Skip to main content

Thank you for visiting nature.com. 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.

  • Protocol Extension
  • Published:

Testing physiologically relevant conditions in minimal inhibitory concentration assays

Nature Protocols volume 16, pages 3761–3774 (2021)Cite this article

Subjects

Abstract

The minimal inhibitory concentration (MIC) assay uses agar or broth dilution methods to measure, under defined test conditions, the lowest effective concentration of an antimicrobial agent that inhibits visible growth of a bacterium of interest. This assay is used to test the susceptibilities of bacterial isolates and of novel antimicrobial drugs, and is typically done in nutrient-rich laboratory media that have little relevance to in vivo conditions. As an extension to our original protocol on MIC assays (also published in Nature Protocols), here we describe the application of the MIC broth microdilution assay to test antimicrobial susceptibility in conditions that are more physiologically relevant to infections observed in the clinic. Specifically, we describe a platform that can be applied to the preparation of medium that mimics lung and wound exudate or blood conditions for the growth and susceptibility testing of bacteria, including ESKAPE pathogens. This protocol can also be applied to most physiologically relevant liquid medium and aerobic pathogens, and takes 3–4 d to complete.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Simplified schematic of broth microdilution MIC assay using host-mimicking media.
Fig. 2: Outline of a 96-well plate MIC assay with eight different antibiotics.

Similar content being viewed by others

References

  1. Wiegand, I., Hilpert, K. & Hancock, R. E. W. Agar and broth dilution methods to determine the minimal inhibitory concentration (MIC) of antimicrobial substances. Nat. Protoc. 3, 163–175 (2008).

    Article  CAS  Google Scholar 

  2. Clinical Laboratory Standards Institute. Performance standards for antimicrobial susceptibility testing (CLSI document M100, 2020).

  3. Nizet, V. The accidental orthodoxy of Drs. Mueller and Hinton. EBioMedicine 22, 26–27 (2017).

    Article  Google Scholar 

  4. Cornforth, D. M., Diggle, F. L., Melvin, J. A., Bomberger, J. M. & Whiteley, M. Quantitative framework for model evaluation in microbiology research using Pseudomonas aeruginosa and cystic fibrosis infection as a test case. mBio 11, e03042-19 (2020).

    Article  Google Scholar 

  5. Tata, M. et al. RNASeq based transcriptional profiling of Pseudomonas aeruginosa PA14 after short- and long-term anoxic cultivation in synthetic cystic fibrosis sputum medium. PLoS One 11, e0147811 (2016).

    Article  Google Scholar 

  6. Palmer, K. L., Mashburn, L. M., Singh, P. K. & Whiteley, M. Cystic fibrosis sputum supports growth and cues key aspects of Pseudomonas aeruginosa physiology. J. Bacteriol 187, 5267–5277 (2005).

    Article  CAS  Google Scholar 

  7. Kruczek, C. et al. Major transcriptome changes accompany the growth of Pseudomonas aeruginosa in blood from patients with severe thermal injuries. PLoS One 11, e0149229 (2016).

    Article  Google Scholar 

  8. Umland, T. C. et al. In vivo-validated essential genes identified in Acinetobacter baumannii by using human ascites overlap poorly with essential genes detected on laboratory media. mBio 3, e00113-12 (2012).

    Article  Google Scholar 

  9. Thulin, E., Thulin, M. & Andersson, D. I. Reversion of high-level mecillinam resistance to susceptibility in Escherichia coli during growth in urine. EBioMedicine 23, 111–118 (2017).

    Article  Google Scholar 

  10. Lee, S. A. et al. General and condition-specific essential functions of Pseudomonas aeruginosa. Proc. Natl Acad. Sci. USA 112, 5189–5194 (2015).

    Article  CAS  Google Scholar 

  11. Ersoy, S. C. et al. Correcting a fundamental flaw in the paradigm for antimicrobial susceptibility testing. EBioMedicine 20, 173–181 (2017).

    Article  Google Scholar 

  12. Hancock, R. E. W. Rethinking the antibiotic discovery paradigm. EBioMedicine 2, 629–630 (2015).

    Article  Google Scholar 

  13. Kubicek-Sutherland, J. Z. et al. Host-dependent induction of transient antibiotic resistance: a prelude to treatment failure. EBioMedicine 2, 1169–1178 (2015).

    Article  Google Scholar 

  14. World Health Organization. Global priority list of antibiotic-resistant bacteria to guide research, discovery, and development of new antibiotics. http://www.who.int/medicines/publications/global-priority-list-antibiotic-resistant-bacteria/en/ (WHO, 2017).

  15. De Oliveira, D. M. P. et al. Antimicrobial resistance in ESKAPE pathogens. Clin. Microbiol. Rev. 33, e00181-19 (2020).

    Article  Google Scholar 

  16. Turner, K. H., Wessel, A. K., Palmer, G. C., Murray, J. L. & Whiteley, M. Essential genome of Pseudomonas aeruginosa in cystic fibrosis sputum. Proc. Natl Acad. Sci. 112, 4110–4115 (2015).

    Article  CAS  Google Scholar 

  17. Malachowa, N. et al. Global changes in Staphylococcus aureus gene expression in human blood. PLoS One 6, e18617 (2011).

    Article  CAS  Google Scholar 

  18. Quinn, B. et al. Human serum albumin alters specific genes that can play a role in survival and persistence in Acinetobacter baumannii. Sci. Rep. 8, 14741 (2018).

    Article  Google Scholar 

  19. Mäder, U. et al. Staphylococcus aureus transcriptome architecture: from laboratory to infection-mimicking conditions. PLoS Genet 12, e1005962 (2016).

    Article  Google Scholar 

  20. Belanger, C. R. et al. Identification of novel targets of azithromycin activity against Pseudomonas aeruginosa grown in physiologically relevant media. Proc. Natl Acad. Sci. 117, 33519–33529 (2020).

    Article  CAS  Google Scholar 

  21. Winsor, G. L. et al. Enhanced annotations and features for comparing thousands of Pseudomonas genomes in the Pseudomonas genome database. Nucleic Acids Res. 44, D646–D653 (2016).

    Article  CAS  Google Scholar 

  22. Fung, C. et al. Gene expression of Pseudomonas aeruginosa in a mucin-containing synthetic growth medium mimicking cystic fibrosis lung sputum. J. Med. Microbiol. 59, 1089–1100 (2010).

    Article  CAS  Google Scholar 

  23. Lin, L. et al. Azithromycin synergizes with cationic antimicrobial peptides to exert bactericidal and therapeutic activity against highly multidrug-resistant Gram-negative bacterial pathogens. EBioMedicine 2, 690–698 (2015).

    Article  Google Scholar 

  24. Colquhoun, J. M., Wozniak, R. A. F. & Dunman, P. M. Clinically relevant growth conditions alter Acinetobacter baumannii antibiotic susceptibility and promote identification of novel antibacterial agents. PLoS One 10, e0143033 (2015).

    Article  Google Scholar 

  25. Yeaman, M. R., Gank, K. D., Bayer, A. S. & Brass, E. P. Synthetic peptides that exert antimicrobial activities in whole blood and blood-derived matrices. Antimicrob. Agents Chemother. 46, 3883–3891 (2002).

    Article  CAS  Google Scholar 

  26. Weber, B. S. et al. Genetic and chemical screening in human blood serum reveals unique antibacterial targets and compounds against Klebsiella pneumoniae. Cell Rep 32, 107927 (2020).

    Article  CAS  Google Scholar 

  27. Palmer, K. L., Aye, L. M. & Whiteley, M. Nutritional cues control Pseudomonas aeruginosa multicellular behavior in cystic fibrosis sputum. J. Bacteriol. 189, 8079–8087 (2007).

    Article  CAS  Google Scholar 

  28. Kirchner, S. et al. Use of artificial sputum medium to test antibiotic efficacy against Pseudomonas aeruginosa in conditions more relevant to the cystic fibrosis lung. J. Vis. Exp. https://doi.org/10.3791/3857 (2012).

  29. Sriramulu, D. D., Lünsdorf, H., Lam, J. S. & Römling, U. Microcolony formation: a novel biofilm model of Pseudomonas aeruginosa for the cystic fibrosis lung. J. Med. Microbiol. 54, 667–676 (2005).

    Article  Google Scholar 

  30. Yeung, A. T. Y., Parayno, A. & Hancock, R. E. W. Mucin promotes rapid surface motility in Pseudomonas aeruginosa. mBio 3, e00073–12 (2012).

    Article  Google Scholar 

  31. Kreda, S. M., Davis, C. W. & Rose, M. C. CFTR, mucins, and mucus obstruction in cystic fibrosis. Cold Spring Harb. Perspect. Med. 2, a009589 (2012).

    Article  Google Scholar 

  32. Kirchner, K. K., Wagener, J. S., Khan, T. Z., Copenhaver, S. C. & Accurso, F. J. Increased DNA levels in bronchoalveolar lavage fluid obtained from infants with cystic fibrosis. Am. J. Respir. Crit. Care Med. 154, 1426–1429 (1996).

    Article  CAS  Google Scholar 

  33. Haney, E., Trimble, M., Cheng, J., Vallé, Q. & Hancock, R. Critical assessment of methods to quantify biofilm growth and evaluate antibiofilm activity of host defence peptides. Biomolecules 8, 29 (2018).

    Article  Google Scholar 

Download references

Acknowledgements

Our own antibiotics research is funded by the Canadian Institutes for Health Research Foundation grant FDN-154287. C.R.B. received a Doctoral Studentship Award from Cystic Fibrosis Canada. R.E.W.H. holds a Canada Research Chair in Health and Genomics and a UBC Killam Professorship.

Author information

Authors and Affiliations

  1. Centre for Microbial Diseases and Immunity Research, Department of Microbiology and Immunology, University of British Columbia, Vancouver, British Columbia, Canada

    Corrie R. Belanger & Robert E. W. Hancock

Authors
  1. Corrie R. Belanger
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Robert E. W. Hancock
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Robert E. W. Hancock.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Peer review information Nature Protocols thanks Fekade Sime and the other, anonymous reviewer(s) for their contribution to the peer review of this work.

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

Related links

Key reference using this protocol

Belanger C. R. et al. Proc. Natl Acad. Sci. USA 117, 33519–33529 (2020): https://doi.org/10.1073/pnas.2007626117

This protocol is an extension to: Nat. Protoc. 3, 163–175 (2008): https://doi.org/10.1038/nprot.2007.521

Supplementary information

Supplementary Information

Supplementary Table 1.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Belanger, C.R., Hancock, R.E.W. Testing physiologically relevant conditions in minimal inhibitory concentration assays. Nat Protoc 16, 3761–3774 (2021). https://doi.org/10.1038/s41596-021-00572-8

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41596-021-00572-8

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Advanced search

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