Skip to main content

Thank you for visiting You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

The high prevalence of antibiotic heteroresistance in pathogenic bacteria is mainly caused by gene amplification


When choosing antibiotics to treat bacterial infections, it is assumed that the susceptibility of the target bacteria to an antibiotic is reflected by laboratory estimates of the minimum inhibitory concentration (MIC) needed to prevent bacterial growth. A caveat of using MIC data for this purpose is heteroresistance, the presence of a resistant subpopulation in a main population of susceptible cells. We investigated the prevalence and mechanisms of heteroresistance in 41 clinical isolates of the pathogens Escherichia coli, Salmonella enterica, Klebsiella pneumoniae and Acinetobacter baumannii against 28 different antibiotics. For the 766 bacteria–antibiotic combinations tested, as much as 27.4% of the total was heteroresistant. Genetic analysis demonstrated that a majority of heteroresistance cases were unstable, with an increased resistance of the subpopulations resulting from spontaneous tandem amplifications, typically including known resistance genes. Using mathematical modelling, we show how heteroresistance in the parameter range estimated in this study can result in the failure of antibiotic treatment of infections with bacteria that are classified as antibiotic susceptible. The high prevalence of heteroresistance with the potential for treatment failure highlights the limitations of MIC as the sole criterion for susceptibility determinations. These results call for the development of facile and rapid protocols to identify heteroresistance in pathogens.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Fig. 1: A schematic outline of methods used to determine HR frequency and the genetics behind unstable susceptibility/resistance to antibiotics among clinical E. coli, S. Typhimurium, A. baumannii and K. pneumoniae
Fig. 2: Summary of tests performed and main results.
Fig. 3: Overexpression of potential genes involved in unstable HR.
Fig. 4: Molecular mechanisms of HR described in this work.

Data availability

Chromosomes and plasmids are deposited at NCBI under the following accession numbers: DA33098 (CP029569–CP029573), DA33133 (CP029574 and CP029575), DA33135 (CP029576–CP029578), DA33137 (CP029579–CP029581), DA33140 (CP029582–CP029586), DA33141 (CP029587–CP029589), DA33144 (CP029590–CP029592), DA33145 (CP029597–CP029599), DA33382 (CP030106–CP030109), DA34821 (CP029567), DA34827 (CP029593 and CP029594), DA34833 (CP029595 and CP029596) and DA34837 (CP029568). Additional data supporting the findings of this study, such as raw data and bacterial strains, are available upon request.


  1. 1.

    The Bacterial Challenge – Time to React ECDC/ EMEA Joint Technical Report (ECDC/EMEA Joint Working Group, 2009).

  2. 2.

    Antibiotic Resistance Threats in the United States (Centers for Disease Control and Prevention, Office of Infectious Diseases, 2013).

  3. 3.

    Frimodt-Møller, N. How predictive is PK/PD for antibacterial agents? Int. J. Antimicrob. Agents 19, 333–339 (2002).

    Article  PubMed  Google Scholar 

  4. 4.

    Asín-Prieto, E., Rodríguez-Gascón, A. & Isla, A. Applications of the pharmacokinetic/pharmacodynamic (PK/PD) analysis of antimicrobial agents. J. Infect. Chemother. 21, 319–329 (2015).

    Article  PubMed  Google Scholar 

  5. 5.

    Nielsen, E. I. & Friberg, L. E. Pharmacokinetic-pharmacodynamic modeling of antibacterial drugs. Pharmacol. Rev. 65, 1053–1090 (2013).

    Article  PubMed  Google Scholar 

  6. 6.

    El-Halfawy, O. M. & Valvano, M. A. Antimicrobial heteroresistance: an emerging field in need of clarity. Clin. Microbiol. Rev. 28, 191–207 (2015).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  7. 7.

    Alexander, H. E. & Leidy, G. Mode of action of streptomycin on type b Haemophilus influenzae: I. Origin of resistant organisms. J. Exp. Med. 85, 329–338 (1947).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  8. 8.

    Band, V. I. et al. Antibiotic failure mediated by a resistant subpopulation in Enterobacter cloacae. Nat. Microbiol. 1, 16053 (2016).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  9. 9.

    Higgins, P. G., Schneiders, T., Hamprecht, A. & Seifert, H. In vivo selection of a missense mutation in adeR and conversion of the novel blaOXA-164 gene into blaOXA-58 in carbapenem-resistant Acinetobacter baumannii isolates from a hospitalized patient. Antimicrob. Agents Chemother. 54, 5021–5027 (2010).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  10. 10.

    Hernan, R. C. et al. Selection of colistin-resistant Acinetobacter baumannii isolates in postneurosurgical meningitis in an intensive care unit with high presence of heteroresistance to colistin. Diagn. Microbiol. Infect. Dis. 65, 188–191 (2009).

    Article  PubMed  Google Scholar 

  11. 11.

    Tan, C.-H., Li, J. & Nation, R. L. Activity of colistin against heteroresistant Acinetobacter baumannii and emergence of resistance in an in vitro pharmacokinetic/pharmacodynamic model. Antimicrob. Agents Chemother. 51, 3413–3415 (2007).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  12. 12.

    Band, V. I. et al. Carbapenem-resistant Klebsiella pneumoniae exhibiting clinically undetected colistin heteroresistance leads to treatment failure in a murine model of infection. mBio 9, e02448–17 (2018).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  13. 13.

    Moore, M. R., Perdreau-Remington, F. & Chambers, H. F. Vancomycin treatment failure associated with heterogeneous vancomycin-intermediate Staphylococcus aureus in a patient with endocarditis and in the rabbit model of endocarditis. Antimicrob. Agents Chemother. 47, 1262–1266 (2003).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  14. 14.

    Yau, W. et al. Colistin hetero-resistance in multidrug-resistant Acinetobacter baumannii clinical isolates from the Western Pacific region in the SENTRY antimicrobial surveillance programme. J. Infect. 58, 138–144 (2009).

    Article  PubMed  Google Scholar 

  15. 15.

    Meletis, G., Tzampaz, E., Sianou, E., Tzavaras, I. & Sofianou, D. Colistin heteroresistance in carbapenemase-producing Klebsiella pneumoniae. J. Antimicrob. Chemother. 66, 946–947 (2011).

    CAS  Article  PubMed  Google Scholar 

  16. 16.

    Sun, J. D. et al. Impact of carbapenem heteroresistance among clinical isolates of invasive Escherichia coli in Chongqing, southwestern China. Clin. Microbiol. Infect. 21, 469.e1–469.e10 (2015).

    CAS  Article  Google Scholar 

  17. 17.

    Mei, S., Gao, Y., Zhu, C., Dong, C. & Chen, Y. Research of the heteroresistance of Pseudomonas aeruginosa to imipenem. Int. J. Clin. Exp. Med. 8, 6129–6132 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  18. 18.

    van Hal, S. J. et al. Performance of various testing methodologies for detection of heteroresistant vancomycin-intermediate Staphylococcus aureus in bloodstream isolates. J. Clin. Microbiol. 49, 1489–1494 (2011).

    Article  PubMed  PubMed Central  Google Scholar 

  19. 19.

    Lo-Ten-Foe, J. R., de Smet, A. M. G. A., Diederen, B. M. W., Kluytmans, J. A. J. W. & van Keulen, P. H. J. Comparative evaluation of the VITEK 2, disk diffusion, etest, broth microdilution, and agar dilution susceptibility testing methods for colistin in clinical isolates, including heteroresistant Enterobacter cloacae and Acinetobacter baumannii. Antimicrob. Agents Chemother. 51, 3726–3730 (2007).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  20. 20.

    Lee, H.-Y. et al. Imipenem heteroresistance induced by imipenem in multidrug-resistant Acinetobacter baumannii: mechanism and clinical implications. Int. J. Antimicrob. Agents 37, 302–308 (2011).

    CAS  Article  PubMed  Google Scholar 

  21. 21.

    Hjort, K., Nicoloff, H. & Andersson, D. I. Unstable tandem gene amplification generates heteroresistance (variation in resistance within a population) to colistin in Salmonella enterica. Mol. Microbiol. 102, 274–289 (2016).

    CAS  Article  PubMed  Google Scholar 

  22. 22.

    Halaby, T. et al. Genomic characterization of colistin heteroresistance in Klebsiella pneumoniae during a nosocomial outbreak. Antimicrob. Agents Chemother. 60, 6837–6843 (2016).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  23. 23.

    Jayol, A., Nordmann, P., Brink, A. & Poirel, L. Heteroresistance to colistin in Klebsiella pneumoniae associated with alterations in the PhoPQ regulatory system. Antimicrob. Agents Chemother. 59, 2780–2784 (2015).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  24. 24.

    Schechter, L. M. et al. Extensive gene amplification as a mechanism for piperacillin-tazobactam resistance in Escherichia coli. mBio 9, e00583-18 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  25. 25.

    Williams, A. B. Spontaneous mutation rates come into focus in Escherichia coli. DNA Repair 24, 73–79 (2014).

    CAS  Article  PubMed  Google Scholar 

  26. 26.

    Lee, J.-Y., Choi, M.-J., Choi, H. J. & Ko, K. S. Preservation of acquired colistin resistance in Gram-negative bacteria. Antimicrob. Agents Chemother. 60, 609–612 (2015).

    Article  PubMed  PubMed Central  Google Scholar 

  27. 27.

    Barin, J., Martins, A. F., Heineck, B. L., Barth, A. L. & Zavascki, A. P. Hetero- and adaptive resistance to polymyxin B in OXA-23-producing carbapenem-resistant Acinetobacter baumannii isolates. Ann. Clin. Microbiol. Antimicrob. 12, 15 (2013).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  28. 28.

    Poudyal, A. et al. In vitro pharmacodynamics of colistin against multidrug-resistant Klebsiella pneumoniae. J. Antimicrob. Chemother. 62, 1311–1318 (2008).

    CAS  Article  PubMed  Google Scholar 

  29. 29.

    Cai, Y., Chai, D., Wang, R., Liang, B. & Bai, N. Colistin resistance of Acinetobacter baumannii: clinical reports, mechanisms and antimicrobial strategies. J. Antimicrob. Chemother. 67, 1607–1615 (2012).

    CAS  Article  PubMed  Google Scholar 

  30. 30.

    Reams, A. B. & Roth, J. R. Mechanisms of gene duplication and amplification. Cold Spring Harb. Perspect. Biol. 7, a016592 (2015).

    Article  PubMed  PubMed Central  Google Scholar 

  31. 31.

    Ramiro, R. S., Costa, H. & Gordo, I. Macrophage adaptation leads to parallel evolution of genetically diverse Escherichia coli small-colony variants with increased fitness in vivo and antibiotic collateral sensitivity. Evol. Appl. 9, 994–1004 (2016).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  32. 32.

    Paradise, M. R., Cook, G., Poole, R. K. & Rather, P. N. Mutations in aarE, the ubiA homolog of Providencia stuartii, result in high-level aminoglycoside resistance and reduced expression of the chromosomal aminoglycoside 2′-N-acetyltransferase. Antimicrob. Agents Chemother. 42, 959–962 (1998).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  33. 33.

    Gallagher, L. A., Lee, S. A. & Manoil, C. Importance of core genome functions for an extreme antibiotic resistance trait. mBio 8, e01655-17 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  34. 34.

    Shan, Y., Lazinski, D., Rowe, S., Camilli, A. & Lewis, K. Genetic basis of persister tolerance to aminoglycosides in Escherichia coli. mBio 6, e00078-15 (2015).

    Article  PubMed  PubMed Central  Google Scholar 

  35. 35.

    Tian, Z.-X., Yi, X.-X., Cho, A., O’Gara, F. & Wang, Y.-P. CpxR activates MexAB-OprM efflux pump expression and enhances antibiotic resistance in both laboratory and clinical nalB-type isolates of Pseudomonas aeruginosa. PLoS Pathog. 12, e1005932 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  36. 36.

    Weatherspoon-Griffin, N., Yang, D., Kong, W., Hua, Z. & Shi, Y. The CpxR/CpxA two-component regulatory system up-regulates the multidrug resistance cascade to facilitate Escherichia coli resistance to a model antimicrobial peptide. J. Biol. Chem. 289, 32571–32582 (2014).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  37. 37.

    Shalel-Levanon, S., San, K.-Y. & Bennett, G. N. Effect of oxygen, and ArcA and FNR regulators on the expression of genes related to the electron transfer chain and the TCA cycle in Escherichia coli. Metab. Eng. 7, 364–374 (2005).

    CAS  Article  PubMed  Google Scholar 

  38. 38.

    Coyne, S., Courvalin, P. & Périchon, B. Efflux-mediated antibiotic resistance in Acinetobacter spp. Antimicrob. Agents Chemother. 55, 947–953 (2011).

    CAS  Article  PubMed  Google Scholar 

  39. 39.

    El Meouche, I., Siu, Y. & Dunlop, M. J. Stochastic expression of a multiple antibiotic resistance activator confers transient resistance in single cells. Sci. Rep. 6, 19538 (2016).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  40. 40.

    Sanchez-Romero, M. A. & Casadesus, J. Contribution of phenotypic heterogeneity to adaptive antibiotic resistance. Proc. Natl Acad. Sci. USA 111, 355–360 (2014).

    Article  PubMed  Google Scholar 

  41. 41.

    Allen, R. C., Engelstädter, J., Bonhoeffer, S., McDonald, B. A. & Hall, A. R. Reversing resistance: different routes and common themes across pathogens. P. Roy. Soc. B-Biol. Sci. 284, 20171619 (2017).

    Article  Google Scholar 

  42. 42.

    Anderson, P. & Roth, J. Spontaneous tandem genetic duplications in Salmonella typhimurium arise by unequal recombination between rRNA (rrn) cistrons. Proc. Natl Acad. Sci. USA 78, 3113–3117 (1981).

    CAS  Article  PubMed  Google Scholar 

  43. 43.

    Sandegren, L. & Andersson, D. I. Bacterial gene amplification: implications for the evolution of antibiotic resistance. Nat. Rev. Microbiol. 7, 578–588 (2009).

    CAS  Article  PubMed  Google Scholar 

  44. 44.

    Reams, A. B., Kofoid, E., Savageau, M. & Roth, J. R. Duplication frequency in a population of Salmonella enterica rapidly approaches steady state with or without recombination. Genetics 184, 1077–1094 (2010).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  45. 45.

    Adler, M., Anjum, M., Berg, O. G., Andersson, D. I. & Sandegren, L. High fitness costs and instability of gene duplications reduce rates of evolution of new genes by duplication-divergence mechanisms. Mol. Biol. Evol. 31, 1526–1535 (2014).

    CAS  Article  PubMed  Google Scholar 

  46. 46.

    Sun, S., Berg, O. G., Roth, J. R. & Andersson, D. I. Contribution of gene amplification to evolution of increased antibiotic resistance in Salmonella typhimurium. Genetics 182, 1183–1195 (2009).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  47. 47.

    Regoes, R. R. et al. Pharmacodynamic functions: a multiparameter approach to the design of antibiotic treatment regimens. Antimicrob. Agents Chemother. 48, 3670–3676 (2004).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  48. 48.

    Kussell, E., Kishony, R., Balaban, N. Q. & Leibler, S. Bacterial persistence: a model of survival in changing environments. Genetics 169, 1807–1814 (2005).

    Article  PubMed  PubMed Central  Google Scholar 

  49. 49.

    Johnson, P. J. T. & Levin, B. R. Pharmacodynamics, population dynamics, and the evolution of persistence in Staphylococcus aureus. PLoS Genet. 9, e1003123 (2013).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  50. 50.

    Levin, B. R., Concepción-Acevedo, J. & Udekwu, K. I. Persistence: a copacetic and parsimonious hypothesis for the existence of non-inherited resistance to antibiotics. Curr. Opin. Microbiol. 21, 18–21 (2014).

    CAS  Article  PubMed  Google Scholar 

  51. 51.

    Anderson, S. E., Sherman, E. X., Weiss, D. S. & Rather, P. N. Aminoglycoside heteroresistance in Acinetobacter baumannii AB5075. mSphere 3, e00271-18 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  52. 52.

    Levin, B. R., Baquero, F., Ankomah, P. & McCall, I. C. Phagocytes, antibiotics, and self-limiting bacterial infections. Trends Microbiol. 25, 878–892 (2017).

    CAS  Article  PubMed  Google Scholar 

  53. 53.

    Wootton, M. et al. A modified population analysis profile (PAP) method to detect hetero-resistance to vancomycin in Staphylococcus aureus in a UK hospital. J. Antimicrob. Chemother. 47, 399–403 (2001).

    CAS  Article  PubMed  Google Scholar 

  54. 54.

    Sutton, S. Measurement of microbial cells by optical density. J. Valid. Technol. 17, 46–49 (2011).

    Google Scholar 

  55. 55.

    Udekwu, K. I., Parrish, N., Ankomah, P., Baquero, F. & Levin, B. R. Functional relationship between bacterial cell density and the efficacy of antibiotics. J. Antimicrob. Chemother. 63, 745–757 (2009).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  56. 56.

    Thulin, M. BAT v.2.0 (Uppsala University, 2018);

  57. 57.

    Nicoloff, H. & Andersson, D. I. Indirect resistance to several classes of antibiotics in cocultures with resistant bacteria expressing antibiotic-modifying or -degrading enzymes. J. Antimicrob. Chemother. 71, 100–110 (2016).

    CAS  Article  PubMed  Google Scholar 

  58. 58.

    Datsenko, K. A. & Wanner, B. L. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc. Natl Acad. Sci. USA 97, 6640–6645 (2000).

    CAS  Article  PubMed  Google Scholar 

  59. 59.

    Zankari, E. et al. Identification of acquired antimicrobial resistance genes. J. Antimicrob. Chemother. 67, 2640–2644 (2012).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

Download references


We thank F.F. Cuenca, P.G. Higgins and C.-H. Chiu for the A. baumannii isolates, L. Sandegren for the E. coli isolates, Å. Melhus and B. Lytsy for some K. pneumoniae isolates and C. Järnberg at The Public Health Agency, Sweden for the S. Typhimurium isolates. We also would like to thank O. Warsi and U. Lustig for helping with the MiSeq sequencer and DNA libraries preparations. This work was supported by grants from the Swedish Research Council, no. 2017-01527 (DIA), and the US National Institutes of General Medical Sciences, no. GM091875 (BRL).

Author information




H.N., K.H. and D.I.A. designed the study. H.N. and K.H. performed the experiments and B.R.L. the mathematical modelling. H.N., K.H., B.R.L. and D.I.A. wrote the manuscript.

Corresponding author

Correspondence to Dan I. Andersson.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Information

Supplementary Figures 1–12, Supplementary Tables 1–5, Supplementary Methods and Supplementary References.

Reporting Summary

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Nicoloff, H., Hjort, K., Levin, B.R. et al. The high prevalence of antibiotic heteroresistance in pathogenic bacteria is mainly caused by gene amplification. Nat Microbiol 4, 504–514 (2019).

Download citation

Further reading


Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing