinolones in Escherichia coli. Proc. Natl. Acad. Sci. USA 92:11801-11805. [PMC free article] [PubMed]
11. 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. [PMC free article] [PubMed]
12. Li, X., N. Mariano, J. J. Rahal, C. M. Urban, and K. Drlica. 2004. Quinolone-resistant Haemophilus influenzae: determination of mutant selection window for ciprofloxacin, garenoxacin, levofloxacin, and moxifloxacin. Antimicrob. Agents Chemother. 48:4460-4462. [PMC free article] [PubMed]
13. Linder, J. A., E. S. Huang, M. A. Steinman, R. Gonzales, and R. S. Stafford. 2005. Fluoroquinolone prescribing in the United States: 1995 to 2002. Am. J. Med. 118:259-268. [PubMed]
14. Lu, T., X. Zhao, and K. Drlica. 1999. Gatifloxacin activity against quinolone-resistant gyrase: allele-specific enhancement of bacteriostatic and bactericidal activities by the C-8-methoxy group. Antimicrob. Agents Chemother. 43:2969-2974. [PMC free article] [PubMed]
15. National Committee for Clinical Laboratory Standards. 2003. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically. Approved standard, 6th ed. M7-A6. National Committee for Clinical Laboratory Standards, Wayne, PA.
16. Ruiz, J. 2003. Mechanisms of resistance to quinolones: target alterations, decreased accumulation and DNA gyrase protection. J. Antimicrob. Chemother. 51:1109-1117. [PubMed]
17. Schulte, A., and P. Heisig. 2000. In vitro activity of gemifloxacin and five other fluoroquinolones against defined isogenic mutants of Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus. J. Antimicrob. Chemother. 46:1037-1038. [PubMed]
18. Turner, A. K., S. Nair, and J. Wain. 2006. The acquisition of full fluoroquinolone resistance in Salmonella Typhi by accumulation of point mutations in the topoisomerase targets. J. Antimicrob. Chemother. 58:733-740. [PubMed]
19. Vila, J., J. Ruiz, P. Goni, and M. T. De Anta. 1996. Detection of mutations in parC in quinolone-resistant clinical isolates of Escherichia coli. Antimicrob. Agents Chemother. 40:491-493. [PMC free article] [PubMed]
20. Reference deleted.
21. Zechiedrich, E. L., and N. R. Cozzarelli. 1995. Roles of topoisomerase IV and DNA gyrase in DNA unlinking during replication in Escherichia coli. Genes Dev. 9:2859-2869. [PubMed]
22. 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. [PMC free article] [PubMed]

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