Journal of Microbiology

, Volume 57, Issue 12, pp 1056–1064 | Cite as

Soft sweep development of resistance in Escherichia coli under fluoroquinolone stress

  • Xianxing Xie
  • Ruichen Lv
  • Chao Yang
  • Yajun Song
  • Yanfeng Yan
  • Yujun Cui
  • Ruifu YangEmail author
Microbial Systematics and Evolutionary Microbiology


We employed a stepwise selection model for investigating the dynamics of antibiotic-resistant variants in Escherichia coli K-12 treated with increasing concentrations of ciprofloxacin (CIP). Firstly, we used Sanger sequencing to screen the variations in the fluoquinolone target genes, then, employed Illumina NGS sequencing for amplicons targeted regions with variations. The results demonstrated that variations G81C in gyrA and K276N and K277L in parC are standing resistance variations (SRVs), while S83A and S83L in gyrA and G78C in parC were emerging resistance variations (ERVs). The variants containing SRVs and/or ERVs were selected successively based on their sensitivities to CIP. Variant strain 1, containing substitution G81C in gyrA, was immediately selected following ciprofloxacin exposure, with obvious increases in the parC SRV, and parC and gyrA ERV allele frequencies. Variant strain 2, containing the SRVs, then dominated the population following a 20× increase in ciprofloxacin concentration, with other associated allele frequencies also elevated. Variant strains 3 and 4, containing ERVs in gyrA and parC, respectively, were then selected at 40× and 160× antibiotic concentrations. Two variants, strains 5 and 6, generated in the selection procedure, were lost because of higher fitness costs or a lower level of resistance compared with variants 3 and 4. For the second induction, all variations/indels were already present as SRVs and selected out step by step at different passages. Whatever the first induction or second induction, our results confirmed the soft selective sweep hypothesis and provided critical information for guiding clinical treatment of pathogens containing SRVs.


soft sweeps stepwise selection Escherichia coli ciprofloxacin fitness cost 


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We thank Tamsin Sheen, PhD, from Liwen Bianji, Edanz Editing China (, for editing the English text of a draft of this manuscript.

This work was supported by the National Special Project on Research and Development of Key Biosafety Technologies (contract no. 2016YFC1200100) and the 973 Project of MOST (contract no. 2015CB554202).

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  1. Andersson, D.I. and Hughes, D. 2010. Antibiotic resistance and its cost: is it possible to reverse resistance? Nat. Rev. Microbiol.8, 260–271.PubMedGoogle Scholar
  2. Baltekin, O., Boucharin, A., Tano, E., Andersson, D.I., and Elf, J. 2017. Antibiotic susceptibility testing in less than 30 min using direct single-cell imaging. Proc. Natl. Acad. Sci. USA114, 9170–9175.PubMedGoogle Scholar
  3. Belland, R.J., Morrison, S.G., Ison, C., and Huang, W.M. 1994. Neisseria gonorrhoeae acquires mutations in analogous regions of gyrA and parC in fluoroquinolone-resistant isolates. Mol. Microbiol.14, 371–380.PubMedGoogle Scholar
  4. Caporaso J.G., Kuczynski, J., Stombaugh, J., Bittinger, K., Bushman, F.D., Costello, E.K., Fierer, N., Peña, A.G., Goodrich, J.K., Gordon, J.I., et al. 2010. QIIME allows analysis of high-throughput community sequencing data. Nat Methods7, 335–336.PubMedPubMedCentralGoogle Scholar
  5. Clinical and Laboratory Standards Institute. 2009. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically: approved standard. CLSI publication M07-A8. Wayne: Clinical and Laboratory Standards Institute.Google Scholar
  6. Conrad, S., Oethinger, M., Kaifel, K., Klotz, G., Marre, R., and Kern, W.V. 1996. gyrA mutations in high-level fluoroquinolone-resistant clinical isolates of Escherichia coli. J. Antimicrob. Chemother.38, 443–455.PubMedGoogle Scholar
  7. Cui, Y., Yu, C., Yan, Y., Li, D., Li, Y., Jombart, T., Weinert, L.A., Wang, Z., Guo, Z., Xu, L., et al. 2013. Historical variations in mutation rate in an epidemic pathogen, Yersinia pestis. Proc. Natl. Acad. Sci. USA110, 577–582.PubMedGoogle Scholar
  8. DePristo, M.A., Banks, E., Poplin, R., Garimella, K.V., Maguire, J.R., Hartl, C., Philippakis, A.A., del Angel, G., Rivas, M.A., Hanna, M., et al. 2011. A framework for variation discovery and genotyping using next-generation DNA sequencing data. Nat. Genet.43, 491–498.PubMedPubMedCentralGoogle Scholar
  9. Drake, J.W., Charlesworth, B., Charlesworth, D., and Crow, J.F. 1998. Rates of spontaneous mutation. Genetics148, 1667–1686.PubMedPubMedCentralGoogle Scholar
  10. Drlica, K., Hiasa, H., Kerns, R., Malik, M., Mustaev, A., and Zhao, X. 2009. Quinolones: action and resistance updated. Curr. Top. Med. Chem.9, 981–998.PubMedPubMedCentralGoogle Scholar
  11. Drlica, K. and Zhao, X. 1997. DNA gyrase, topoisomerase IV, and the 4-quinolones. Microbiol. Mol. Biol. Rev.61, 377–392.PubMedPubMedCentralGoogle Scholar
  12. Edgar, R.C., Haas, B.J., Clemente, J.C., Quince, C., and Knight, R. 2011. UCHIME improves sensitivity and speed of chimera detection. Bioinformatics27, 2194–2200.PubMedPubMedCentralGoogle Scholar
  13. Ferrero, L., Cameron, B., and Crouzet, J. 1995. Analysis of gyrA and grlA mutations in stepwise-selected ciprofloxacin-resistant mutants of Staphylococcus aureus. Antimicrob. Agents Chemother.39, 1554–1558.PubMedPubMedCentralGoogle Scholar
  14. Furusawa, C., Horinouchi, T., and Maeda, T. 2018. Toward prediction and control of antibiotic-resistance evolution. Curr. Opin. Biotechnol.54, 45–49.PubMedGoogle Scholar
  15. Harkins, C.P., Pichon, B., Doumith, M., Parkhill, J., Westh, H., Tomasz, A., de Lencastre, H., Bentley, S.D., Kearns, A.M., and Holden, M.T.G. 2017. Methicillin-resistant Staphylococcus aureus emerged long before the introduction of methicillin into clinical practice. Genome Biol.18, 130.PubMedPubMedCentralGoogle Scholar
  16. Hermisson, J. and Pennings, P.S. 2005. Soft sweeps: molecular population genetics of adaptation from standing genetic variation. Genetics169, 2335–1352.PubMedPubMedCentralGoogle Scholar
  17. Hooper, D.C. 2000. Mechanisms of action and resistance of older and newer fluoroquinolones. Clin. Infect. Dis.31, S24–28.PubMedGoogle Scholar
  18. Hooper, D.C. 2001. Emerging mechanisms of fluoroquinolone resistance. Emerg. Infect. Dis.7, 337–341.PubMedPubMedCentralGoogle Scholar
  19. Huang, T., Zheng, Y., Yan, Y., Yang, L., Yao, Y., Zheng, J., Wu, L., Wang, X., Chen, Y., Xing, J., et al. 2016. Probing minority population of antibiotic-resistant bacteria. Biosens. Bioelectron.80, 323–330.PubMedGoogle Scholar
  20. Hughes, D. and Andersson, D.I. 2015. Evolutionary consequences of drug resistance: shared principles across diverse targets and organisms. Nat. Rev. Genet.16, 459–471.PubMedGoogle Scholar
  21. Jaskólska, M. and Gerdes, K. 2015. CRP-dependent positive autoregulation and proteolytic degradation regulate competence activator Sxy of Escherichia coli. Mol. Microbiol.95, 833–845.PubMedGoogle Scholar
  22. Jee, J., Rasouly, A., Shamovsky, I., Akivis, Y., Steinman, S.R., Mishra, B., and Nudler, E. 2016. Rates and mechanisms of bacterial mutagenesis from maximum-depth sequencing. Nature534, 693–696.PubMedPubMedCentralGoogle Scholar
  23. Jensen, J.D. 2014. On the unfounded enthusiasm for soft selective sweeps. Nat. Commun.5, 5281.PubMedGoogle Scholar
  24. Kim, J., Jeon, S., Kim, H., Park, M., Kim, S., and Kim, S. 2012. Multiplex real-time polymerase chain reaction-based method for the rapid detection of gyrA and parC mutations in quinolone-resistant Escherichia coli and Shigella spp. Osong Public Health Res. Perspect.3, 113–117.PubMedPubMedCentralGoogle Scholar
  25. Koch, L. 2017. Pathogen genetics: evolutionary dynamics driving drug resistance. Nat. Rev. Genet.18, 578–579.PubMedGoogle Scholar
  26. Komp Lindgren, P., Karlsson, A., and Hughes, D. 2003. Mutation rate and evolution of fluoroquinolone resistance in Escherichia coli isolates from patients with urinary tract infections. Antimicrob. Agents Chemother.47, 3222–3232.PubMedPubMedCentralGoogle Scholar
  27. Kurtz, S., Phillippy, A., Delcher, A.L., Smoot, M., Shumway, M., Antonescu, C., and Salzberg, S.L. 2004. Versatile and open software for comparing large genomes. Genome Biol.5, R12.PubMedPubMedCentralGoogle Scholar
  28. Lenski, R.E. 2017. Experimental evolution and the dynamics of adaptation and genome evolution in microbial populations. ISME J.11, 2181–2194.PubMedPubMedCentralGoogle Scholar
  29. Li, X., Mariano, N., Rahal, J.J., Urban, C.M., and Drlica, K. 2004. Quinolone-resistant Haemophilus influenzae: determination of mutant selection window for ciprofloxacin, garenoxacin, levofloxacin, and moxifloxacin. Antimicrob. Agents Chemother.48, 4460–4462.PubMedPubMedCentralGoogle Scholar
  30. Li, R., Zhu, H., Ruan, J., Qian, W., Fang, X., Shi, Z., Li, Y., Li, S., Shan, G., Kristiansen, K., et al. 2009. De novo assembly of human genomes with massively parallel short read sequencing. Genome Res.20, 265–272.PubMedGoogle Scholar
  31. Lin, W., Zeng, J., Wan, K., Lv, L., Guo, L., Li, X., and Yu, X. 2018. Reduction of the fitness cost of antibiotic resistance caused by chromosomal mutations under poor nutrient conditions. Environ. Int.120, 63–71.PubMedGoogle Scholar
  32. Lo, S.W., Kumar, N., and Wheeler, N.E. 2018. Breaking the code of antibiotic resistance. Nat. Rev. Microbiol.16, 262.PubMedGoogle Scholar
  33. Long, H., Miller, S.F., Strauss, C., Zhao, C., Cheng, L., Ye, Z., Griffin, K., Te, R., Lee, H., Chen, C.C., et al. 2016. Antibiotic treatment enhances the genome-wide mutation rate of target cells. Proc. Natl. Acad. Sci. USA113, E2498–2505.PubMedGoogle Scholar
  34. López, E., Elez, M., Matic, I., and Blázquez, J. 2007. Antibiotic-mediated recombination: ciprofloxacin stimulates SOS-independent recombination of divergent sequences in Escherichia coli. Mol. Microbiol.64, 83–93.PubMedGoogle Scholar
  35. Magoc, T. and Salzberg, S.L. 2011. FLASH: fast length adjustment of short reads to improve genome assemblies. Bioinformatics27, 2957–2963.PubMedPubMedCentralGoogle Scholar
  36. Marcusson, L.L., Frimodt-Moller, N., and Hughes, D. 2009. Interplay in the selection of fluoroquinolone resistance and bacterial fitness. PLoS Pathog.5, e1000541.PubMedPubMedCentralGoogle Scholar
  37. Martinez, J.L., Baquero, F., and Andersson, D.I. 2011. Beyond serial passages: new methods for predicting the emergence of resistance to novel antibiotics. Curr. Opin. Pharmacol.11, 439–445.PubMedGoogle Scholar
  38. Messer, P.W. and Petrov, D.A. 2013. Population genomics of rapid adaptation by soft selective sweeps. Trends Ecol. Evol.28, 659–669.PubMedGoogle Scholar
  39. Mezger, A., Gullberg, E., Goransson, J., Zorzet, A., Herthnek, D., Tano, E., Nilsson, M., Andersson, D.I. 2015. A general method for rapid determination of antibiotic susceptibility and species in bacterial infections. J. Clin. Microbiol.53, 425–432.PubMedPubMedCentralGoogle Scholar
  40. O’Toole, D.K. 2014. The natural environment may be the most important source of antibiotic resistance genes. MBio5, e01285–14.PubMedPubMedCentralGoogle Scholar
  41. Pallecchi, L., Bartoloni, A., Riccobono, E., Fernandez, C., Mantella, A., Magnelli, D., Mannini, D., Strohmeyer, M., Bartalesi, F., Rodriguez, H., et al. 2012. Quinolone resistance in absence of selective pressure: the experience of a very remote community in the Amazon forest. PLoS Negl. Trop. Dis.6, e1790.PubMedPubMedCentralGoogle Scholar
  42. Pennings, P.S. and Hermisson, J. 2006a. Soft sweeps II-molecular population genetics of adaptation from recurrent mutation or migration. Mol. Biol. Evol.23, 1076–1084.PubMedGoogle Scholar
  43. Pennings, P.S. and Hermisson, J. 2006b. Soft sweeps III: the signature of positive selection from recurrent mutation. PLoS Genet.2, e186.PubMedPubMedCentralGoogle Scholar
  44. Poteete, A.R., Sinha, S., and Redfield, R.J. 2012. Natural DNA uptake by Escherichia coli. PLoS One7, e35620.Google Scholar
  45. Redgrave, L.S., Sutton, S.B., Webber, M.A., and Piddock, L.J. 2014. Fluoroquinolone resistance: mechanisms, impact on bacteria, and role in evolutionary success. Trends Microbiol.22, 438–445.PubMedGoogle Scholar
  46. Ruiz, J., Gomez, J., Navia, M.M., Ribera, A., Sierra, J.M., Marco, F., Mensa, J., Vila, J., et al. 2002. High prevalence of nalidixic acid resistant, ciprofloxacin susceptible phenotype among clinical isolates of Escherichia coli and other Enterobacteriaceae. Diagn. Microbiol. Infect. Dis.42, 257–261.PubMedGoogle Scholar
  47. Schmitt, M.W., Kennedy, S.R., Salk, J.J., Fox, E.J., Hiatt, J.B., and Loeb, L.A. 2012. Detection of ultra-rare mutations by next-generation sequencing. Proc. Natl. Acad. Sci. USA109, 14508–14513.PubMedGoogle Scholar
  48. Solomon, J. and Grossman, A. 1996. Who’s competent and when: regulation of natural genetic competence in bacteria. Trends Genet.12, 150–155.PubMedGoogle Scholar
  49. Wichmann, F., Udikovik-Kolic, N., Andrew, S., and Handelsman, J. 2014. Reply to “The natural environment may be the most important source of antibiotic resistance genes”. MBio5, e01421–14.PubMedPubMedCentralGoogle Scholar
  50. Wilson, B.A., Pennings, P.S., and Petrov, D.A. 2017. Soft selective sweeps in evolutionary rescue. Genetics205, 1573–1586.PubMedPubMedCentralGoogle Scholar
  51. Yamada, K., Saito, R., Muto, S., Kashiwa, M., Tamamori, Y., and Fujisaki, S. 2017. Molecular characterization of fluoroquinolone-resistant Moraxella catarrhalis variants generated in vitro by step-wise selection. Antimicrob. Agents Chemother.61, e01336–17.PubMedPubMedCentralGoogle Scholar
  52. Zhang, G., Wang, C., Sui, Z., and Feng, J. 2015. Insights into the evolutionary trajectories of fluoroquinolone resistance in Streptococcus pneumoniae. J. Antimicrob. Chemother.70, 2499–2506.PubMedGoogle Scholar
  53. Zhao, X. and Drlica, K. 2001. Restricting the selection of antibiotic-resistant mutants: a general strategy derived from fluoroquinolone studies. Clin. Infect. Dis.33, S147–156.PubMedGoogle Scholar

Copyright information

© The Microbiological Society of Korea 2019

Authors and Affiliations

  • Xianxing Xie
    • 1
  • Ruichen Lv
    • 1
    • 2
  • Chao Yang
    • 1
  • Yajun Song
    • 1
  • Yanfeng Yan
    • 1
  • Yujun Cui
    • 1
  • Ruifu Yang
    • 1
    Email author
  1. 1.State Key Laboratory of Pathogen and BiosecurityBeijing Institute of Microbiology and EpidemiologyBeijingP. R. China
  2. 2.Huadong Research Institute for Medicine and BiotechnicsNanjingP. R. China

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