Skip to main content
SpringerLink
Log in

Soft sweep development of resistance in Escherichia coli under fluoroquinolone stress

  • Microbial Systematics and Evolutionary Microbiology
  • Published:
Journal of Microbiology Aims and scope Submit manuscript

Abstract

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.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  • Andersson, D.I. and Hughes, D. 2010. Antibiotic resistance and its cost: is it possible to reverse resistance? Nat. Rev. Microbiol.8, 260–271.

    Article  CAS  PubMed  Google Scholar 

  • 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.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • 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.

    Article  CAS  PubMed  Google Scholar 

  • 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.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • 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 

  • 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.

    Article  CAS  PubMed  Google Scholar 

  • 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.

    Article  CAS  PubMed  Google Scholar 

  • 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.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Drake, J.W., Charlesworth, B., Charlesworth, D., and Crow, J.F. 1998. Rates of spontaneous mutation. Genetics148, 1667–1686.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • 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.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Drlica, K. and Zhao, X. 1997. DNA gyrase, topoisomerase IV, and the 4-quinolones. Microbiol. Mol. Biol. Rev.61, 377–392.

    CAS  PubMed  PubMed Central  Google Scholar 

  • 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.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • 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.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Furusawa, C., Horinouchi, T., and Maeda, T. 2018. Toward prediction and control of antibiotic-resistance evolution. Curr. Opin. Biotechnol.54, 45–49.

    Article  CAS  PubMed  Google Scholar 

  • 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.

    Article  PubMed  PubMed Central  Google Scholar 

  • Hermisson, J. and Pennings, P.S. 2005. Soft sweeps: molecular population genetics of adaptation from standing genetic variation. Genetics169, 2335–1352.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Hooper, D.C. 2000. Mechanisms of action and resistance of older and newer fluoroquinolones. Clin. Infect. Dis.31, S24–28.

    Article  CAS  PubMed  Google Scholar 

  • Hooper, D.C. 2001. Emerging mechanisms of fluoroquinolone resistance. Emerg. Infect. Dis.7, 337–341.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • 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.

    Article  CAS  PubMed  Google Scholar 

  • 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.

    Article  CAS  PubMed  Google Scholar 

  • 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.

    Article  PubMed  CAS  Google Scholar 

  • 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.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Jensen, J.D. 2014. On the unfounded enthusiasm for soft selective sweeps. Nat. Commun.5, 5281.

    Article  CAS  PubMed  Google Scholar 

  • 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.

    Article  PubMed  PubMed Central  Google Scholar 

  • Koch, L. 2017. Pathogen genetics: evolutionary dynamics driving drug resistance. Nat. Rev. Genet.18, 578–579.

    Article  CAS  PubMed  Google Scholar 

  • 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.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • 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.

    Article  PubMed  PubMed Central  Google Scholar 

  • Lenski, R.E. 2017. Experimental evolution and the dynamics of adaptation and genome evolution in microbial populations. ISME J.11, 2181–2194.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • 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.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • 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.

    Article  PubMed  CAS  Google Scholar 

  • 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.

    Article  CAS  PubMed  Google Scholar 

  • Lo, S.W., Kumar, N., and Wheeler, N.E. 2018. Breaking the code of antibiotic resistance. Nat. Rev. Microbiol.16, 262.

    Article  CAS  PubMed  Google Scholar 

  • 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.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • 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.

    Article  PubMed  CAS  Google Scholar 

  • Magoc, T. and Salzberg, S.L. 2011. FLASH: fast length adjustment of short reads to improve genome assemblies. Bioinformatics27, 2957–2963.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Marcusson, L.L., Frimodt-Moller, N., and Hughes, D. 2009. Interplay in the selection of fluoroquinolone resistance and bacterial fitness. PLoS Pathog.5, e1000541.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • 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.

    Article  CAS  PubMed  Google Scholar 

  • Messer, P.W. and Petrov, D.A. 2013. Population genomics of rapid adaptation by soft selective sweeps. Trends Ecol. Evol.28, 659–669.

    Article  PubMed  Google Scholar 

  • 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.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • O’Toole, D.K. 2014. The natural environment may be the most important source of antibiotic resistance genes. MBio5, e01285–14.

    Article  PubMed  PubMed Central  Google Scholar 

  • 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.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • 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.

    Article  CAS  PubMed  Google Scholar 

  • Pennings, P.S. and Hermisson, J. 2006b. Soft sweeps III: the signature of positive selection from recurrent mutation. PLoS Genet.2, e186.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Poteete, A.R., Sinha, S., and Redfield, R.J. 2012. Natural DNA uptake by Escherichia coli. PLoS One7, e35620.

    Article  CAS  Google Scholar 

  • 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.

    Article  CAS  PubMed  Google Scholar 

  • 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.

    Article  CAS  PubMed  Google Scholar 

  • 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.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Solomon, J. and Grossman, A. 1996. Who’s competent and when: regulation of natural genetic competence in bacteria. Trends Genet.12, 150–155.

    Article  CAS  PubMed  Google Scholar 

  • 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.

    PubMed  PubMed Central  Google Scholar 

  • Wilson, B.A., Pennings, P.S., and Petrov, D.A. 2017. Soft selective sweeps in evolutionary rescue. Genetics205, 1573–1586.

    Article  PubMed  PubMed Central  Google Scholar 

  • 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.

    CAS  PubMed  PubMed Central  Google Scholar 

  • 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.

    Article  CAS  PubMed  Google Scholar 

  • 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.

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank Tamsin Sheen, PhD, from Liwen Bianji, Edanz Editing China (www.liwenbianji.cn/ac), 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).

Author information

Authors and Affiliations

  1. State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing, P. R. China

    Xianxing Xie, Ruichen Lv, Chao Yang, Yajun Song, Yanfeng Yan, Yujun Cui & Ruifu Yang

  2. Huadong Research Institute for Medicine and Biotechnics, Nanjing, P. R. China

    Ruichen Lv

Authors
  1. Xianxing Xie
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Ruichen Lv
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Chao Yang
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Yajun Song
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Yanfeng Yan
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Yujun Cui
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Ruifu Yang
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Ruifu Yang.

Additional information

Conflict of Interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Supplemental material for this article may be found at http://www.springerlink.com/content/120956.

Supporting Information

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Xie, X., Lv, R., Yang, C. et al. Soft sweep development of resistance in Escherichia coli under fluoroquinolone stress. J Microbiol. 57, 1056–1064 (2019). https://doi.org/10.1007/s12275-019-9177-5

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12275-019-9177-5

Keywords

Search

Navigation