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Volume 166, Issue 12

Editor's Choice Effect of host-mimicking medium and biofilm growth on the ability of colistin to kill Open Access

Abstract

biofilms cause recalcitrant infections with extensive and unpredictable antibiotic tolerance. Here, we demonstrate increased tolerance of colistin by when grown in medium that mimics cystic fibrosis (CF) sputum versus standard medium in biofilm assays, and drastically increased tolerance when grown in an CF model versus the assay. We used colistin conjugated to the fluorescent dye BODIPY to assess the penetration of the antibiotic into biofilms and showed that poor penetration partly explains the high doses of drug necessary to kill bacteria in these biofilms. The ability of antibiotics to penetrate the biofilm matrix is key to their clinical success, but hard to measure. Our results demonstrate both the importance of reduced entry into the matrix in -like biofilm, and the tractability of using a fluorescent tag and benchtop fluorimeter to assess antibiotic entry into biofilms. This method could be a relatively quick, cheap and useful addition to diagnostic and drug development pipelines, allowing the assessment of drug entry into biofilms, in -like conditions, prior to more detailed tests of biofilm killing.

  • Received:
  • Accepted:
  • Published Online:
Keyword(s): antibiotic tolerance , antibiotics , biofilm , cystic fibrosis and infection models
Funding
This study was supported by the:
  • Medical Research Council (Award MR/N014103/1)
    • Principle Award Recipient: AkshaySabnis
  • Medical Research Council (Award MR/R001898/1)
    • Principle Award Recipient: FreyaHarrison
© 2020 The Authors
  • This is an open-access article distributed under the terms of the Creative Commons Attribution License.
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2020-11-30
2024-04-26
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References

  1. Penesyan A, Gillings M, Paulsen IT. Antibiotic discovery: combatting bacterial resistance in cells and in biofilm communities. Molecules 2015; 20:5286 [View Article][PubMed]
     [Google Scholar]
  2. Bjarnsholt T, Jensen Peter Østrup, Fiandaca MJ, Pedersen J, Hansen CR et al. Pseudomonas aeruginosa biofilms in the respiratory tract of cystic fibrosis patients. Pediatr Pulmonol 2009; 44:547–558 [View Article][PubMed]
     [Google Scholar]
  3. Elborn JS. Cystic fibrosis. The Lancet 2016; 388:2519–2531
     [Google Scholar]
  4. Angelis A, Kanavos P, López-Bastida J, Linertová R, Nicod E et al. Social and economic costs and health-related quality of life in non-institutionalised patients with cystic fibrosis in the United Kingdom. BMC Health Serv Res 2015; 15:428 [View Article][PubMed]
     [Google Scholar]
  5. Eidt-Koch D, Wagner TOF, Mittendorf T, Graf von der Schulenburg JMG. Outpatient medication costs of patients with cystic fibrosis in Germany. Appl Health Econ Health Policy 2010; 8:111–118 [View Article][PubMed]
     [Google Scholar]
  6. Trust CF Annual data report 2018. 2019
  7. Smith AL, Fiel SB, Mayer-Hamblett N, Ramsey B, Burns JL. Susceptibility testing of Pseudomonas aeruginosa isolates and clinical response to parenteral antibiotic administration: lack of association in cystic fibrosis. Chest 2003; 123:1495–1502 [View Article][PubMed]
     [Google Scholar]
  8. Waters V, Ratjen F. Standard versus biofilm antimicrobial susceptibility testing to guide antibiotic therapy in cystic fibrosis. Cochrane Database Syst Rev 2017; 10:CD009528 [View Article][PubMed]
     [Google Scholar]
  9. Ceri H, Olson ME, Stremick C, Read RR, Morck D et al. The Calgary biofilm device: new technology for rapid determination of antibiotic susceptibilities of bacterial biofilms. J Clin Microbiol 1999; 37:1771–1776 [View Article][PubMed]
     [Google Scholar]
  10. Moskowitz SM, Foster JM, Emerson J, Burns JL. Clinically feasible biofilm susceptibility assay for isolates of Pseudomonas aeruginosa from patients with cystic fibrosis. J Clin Microbiol 2004; 42:1915–1922 [View Article][PubMed]
     [Google Scholar]
  11. Ersoy SC, Heithoff DM, Barnes L, Tripp GK, House JK et al. Correcting a fundamental Flaw in the paradigm for antimicrobial susceptibility testing. EBioMedicine 2017; 20:173–181 [View Article][PubMed]
     [Google Scholar]
  12. Kubicek-Sutherland JZ, Heithoff DM, Ersoy SC, Shimp WR, House JK et al. Host-dependent induction of transient antibiotic resistance: a prelude to treatment failure. EBioMedicine 2015; 2:1169–1178 [View Article][PubMed]
     [Google Scholar]
  13. Kirchner S, Fothergill JL, Wright EA, James CE, Mowat E 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 2012; 64:3857 [View Article][PubMed]
     [Google Scholar]
  14. Drevinek P, Holden MTG, Ge Z, Jones AM, Ketchell I et al. Gene expression changes linked to antimicrobial resistance, oxidative stress, iron depletion and retained motility are observed when Burkholderia cenocepacia grows in cystic fibrosis sputum. BMC Infect Dis 2008; 8:121 [View Article][PubMed]
     [Google Scholar]
  15. Billings N, Birjiniuk A, Samad TS, Doyle PS, Ribbeck K. Material properties of biofilms-a review of methods for understanding permeability and mechanics. Rep Prog Phys 2015; 78:036601 [View Article][PubMed]
     [Google Scholar]
  16. Coenye T, Nelis HJ. In vitro and in vivo model systems to study microbial biofilm formation. J Microbiol Methods 2010; 83:89–105 [View Article][PubMed]
     [Google Scholar]
  17. Roberts AEL, Kragh KN, Bjarnsholt T, Diggle SP. The limitations of in vitro experimentation in understanding biofilms and chronic infection. J Mol Biol 2015; 427:3646–3661 [View Article][PubMed]
     [Google Scholar]
  18. Kragh KN, Alhede M, Kvich L, Bjarnsholt T. Into the well—A close look at the complex structures of a microtiter biofilm and the crystal violet assay. Biofilm 2019; 100006:
     [Google Scholar]
  19. Sabnis A, Klöckner A, Becce M, Hagart KLH, Evans LE et al. Colistin kills bacteria by targeting lipopolysaccharide in the cytoplasmic membrane. bioRxiv 2020; 479618:
     [Google Scholar]
  20. Billings N, Millan M, Caldara M, Rusconi R, Tarasova Y et al. The extracellular matrix component Psl provides fast-acting antibiotic defense in Pseudomonas aeruginosa biofilms. PLoS Pathog 2013; 9:e1003526 [View Article][PubMed]
     [Google Scholar]
  21. Yokota S-I, Hakamada H, Yamamoto S, Sato T, Shiraishi T et al. Release of large amounts of lipopolysaccharides from Pseudomonas aeruginosa cells reduces their susceptibility to colistin. Int J Antimicrob Agents 2018; 51:888–896 [View Article][PubMed]
     [Google Scholar]
  22. Manning AJ, Kuehn MJ. Contribution of bacterial outer membrane vesicles to innate bacterial defense. BMC Microbiol 2011; 11:258 [View Article][PubMed]
     [Google Scholar]
  23. Palmer KL, Aye LM, Whiteley M. Nutritional cues control Pseudomonas aeruginosa multicellular behavior in cystic fibrosis sputum. J Bacteriol 2007; 189:8079–8087 [View Article][PubMed]
     [Google Scholar]
  24. Harrington NE, Sweeney E, Harrison F. Building a better biofilm - Formation of in vivo-like biofilm structures by Pseudomonas aeruginosa in a porcine model of cystic fibrosis lung infection. Biofilm 2020; 2:100024
     [Google Scholar]
  25. Harrison F, Diggle SP. An ex vivo lung model to study bronchioles infected with Pseudomonas aeruginosa biofilms. Microbiology 2016; 162:1755–1760 [View Article][PubMed]
     [Google Scholar]
  26. Darch S, McNally A, Harrison F, Corander J, Barr H et al. Recombination is a key driver of genomic and phenotypic diversity in a Pseudomonas aeruginosa population during cystic fibrosis infection. Nature Scientific Reports 2015; 5:764
     [Google Scholar]
  27. Cornforth DM, Dees JL, Ibberson CB, Huse HK, Mathiesen IH et al. Pseudomonas aeruginosa transcriptome during human infection. Proc Natl Acad Sci U S A 2018; 115:E5125–E5134 [View Article][PubMed]
     [Google Scholar]
  28. Hogardt M, Trebesius K, Geiger AM, Hornef M, Rosenecker J et al. Specific and rapid detection by fluorescent in situ hybridization of bacteria in clinical samples obtained from cystic fibrosis patients. J Clin Microbiol 2000; 38:818–825 [View Article][PubMed]
     [Google Scholar]
  29. Lam JC, Somayaji R, Surette MG, Rabin HR, Parkins MD. Reduction in Pseudomonas aeruginosa sputum density during a cystic fibrosis pulmonary exacerbation does not predict clinical response. BMC Infect Dis 2015; 15:145 [View Article][PubMed]
     [Google Scholar]
  30. Stressmann FA, Rogers GB, Marsh P, Lilley AK, Daniels TW et al. Does bacterial density in cystic fibrosis sputum increase prior to pulmonary exacerbation?. J Cyst Fibros 2011; 10:357–365 [View Article][PubMed]
     [Google Scholar]
  31. Caceres SM, Malcolm KC, Taylor-Cousar JL, Nichols DP, Saavedra MT et al. Enhanced in vitro formation and antibiotic resistance of nonattached Pseudomonas aeruginosa aggregates through incorporation of neutrophil products. Antimicrob Agents Chemother 2014; 58:6851–6860 [View Article][PubMed]
     [Google Scholar]
  32. DePas WH, Starwalt-Lee R, Van Sambeek L, Ravindra Kumar S, Gradinaru V et al. Exposing the three-dimensional biogeography and metabolic states of pathogens in cystic fibrosis sputum via hydrogel embedding, clearing, and rRNA labeling. mBio 2016; 7:e00796-16 [View Article][PubMed]
     [Google Scholar]
  33. Rueden CT, Schindelin J, Hiner MC, DeZonia BE, Walter AE et al. ImageJ2: imageJ for the next generation of scientific image data. BMC Bioinformatics 2017; 18:529 [View Article][PubMed]
     [Google Scholar]
  34. Couet W, Grégoire N, Marchand S, Mimoz O. Colistin pharmacokinetics: the fog is lifting. Clin Microbiol Infect 2012; 18:30–39 [View Article][PubMed]
     [Google Scholar]
  35. Karvanen M, Malmberg C, Lagerbäck P, Friberg LE, Cars O. Colistin Is Extensively Lost during Standard In Vitro Experimental Conditions. Antimicrob Agents Chemother 2017; 61:e00857–17 [View Article][PubMed]
     [Google Scholar]
  36. Walters MC, Roe F, Bugnicourt A, Franklin MJ, Stewart PS. Contributions of antibiotic penetration, oxygen limitation, and low metabolic activity to tolerance of Pseudomonas aeruginosa biofilms to ciprofloxacin and tobramycin. Antimicrob Agents Chemother 2003; 47:317–323 [View Article][PubMed]
     [Google Scholar]
  37. Ratjen F, Rietschel E, Kasel D, Schwiertz R, Starke K et al. Pharmacokinetics of inhaled colistin in patients with cystic fibrosis. J Antimicrob Chemother 2006; 57:306–311 [View Article][PubMed]
     [Google Scholar]
  38. Yapa S, Li J, Patel K, Wilson JW, Dooley MJ et al. Pulmonary and systemic pharmacokinetics of inhaled and intravenous colistin methanesulfonate in cystic fibrosis patients: targeting advantage of inhalational administration. Antimicrob Agents Chemother 2014; 58:2570–2579 [View Article][PubMed]
     [Google Scholar]
  39. Turner KH, Wessel AK, Palmer GC, Murray JL, Whiteley M. Essential genome of Pseudomonas aeruginosa in cystic fibrosis sputum. Proc Natl Acad Sci U S A 2015; 112:4110–4115 [View Article][PubMed]
     [Google Scholar]
  40. Huang JX, Blaskovich MAT, Pelingon R, Ramu S, Kavanagh A et al. Mucin binding reduces colistin antimicrobial activity. Antimicrob Agents Chemother 2015; 59:5925–5931 [View Article][PubMed]
     [Google Scholar]
  41. Hunter JD. Ventilator associated pneumonia. BMJ 2012; 344:e3325 [View Article]
     [Google Scholar]
  42. Wilson R, Sethi S, Anzueto A, Miravitlles M. Antibiotics for treatment and prevention of exacerbations of chronic obstructive pulmonary disease. J Infect 2013; 67:497–515 [View Article][PubMed]
     [Google Scholar]
  43. Schultz G, Bjarnsholt T, James GA, Leaper DJ, McBain AJ et al. Consensus guidelines for the identification and treatment of biofilms in chronic nonhealing wounds. Wound Repair Regen 2017; 25:744–757 [View Article][PubMed]
     [Google Scholar]
  44. Percival SL, Suleman L, Vuotto C, Donelli G. Healthcare-associated infections, medical devices and biofilms: risk, tolerance and control. J Med Microbiol 2015; 64:323–334 [View Article][PubMed]
     [Google Scholar]
  45. Rumbaugh KP. How well are we translating biofilm research from bench-side to bedside?. Biofilm 2020; 2:100028
     [Google Scholar]
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Effect of host-mimicking medium and biofilm growth on the ability of colistin to kill Pseudomonas aeruginosa
Microbiology 166, 1171 (2020); https://doi.org/10.1099/mic.0.000995
/content/journal/micro/10.1099/mic.0.000995
/content/journal/micro/10.1099/mic.0.000995
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