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  • Review Article
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Tolerance and resistance of microbial biofilms

Nature Reviews Microbiology volume 20, pages 621–635 (2022)Cite this article

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Abstract

Chronic infections caused by microbial biofilms represent an important clinical challenge. The recalcitrance of microbial biofilms to antimicrobials and to the immune system is a major cause of persistence and clinical recurrence of these infections. In this Review, we present the extent of the clinical problem, and the mechanisms underlying the tolerance of biofilms to antibiotics and to host responses. We also explore the role of biofilms in the development of antimicrobial resistance mechanisms.

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Fig. 1: Tolerance of microbial biofilms to host immune responses.
Fig. 2: The mechanisms of antimicrobial tolerance of a biofilm.
Fig. 3: The development of antimicrobial resistance in planktonic and biofilm-growing bacterial populations.

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References

  1. Davey, M. E. & O’Toole, G. A. Microbial biofilms: from ecology to molecular genetics. Microbiol. Mol. Biol. Rev. 64, 847–867 (2000).

    CAS  PubMed  PubMed Central  Google Scholar 

  2. Rinaudi, L. V. & Giordano, W. An integrated view of biofilm formation in rhizobia. FEMS Microbiol. Lett. 304, 1–11 (2010).

    CAS  PubMed  Google Scholar 

  3. Brandwein, M., Steinberg, D. & Meshner, S. Microbial biofilms and the human skin microbiome. NPJ Biofilms. Microbiomes. 2, 3 (2016).

    PubMed  PubMed Central  Google Scholar 

  4. Hardy, L., Cerca, N., Jespers, V., Vaneechoutte, M. & Crucitti, T. Bacterial biofilms in the vagina. Res. Microbiol. 168, 865–874 (2017).

    PubMed  Google Scholar 

  5. Motta, J. P., Wallace, J. L., Buret, A. G., Deraison, C. & Vergnolle, N. Gastrointestinal biofilms in health and disease. Nat. Rev. Gastroenterol. Hepatol. 18, 314–334 (2021).

    PubMed  Google Scholar 

  6. Costerton, J. W. et al. Bacterial biofilms in nature and disease. Annu. Rev. Microbiol. 41, 435–464 (1987).

    CAS  PubMed  Google Scholar 

  7. Bjarnsholt, T. et al. The in vivo biofilm. Trends Microbiol. 21, 466–474 (2013).

    CAS  PubMed  Google Scholar 

  8. Lebeaux, D., Ghigo, J. M. & Beloin, C. Biofilm-related infections: bridging the gap between clinical management and fundamental aspects of recalcitrance toward antibiotics. Microbiol. Mol. Biol. Rev. 78, 510–543 (2014).

    PubMed  PubMed Central  Google Scholar 

  9. Rupp, M. E. & Karnatak, R. Intravascular catheter-related bloodstream infections. Infect. Dis. Clin. North Am. 32, 765–787 (2018).

    PubMed  Google Scholar 

  10. Kurtz, S., Ong, K., Lau, E., Mowat, F. & Halpern, M. Projections of primary and revision hip and knee arthroplasty in the United States from 2005 to 2030. J. Bone Jt. Surg. Am. 89, 780–785 (2007).

    Google Scholar 

  11. James, G. A. et al. Biofilms in chronic wounds. Wound Repair. Regen. 16, 37–44 (2008).

    PubMed  Google Scholar 

  12. Moser, C. et al. Biofilms and host response-helpful or harmful. APMIS 125, 320–338 (2017).

    PubMed  Google Scholar 

  13. Hoiby, N. et al. ESCMID guideline for the diagnosis and treatment of biofilm infections 2014. Clin. Microbiol. Infect. 21 (Suppl. 1), 1–25 (2015).

    Google Scholar 

  14. Moser, C. et al. Immune responses to Pseudomonas aeruginosa biofilm infections. Front. Immunol. 12, 625597 (2021).

    CAS  PubMed  PubMed Central  Google Scholar 

  15. Werdan, K. et al. Mechanisms of infective endocarditis: pathogen-host interaction and risk states. Nat. Rev. Cardiol. 11, 35–50 (2014).

    CAS  PubMed  Google Scholar 

  16. Foster, T. J., Geoghegan, J. A., Ganesh, V. K. & Hook, M. Adhesion, invasion and evasion: the many functions of the surface proteins of Staphylococcus aureus. Nat. Rev. Microbiol. 12, 49–62 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  17. Arciola, C. R., Campoccia, D. & Montanaro, L. Implant infections: adhesion, biofilm formation and immune evasion. Nat. Rev. Microbiol. 16, 397–409 (2018).

    CAS  PubMed  Google Scholar 

  18. Tattevin, P. et al. Risk factors and prognostic impact of left ventricular assist device-associated infections. Am. Heart J. 214, 69–76 (2019).

    PubMed  Google Scholar 

  19. Trautner, B. W. & Darouiche, R. O. Role of biofilm in catheter-associated urinary tract infection. Am. J. Infect. Control. 32, 177–183 (2004).

    PubMed  PubMed Central  Google Scholar 

  20. Pitts, N. B. et al. Dental caries. Nat. Rev. Dis. Prim. 3, 17030 (2017).

    PubMed  Google Scholar 

  21. Kimbrell, D. A. & Beutler, B. The evolution and genetics of innate immunity. Nat. Rev. Genet. 2, 256–267 (2001).

    CAS  PubMed  Google Scholar 

  22. Jensen, E. T. et al. Complement activation by Pseudomonas aeruginosa biofilms. Microb. Pathog. 15, 377–388 (1993).

    CAS  PubMed  Google Scholar 

  23. Rybtke, M., Jensen, P. O., Nielsen, C. H. & Tolker-Nielsen, T. The extracellular polysaccharide matrix of pseudomonas aeruginosa biofilms is a determinant of polymorphonuclear leukocyte responses. Infect. Immun. 89, e00631-20 (2020).

    PubMed  PubMed Central  Google Scholar 

  24. Secor, P. R. et al. Pf bacteriophage and their impact on pseudomonas virulence, mammalian immunity, and chronic infections. Front. Immunol. 11, 244 (2020).

    CAS  PubMed  PubMed Central  Google Scholar 

  25. Herant, M., Heinrich, V. & Dembo, M. Mechanics of neutrophil phagocytosis: experiments and quantitative models. J. Cell Sci. 119, 1903–1913 (2006).

    CAS  PubMed  Google Scholar 

  26. Alhede, M. et al. Bacterial aggregate size determines phagocytosis efficiency of polymorphonuclear leukocytes. Med. Microbiol. Immunol. 209, 669–680 (2020).

    CAS  PubMed  PubMed Central  Google Scholar 

  27. Jones, C. J. & Wozniak, D. J. Psl produced by mucoid pseudomonas aeruginosa contributes to the establishment of biofilms and immune evasion. mBio 8, e00864–17 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  28. Leid, J. G. et al. The exopolysaccharide alginate protects Pseudomonas aeruginosa biofilm bacteria from IFN-gamma-mediated macrophage killing. J. Immunol. 175, 7512–7518 (2005).

    CAS  PubMed  Google Scholar 

  29. de, V. L., Rooijakkers, S. H. M. & van Strijp, J. A. G. Staphylococci evade the innate immune response by disarming neutrophils and forming biofilms. FEBS Lett. 594, 2556–2569 (2020).

    Google Scholar 

  30. Pier, G. B., Coleman, F., Grout, M., Franklin, M. & Ohman, D. E. Role of alginate O acetylation in resistance of mucoid Pseudomonas aeruginosa to opsonic phagocytosis. Infect. Immun. 69, 1895–1901 (2001).

    CAS  PubMed  PubMed Central  Google Scholar 

  31. Bjarnsholt, T. et al. Pseudomonas aeruginosa biofilms in the respiratory tract of cystic fibrosis patients. Pediatr. Pulmonol. 44, 547–558 (2009).

    PubMed  Google Scholar 

  32. Jensen, P. O. et al. Rapid necrotic killing of polymorphonuclear leukocytes is caused by quorum-sensing-controlled production of rhamnolipid by Pseudomonas aeruginosa. Microbiology 153, 1329–1338 (2007).

    CAS  PubMed  Google Scholar 

  33. Alhede, M. et al. Pseudomonas aeruginosa recognizes and responds aggressively to the presence of polymorphonuclear leukocytes. Microbiology 155, 3500–3508 (2009).

    CAS  PubMed  Google Scholar 

  34. Mauch, R. M., Jensen, P. O., Moser, C., Levy, C. E. & Hoiby, N. Mechanisms of humoral immune response against Pseudomonas aeruginosa biofilm infection in cystic fibrosis. J. Cyst. Fibros. 17, 143–152 (2018).

    CAS  PubMed  Google Scholar 

  35. Libraty, D. H., Patkar, C. & Torres, B. Staphylococcus aureus reactivation osteomyelitis after 75 years. N. Engl. J. Med. 366, 481–482 (2012).

    CAS  PubMed  Google Scholar 

  36. Brauner, A., Fridman, O., Gefen, O. & Balaban, N. Q. Distinguishing between resistance, tolerance and persistence to antibiotic treatment. Nat. Rev. Microbiol. 14, 320–330 (2016).

    CAS  PubMed  Google Scholar 

  37. Anwar, H., van, B. T., Dasgupta, M., Lam, K. & Costerton, J. W. Interaction of biofilm bacteria with antibiotics in a novel in vitro chemostat system. Antimicrob. Agents Chemother. 33, 1824–1826 (1989).

    CAS  PubMed  PubMed Central  Google Scholar 

  38. Hengzhuang, W., Wu, H., Ciofu, O., Song, Z. & Hoiby, N. Pharmacokinetics/pharmacodynamics of colistin and imipenem on mucoid and nonmucoid Pseudomonas aeruginosa biofilms. Antimicrob. Agents Chemother. 55, 4469–4474 (2011).

    PubMed  PubMed Central  Google Scholar 

  39. Hoiby, N. et al. Formation of Pseudomonas aeruginosa inhibition zone during tobramycin disk diffusion is due to transition from planktonic to biofilm mode of growth. Int. J. Antimicrob. Agents 53, 564–573 (2019).

    PubMed  Google Scholar 

  40. Cruz, C. D., Shah, S. & Tammela, P. Defining conditions for biofilm inhibition and eradication assays for Gram-positive clinical reference strains. BMC Microbiol. 18, 173 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  41. Mottola, C. et al. Susceptibility patterns of Staphylococcus aureus biofilms in diabetic foot infections. BMC Microbiol. 16, 119 (2016).

    PubMed  PubMed Central  Google Scholar 

  42. Macia, M. D., Rojo-Molinero, E. & Oliver, A. Antimicrobial susceptibility testing in biofilm-growing bacteria. Clin. Microbiol. Infect. 20, 981–990 (2014).

    CAS  PubMed  Google Scholar 

  43. Fernandez-Barat, L. et al. Phenotypic shift in Pseudomonas aeruginosa populations from cystic fibrosis lungs after 2-week antipseudomonal treatment. J. Cyst. Fibros. 16, 222–229 (2017).

    PubMed  Google Scholar 

  44. Cao, B. et al. Antibiotic penetration and bacterial killing in a Pseudomonas aeruginosa biofilm model. J. Antimicrob. Chemother. 70, 2057–2063 (2015).

    CAS  PubMed  Google Scholar 

  45. Cao, B. et al. Diffusion retardation by binding of tobramycin in an alginate biofilm model. PLoS ONE 11, e0153616 (2016).

    PubMed  PubMed Central  Google Scholar 

  46. Ciofu, O. & Tolker-Nielsen, T. Tolerance and resistance of pseudomonas aeruginosa biofilms to antimicrobial agents-how p. aeruginosa can escape antibiotics. Front. Microbiol. 10, 913 (2019).

    PubMed  PubMed Central  Google Scholar 

  47. Sweeney, E., Sabnis, A., Edwards, A. M. & Harrison, F. Effect of host-mimicking medium and biofilm growth on the ability of colistin to kill Pseudomonas aeruginosa. Microbiology 166, 1171–1180 (2020).

    CAS  PubMed  PubMed Central  Google Scholar 

  48. Bagge, N. et al. Pseudomonas aeruginosa biofilms exposed to imipenem exhibit changes in global gene expression and beta-lactamase and alginate production. Antimicrob. Agents Chemother. 48, 1175–1187 (2004).

    CAS  PubMed  PubMed Central  Google Scholar 

  49. Kranjec, C. et al. Staphylococcal biofilms: challenges and novel therapeutic perspectives. Antibiotics 10, 131 (2021).

    CAS  PubMed  PubMed Central  Google Scholar 

  50. Ciofu, O., Beveridge, T. J., Kadurugamuwa, J., Walther-Rasmussen, J. & Hoiby, N. Chromosomal beta-lactamase is packaged into membrane vesicles and secreted from Pseudomonas aeruginosa. J. Antimicrob. Chemother. 45, 9–13 (2000).

    CAS  PubMed  Google Scholar 

  51. Bagge, N. et al. Dynamics and spatial distribution of beta-lactamase expression in Pseudomonas aeruginosa biofilms. Antimicrob. Agents Chemother. 48, 1168–1174 (2004).

    CAS  PubMed  PubMed Central  Google Scholar 

  52. Webb, J. S. et al. Cell death in Pseudomonas aeruginosa biofilm development. J. Bacteriol. 185, 4585–4592 (2003).

    CAS  PubMed  PubMed Central  Google Scholar 

  53. Christophersen, L. et al. In vivo demonstration of Pseudomonas aeruginosa biofilms as independent pharmacological microcompartments. J. Cyst. Fibros. 19, 996–1003 (2020).

    CAS  PubMed  Google Scholar 

  54. Stewart, P. S. & Franklin, M. J. Physiological heterogeneity in biofilms. Nat. Rev. Microbiol. 6, 199–210 (2008).

    CAS  PubMed  Google Scholar 

  55. Pamp, S. J., Gjermansen, M., Johansen, H. K. & Tolker-Nielsen, T. Tolerance to the antimicrobial peptide colistin in Pseudomonas aeruginosa biofilms is linked to metabolically active cells, and depends on the pmr and mexAB-oprM genes. Mol. Microbiol. 68, 223–240 (2008).

    CAS  PubMed  Google Scholar 

  56. Stokes, J. M., Lopatkin, A. J., Lobritz, M. A. & Collins, J. J. Bacterial metabolism and antibiotic efficacy. Cell Metab. 30, 251–259 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  57. Haagensen, J. et al. Spatiotemporal pharmacodynamics of meropenem- and tobramycin-treated Pseudomonas aeruginosa biofilms. J. Antimicrob. Chemother. 72, 3357–3365 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  58. Moskowitz, S. M., Foster, J. M., Emerson, J. & Burns, J. L. Clinically feasible biofilm susceptibility assay for isolates of Pseudomonas aeruginosa from patients with cystic fibrosis. J. Clin. Microbiol. 42, 1915–1922 (2004).

    CAS  PubMed  PubMed Central  Google Scholar 

  59. Levin, B. R. & Rozen, D. E. Non-inherited antibiotic resistance. Nat. Rev. Microbiol. 4, 556–562 (2006).

    CAS  PubMed  Google Scholar 

  60. Greulich, P., Scott, M., Evans, M. R. & Allen, R. J. Growth-dependent bacterial susceptibility to ribosome-targeting antibiotics. Mol. Syst. Biol. 11, 796 (2015).

    PubMed  Google Scholar 

  61. Herrmann, G. et al. Colistin-tobramycin combinations are superior to monotherapy concerning the killing of biofilm Pseudomonas aeruginosa. J. Infect. Dis. 202, 1585–1592 (2010).

    CAS  PubMed  Google Scholar 

  62. Hansen, C. R., Pressler, T. & Hoiby, N. Early aggressive eradication therapy for intermittent Pseudomonas aeruginosa airway colonization in cystic fibrosis patients: 15 years experience. J. Cyst. Fibros. 7, 523–530 (2008).

    CAS  PubMed  Google Scholar 

  63. Zimmerli, W. & Sendi, P. Role of rifampin against staphylococcal biofilm infections in vitro, in animal models, and in orthopedic-device-related infections. Antimicrob. Agents Chemother. 63, e01746-18 (2019).

    PubMed  PubMed Central  Google Scholar 

  64. Zimmerli, W. & Sendi, P. Orthopaedic biofilm infections. APMIS 125, 353–364 (2017).

    PubMed  Google Scholar 

  65. Testa, S. et al. Spatial structure affects phage efficacy in infecting dual-strain biofilms of Pseudomonas aeruginosa. Commun. Biol. 2, 405 (2019).

    PubMed  PubMed Central  Google Scholar 

  66. Henriksen, K. et al. P. aeruginosa flow-cell biofilms are enhanced by repeated phage treatments but can be eradicated by phage-ciprofloxacin combination. Pathog. Dis. 77, ftz011 (2019).

    CAS  PubMed  Google Scholar 

  67. Hansen, M. F., Svenningsen, S. L., Roder, H. L., Middelboe, M. & Burmolle, M. Big impact of the tiny: bacteriophage-bacteria interactions in biofilms. Trends Microbiol. 27, 739–752 (2019).

    CAS  PubMed  Google Scholar 

  68. Balaban, N. Q. et al. Definitions and guidelines for research on antibiotic persistence. Nat. Rev. Microbiol. 17, 441–448 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  69. Defraine, V., Fauvart, M. & Michiels, J. Fighting bacterial persistence: current and emerging anti-persister strategies and therapeutics. Drug Resist. Updat. 38, 12–26 (2018).

    PubMed  Google Scholar 

  70. Fisher, R. A., Gollan, B. & Helaine, S. Persistent bacterial infections and persister cells. Nat. Rev. Microbiol. 15, 453–464 (2017).

    CAS  PubMed  Google Scholar 

  71. Wilmaerts, D., Windels, E. M., Verstraeten, N. & Michiels, J. General mechanisms leading to persister formation and awakening. Trends Genet. 35, 401–411 (2019).

    CAS  PubMed  Google Scholar 

  72. Haussler, S., Tummler, B., Weissbrodt, H., Rohde, M. & Steinmetz, I. Small-colony variants of Pseudomonas aeruginosa in cystic fibrosis. Clin. Infect. Dis. 29, 621–625 (1999).

    CAS  PubMed  Google Scholar 

  73. Vulin, C., Leimer, N., Huemer, M., Ackermann, M. & Zinkernagel, A. S. Prolonged bacterial lag time results in small colony variants that represent a sub-population of persisters. Nat. Commun. 9, 4074 (2018).

    PubMed  PubMed Central  Google Scholar 

  74. Pestrak, M. J. et al. Pseudomonas aeruginosa rugose small-colony variants evade host clearance, are hyper-inflammatory, and persist in multiple host environments. PLoS Pathog. 14, e1006842 (2018).

    PubMed  PubMed Central  Google Scholar 

  75. Tuchscherr, L., Loffler, B. & Proctor, R. A. Persistence of staphylococcus aureus: multiple metabolic pathways impact the expression of virulence factors in small-colony variants (SCVs). Front. Microbiol. 11, 1028 (2020).

    PubMed  PubMed Central  Google Scholar 

  76. Bogut, A. & Magrys, A. The road to success of coagulase-negative staphylococci: clinical significance of small colony variants and their pathogenic role in persistent infections. Eur. J. Clin. Microbiol. Infect. Dis. 40, 2249–2270 (2021).

    CAS  PubMed  PubMed Central  Google Scholar 

  77. Dengler Haunreiter, V. et al. In-host evolution of Staphylococcus epidermidis in a pacemaker-associated endocarditis resulting in increased antibiotic tolerance. Nat. Commun. 10, 1149 (2019).

    PubMed  PubMed Central  Google Scholar 

  78. Wellinghausen, N. et al. Characterization of clinical Enterococcus faecalis small-colony variants. J. Clin. Microbiol. 47, 2802–2811 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  79. Rani, S. A. et al. Spatial patterns of DNA replication, protein synthesis, and oxygen concentration within bacterial biofilms reveal diverse physiological states. J. Bacteriol. 189, 4223–4233 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  80. Sonderholm, M. et al. The consequences of being in an infectious biofilm: microenvironmental conditions governing antibiotic tolerance. Int. J. Mol. Sci. 18, 2688 (2017).

    PubMed Central  Google Scholar 

  81. Stewart, P. S. et al. Contribution of stress responses to antibiotic tolerance in Pseudomonas aeruginosa biofilms. Antimicrob. Agents Chemother. 59, 3838–3847 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  82. Kolpen, M. et al. Reinforcement of the bactericidal effect of ciprofloxacin on Pseudomonas aeruginosa biofilm by hyperbaric oxygen treatment. Int. J. Antimicrob. Agents 47, 163–167 (2016).

    CAS  PubMed  Google Scholar 

  83. Tuomanen, E., Cozens, R., Tosch, W., Zak, O. & Tomasz, A. The rate of killing of Escherichia coli by beta-lactam antibiotics is strictly proportional to the rate of bacterial growth. J. Gen. Microbiol. 132, 1297–1304 (1986).

    CAS  PubMed  Google Scholar 

  84. Brochmann, R. P. et al. Bactericidal effect of colistin on planktonic Pseudomonas aeruginosa is independent of hydroxyl radical formation. Int. J. Antimicrob. Agents 43, 140–147 (2014).

    CAS  PubMed  Google Scholar 

  85. Dwyer, D. J. et al. Antibiotics induce redox-related physiological alterations as part of their lethality. Proc. Natl Acad. Sci. USA 111, E2100–E2109 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  86. Kolpen, M. et al. Polymorphonuclear leucocytes consume oxygen in sputum from chronic Pseudomonas aeruginosa pneumonia in cystic fibrosis. Thorax 65, 57–62 (2010).

    CAS  PubMed  Google Scholar 

  87. Worlitzsch, D. et al. Effects of reduced mucus oxygen concentration in airway Pseudomonas infections of cystic fibrosis patients. J. Clin. Invest. 109, 317–325 (2002).

    CAS  PubMed  PubMed Central  Google Scholar 

  88. Kolpen, M. et al. Nitrous oxide production in sputum from cystic fibrosis patients with chronic Pseudomonas aeruginosa lung infection. PLoS ONE 9, e84353 (2014).

    PubMed  PubMed Central  Google Scholar 

  89. Kragh, K. N. et al. Polymorphonuclear leukocytes restrict growth of Pseudomonas aeruginosa in the lungs of cystic fibrosis patients. Infect. Immun. 82, 4477–4486 (2014).

    PubMed  PubMed Central  Google Scholar 

  90. Kolpen, M. et al. Denitrification by cystic fibrosis pathogens-Stenotrophomonas maltophilia is dormant in sputum. Int. J. Med. Microbiol. 305, 1–10 (2015).

    CAS  PubMed  Google Scholar 

  91. DePas, W. H. et al. Exposing the three-dimensional biogeography and metabolic states of pathogens in cystic fibrosis sputum via hydrogel embedding, clearing, and rRNA labeling. mBio 7, e00796–16 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  92. Chao, Y., Marks, L. R., Pettigrew, M. M. & Hakansson, A. P. Streptococcus pneumoniae biofilm formation and dispersion during colonization and disease. Front. Cell Infect. Microbiol. 4, 194 (2014).

    PubMed  Google Scholar 

  93. Zumft, W. G. Cell biology and molecular basis of denitrification. Microbiol. Mol. Biol. Rev. 61, 533–616 (1997).

    CAS  PubMed  PubMed Central  Google Scholar 

  94. Line, L. et al. Physiological levels of nitrate support anoxic growth by denitrification of Pseudomonas aeruginosa at growth rates reported in cystic fibrosis lungs and sputum. Front. Microbiol. 5, 554 (2014).

    PubMed  PubMed Central  Google Scholar 

  95. Eschbach, M. et al. Long-term anaerobic survival of the opportunistic pathogen Pseudomonas aeruginosa via pyruvate fermentation. J. Bacteriol. 186, 4596–4604 (2004).

    CAS  PubMed  PubMed Central  Google Scholar 

  96. Bernier, S. P., Ha, D. G., Khan, W., Merritt, J. H. & O’Toole, G. A. Modulation of Pseudomonas aeruginosa surface-associated group behaviors by individual amino acids through c-di-GMP signaling. Res. Microbiol. 162, 680–688 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  97. Rossi, E. et al. Pseudomonas aeruginosa adaptation and evolution in patients with cystic fibrosis. Nat. Rev. Microbiol. 19, 331–342 (2021).

    CAS  PubMed  Google Scholar 

  98. Aanaes, K. et al. Decreased mucosal oxygen tension in the maxillary sinuses in patients with cystic fibrosis. J. Cyst. Fibros. 10, 114–120 (2011).

    PubMed  Google Scholar 

  99. Aanaes, K. et al. Secretory IgA as a diagnostic tool for Pseudomonas aeruginosa respiratory colonization. J. Cyst. Fibros. 12, 81–87 (2013).

    CAS  PubMed  Google Scholar 

  100. Ciofu, O. et al. P. aeruginosa in the paranasal sinuses and transplanted lungs have similar adaptive mutations as isolates from chronically infected CF lungs. J. Cyst. Fibros. 12, 729–736 (2013).

    PubMed  Google Scholar 

  101. Schreml, S. et al. Luminescent dual sensors reveal extracellular pH-gradients and hypoxia on chronic wounds that disrupt epidermal repair. Theranostics 4, 721–735 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  102. James, G. A. et al. Microsensor and transcriptomic signatures of oxygen depletion in biofilms associated with chronic wounds. Wound Repair Regen. 24, 373–383 (2016).

    PubMed  PubMed Central  Google Scholar 

  103. Debats, I. B. et al. Infected chronic wounds show different local and systemic arginine conversion compared with acute wounds. J. Surg. Res. 134, 205–214 (2006).

    CAS  PubMed  Google Scholar 

  104. Fazli, M. et al. Quantitative analysis of the cellular inflammatory response against biofilm bacteria in chronic wounds. Wound Repair Regen. 19, 387–391 (2011).

    PubMed  Google Scholar 

  105. Trostrup, H. et al. Pseudomonas aeruginosa biofilm aggravates skin inflammatory response in BALB/c mice in a novel chronic wound model. Wound Repair Regen. 21, 292–299 (2013).

    PubMed  Google Scholar 

  106. Wu, Y., Klapper, I. & Stewart, P. S. Hypoxia arising from concerted oxygen consumption by neutrophils and microorganisms in biofilms. Pathog. Dis. 76, fty043 (2018).

    PubMed Central  Google Scholar 

  107. Hunt, T. K., Zederfeldt, B. & Goldstick, T. K. Oxygen and healing. Am. J. Surg. 118, 521–525 (1969).

    CAS  PubMed  Google Scholar 

  108. Frykberg, R. G. et al. A multinational, multicenter, randomized, double-blinded, placebo-controlled trial to evaluate the efficacy of cyclical topical wound oxygen (TWO2) therapy in the treatment of chronic diabetic foot ulcers: the TWO2 Study. Diabetes Care 43, 616–624 (2020).

    CAS  PubMed  Google Scholar 

  109. Boutte, C. C. & Crosson, S. Bacterial lifestyle shapes stringent response activation. Trends Microbiol. 21, 174–180 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  110. Nguyen, D. et al. Active starvation responses mediate antibiotic tolerance in biofilms and nutrient-limited bacteria. Science 334, 982–986 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  111. Hobbs, J. K. & Boraston, A. B. (p)ppGpp and the stringent response: an emerging threat to antibiotic therapy. ACS Infect. Dis. 5, 1505–1517 (2019).

    CAS  PubMed  Google Scholar 

  112. Kohanski, M. A., Dwyer, D. J. & Collins, J. J. How antibiotics kill bacteria: from targets to networks. Nat. Rev. Microbiol. 8, 423–435 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  113. Blazquez, J., Rodriguez-Beltran, J. & Matic, I. Antibiotic-induced genetic variation: how it arises and how it can be prevented. Annu. Rev. Microbiol. 72, 209–230 (2018).

    CAS  PubMed  Google Scholar 

  114. Nang, S. C., Azad, M. A. K., Velkov, T., Zhou, Q. T. & Li, J. Rescuing the last-line polymyxins: achievements and challenges. Pharmacol. Rev. 73, 679–728 (2021).

    CAS  PubMed  PubMed Central  Google Scholar 

  115. Fernandez, L. & Hancock, R. E. Adaptive and mutational resistance: role of porins and efflux pumps in drug resistance. Clin. Microbiol. Rev. 25, 661–681 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  116. Chiang, W. C., Pamp, S. J., Nilsson, M., Givskov, M. & Tolker-Nielsen, T. The metabolically active subpopulation in Pseudomonas aeruginosa biofilms survives exposure to membrane-targeting antimicrobials via distinct molecular mechanisms. FEMS Immunol. Med. Microbiol. 65, 245–256 (2012).

    CAS  PubMed  Google Scholar 

  117. Poole, K. Stress responses as determinants of antimicrobial resistance in Pseudomonas aeruginosa: multidrug efflux and more. Can. J. Microbiol. 60, 783–791 (2014).

    CAS  PubMed  Google Scholar 

  118. Buroni, S. et al. Differential role of RND efflux pumps in antimicrobial drug resistance of sessile and planktonic Burkholderia cenocepacia cells. Antimicrob. Agents Chemother. 58, 7424–7429 (2014).

    PubMed  PubMed Central  Google Scholar 

  119. Zhang, Y. et al. ampG gene of Pseudomonas aeruginosa and its role in beta-lactamase expression. Antimicrob. Agents Chemother. 54, 4772–4779 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  120. Cornforth, D. M. et al. Pseudomonas aeruginosa transcriptome during human infection. Proc. Natl Acad. Sci. USA 115, E5125–E5134 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  121. Liao, J., Schurr, M. J. & Sauer, K. The MerR-like regulator BrlR confers biofilm tolerance by activating multidrug efflux pumps in Pseudomonas aeruginosa biofilms. J. Bacteriol. 195, 3352–3363 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  122. Liao, J. & Sauer, K. The MerR-like transcriptional regulator BrlR contributes to Pseudomonas aeruginosa biofilm tolerance. J. Bacteriol. 194, 4823–4836 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  123. Poudyal, B. & Sauer, K. The PA3177 gene encodes an active diguanylate cyclase that contributes to biofilm antimicrobial tolerance but not biofilm formation by pseudomonas aeruginosa. Antimicrob. Agents Chemother. 62, e01049–18 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  124. Mah, T. F. et al. A genetic basis for Pseudomonas aeruginosa biofilm antibiotic resistance. Nature 426, 306–310 (2003).

    CAS  PubMed  Google Scholar 

  125. Beaudoin, T., Zhang, L., Hinz, A. J., Parr, C. J. & Mah, T. F. The biofilm-specific antibiotic resistance gene ndvB is important for expression of ethanol oxidation genes in Pseudomonas aeruginosa biofilms. J. Bacteriol. 194, 3128–3136 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  126. Fernandez, L., Rodriguez, A. & Garcia, P. Phage or foe: an insight into the impact of viral predation on microbial communities. ISME J. 12, 1171–1179 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  127. Bjedov, I. et al. Stress-induced mutagenesis in bacteria. Science 300, 1404–1409 (2003).

    CAS  PubMed  Google Scholar 

  128. Conibear, T. C., Collins, S. L. & Webb, J. S. Role of mutation in Pseudomonas aeruginosa biofilm development. PLoS ONE 4, e6289 (2009).

    PubMed  PubMed Central  Google Scholar 

  129. Driffield, K., Miller, K., Bostock, J. M., O’Neill, A. J. & Chopra, I. Increased mutability of Pseudomonas aeruginosa in biofilms. J. Antimicrob. Chemother. 61, 1053–1056 (2008).

    CAS  PubMed  Google Scholar 

  130. Sekowska, A., Wendel, S., Fischer, E. C., Norholm, M. H. H. & Danchin, A. Generation of mutation hotspots in ageing bacterial colonies. Sci. Rep. 6, 2 (2016).

    PubMed  PubMed Central  Google Scholar 

  131. Levin-Reisman, I. et al. Antibiotic tolerance facilitates the evolution of resistance. Science 355, 826–830 (2017).

    CAS  PubMed  Google Scholar 

  132. Levin-Reisman, I., Brauner, A., Ronin, I. & Balaban, N. Q. Epistasis between antibiotic tolerance, persistence, and resistance mutations. Proc. Natl Acad. Sci. USA 116, 14734–14739 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  133. Melnyk, A. H., Wong, A. & Kassen, R. The fitness costs of antibiotic resistance mutations. Evol. Appl. 8, 273–283 (2015).

    PubMed  Google Scholar 

  134. Rainey, P. B., Buckling, A., Kassen, R. & Travisano, M. The emergence and maintenance of diversity: insights from experimental bacterial populations. Trends Ecol. Evol. 15, 243–247 (2000).

    CAS  PubMed  Google Scholar 

  135. Perron, G. G., Gonzalez, A. & Buckling, A. Source-sink dynamics shape the evolution of antibiotic resistance and its pleiotropic fitness cost. Proc. Biol. Sci. 274, 2351–2356 (2007).

    PubMed  PubMed Central  Google Scholar 

  136. Santos-Lopez, A., Marshall, C. W., Scribner, M. R., Snyder, D. J. & Cooper, V. S. Evolutionary pathways to antibiotic resistance are dependent upon environmental structure and bacterial lifestyle. eLife 8, e47612 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  137. Harris, K. B., Flynn, K. M. & Cooper, V. S. Polygenic adaptation and clonal interference enable sustained diversity in experimental Pseudomonas aeruginosa populations. Mol. Biol. Evol. 38, 5359–5375 (2021).

    CAS  PubMed  PubMed Central  Google Scholar 

  138. Ahmed, M. N., Porse, A., Sommer, M. O. A., Hoiby, N. & Ciofu, O. Evolution of antibiotic resistance in biofilm and planktonic pseudomonas aeruginosa populations exposed to subinhibitory levels of ciprofloxacin. Antimicrob. Agents Chemother. 62, e00320-18 (2018).

    PubMed  PubMed Central  Google Scholar 

  139. Ahmed, M. N. et al. Lack of the major multifunctional catalase KatA in pseudomonas aeruginosa accelerates evolution of antibiotic resistance in ciprofloxacin-treated biofilms. Antimicrob. Agents Chemother. 63, e00766-19 (2019).

    PubMed  PubMed Central  Google Scholar 

  140. Ahmed, M. N. et al. The evolutionary trajectories of P. aeruginosa in biofilm and planktonic growth modes exposed to ciprofloxacin: beyond selection of antibiotic resistance. NPJ Biofilms. Microbiomes. 6, 28 (2020).

    CAS  PubMed  PubMed Central  Google Scholar 

  141. Frimodt-Moller, J. et al. Mutations causing low level antibiotic resistance ensure bacterial survival in antibiotic-treated hosts. Sci. Rep. 8, 12512 (2018).

    PubMed  PubMed Central  Google Scholar 

  142. Ciofu, O., Fussing, V., Bagge, N., Koch, C. & Hoiby, N. Characterization of paired mucoid/non-mucoid Pseudomonas aeruginosa isolates from Danish cystic fibrosis patients: antibiotic resistance, beta-lactamase activity and RiboPrinting. J. Antimicrob. Chemother. 48, 391–396 (2001).

    CAS  PubMed  Google Scholar 

  143. Zaborskyte, G., Andersen, J. B., Kragh, K. N. & Ciofu, O. Real-time monitoring of nfxB mutant occurrence and dynamics in P. aeruginosa biofilm exposed to sub-inhibitory concentrations of ciprofloxacin. Antimicrob. Agents Chemother. 61, e02292-16 (2016).

    Google Scholar 

  144. Scribner, M. R., Santos-Lopez, A., Marshall, C. W., Deitrick, C. & Cooper, V. S. Parallel evolution of tobramycin resistance across species and environments. mBio 11, e00932-20 (2020).

    PubMed  PubMed Central  Google Scholar 

  145. Trampari, E. et al. Exposure of Salmonella biofilms to antibiotic concentrations rapidly selects resistance with collateral tradeoffs. NPJ Biofilms Microbiomes. 7, 3 (2021).

    CAS  PubMed  PubMed Central  Google Scholar 

  146. Hausner, M. & Wuertz, S. High rates of conjugation in bacterial biofilms as determined by quantitative in situ analysis. Appl. Environ. Microbiol. 65, 3710–3713 (1999).

    CAS  PubMed  PubMed Central  Google Scholar 

  147. Stalder, T. & Top, E. Plasmid transfer in biofilms: a perspective on limitations and opportunities. NPJ Biofilms. Microbiomes. 2, 16022 (2016).

    PubMed  PubMed Central  Google Scholar 

  148. Stalder, T. et al. Evolving populations in biofilms contain more persistent plasmids. Mol. Biol. Evol. 37, 1563–1576 (2020).

    CAS  PubMed  PubMed Central  Google Scholar 

  149. Savage, V. J., Chopra, I. & O’Neill, A. J. Staphylococcus aureus biofilms promote horizontal transfer of antibiotic resistance. Antimicrob. Agents Chemother. 57, 1968–1970 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  150. Strugeon, E., Tilloy, V., Ploy, M. C. & Da, R. S. The stringent response promotes antibiotic resistance dissemination by regulating integron integrase expression in biofilms. mBio 7, e00868-16 (2016).

    PubMed  PubMed Central  Google Scholar 

  151. Chi, F., Nolte, O., Bergmann, C., Ip, M. & Hakenbeck, R. Crossing the barrier: evolution and spread of a major class of mosaic pbp2x in Streptococcus pneumoniae, S. mitis and S. oralis. Int. J. Med. Microbiol. 297, 503–512 (2007).

    CAS  PubMed  Google Scholar 

  152. Ghigo, J. M. Natural conjugative plasmids induce bacterial biofilm development. Nature 412, 442–445 (2001).

    CAS  PubMed  Google Scholar 

  153. Nolan, L. M. et al. Pseudomonas aeruginosa is capable of natural transformation in biofilms. Microbiology 166, 995–1003 (2020).

    CAS  PubMed  PubMed Central  Google Scholar 

  154. Lerminiaux, N. A. & Cameron, A. D. S. Horizontal transfer of antibiotic resistance genes in clinical environments. Can. J. Microbiol. 65, 34–44 (2019).

    CAS  PubMed  Google Scholar 

  155. Schooling, S. R. & Beveridge, T. J. Membrane vesicles: an overlooked component of the matrices of biofilms. J. Bacteriol. 188, 5945–5957 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  156. Abe, K., Nomura, N. & Suzuki, S. Biofilms: hot spots of horizontal gene transfer (HGT) in aquatic environments, with a focus on a new HGT mechanism. FEMS Microbiol. Ecol. 96, fiaa031 (2020).

    CAS  PubMed  PubMed Central  Google Scholar 

  157. Lebeaux, D., Chauhan, A., Rendueles, O. & Beloin, C. From in vitro to in vivo models of bacterial biofilm-related infections. Pathogens 2, 288–356 (2013).

    PubMed  PubMed Central  Google Scholar 

  158. Kaplan, J. B. Biofilm dispersal: mechanisms, clinical implications, and potential therapeutic uses. J. Dent. Res. 89, 205–218 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  159. Otto, M. Staphylococcal biofilms. Microbiol. Spectr. https://doi.org/10.1128/microbiolspec.GPP3-0023-2018 (2018).

    Article  PubMed  Google Scholar 

  160. Whiteley, M. et al. Gene expression in Pseudomonas aeruginosa biofilms. Nature 413, 860–864 (2001).

    CAS  PubMed  Google Scholar 

  161. Valentini, M., Gonzalez, D., Mavridou, D. A. & Filloux, A. Lifestyle transitions and adaptive pathogenesis of Pseudomonas aeruginosa. Curr. Opin. Microbiol. 41, 15–20 (2018).

    CAS  PubMed  Google Scholar 

  162. Valentini, M. & Filloux, A. Biofilms and cyclic di-GMP (c-di-GMP) signaling: lessons from pseudomonas aeruginosa and other bacteria. J. Biol. Chem. 291, 12547–12555 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  163. Hall-Stoodley, L. et al. Towards diagnostic guidelines for biofilm-associated infections. FEMS Immunol. Med. Microbiol. 65, 127–145 (2012).

    CAS  PubMed  Google Scholar 

  164. Crabbe, A., Jensen, P. O., Bjarnsholt, T. & Coenye, T. Antimicrobial tolerance and metabolic adaptations in microbial biofilms. Trends Microbiol. 27, 850–863 (2019).

    CAS  PubMed  Google Scholar 

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Acknowledgements

C.M. is supported by Novo Nordisk Foundation (Borregaard Clinical Scientist Fellowship in translational research; grant no. NNF17OC0025074).

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Authors and Affiliations

  1. Department of Immunology and Microbiology, Costerton Biofilm Center, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark

    Oana Ciofu, Claus Moser, Peter Østrup Jensen & Niels Høiby

  2. Department of Clinical Microbiology, Rigshospitalet, Copenhagen, Denmark

    Claus Moser, Peter Østrup Jensen & Niels Høiby

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  1. Oana Ciofu
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  2. Claus Moser
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  3. Peter Østrup Jensen
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The authors contributed equally to all aspects of the article.

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Supplementary information

Glossary

Tolerance

A tolerant population is characterized by a slow rate of killing due to slow growth and low metabolic activity of the bacterial cells, requiring a longer time to kill 99% of the bacterial population than a susceptible population despite the similar values of the minimum inhibitory concentration of antibiotics against both populations.

Resistance

A resistant population is characterized by the lack of killing by antibiotic concentrations above the minimum inhibitory concentration of antibiotics against the susceptible bacterial population.

Aggregates

An assemblage of bacterial cells that can develop into structured biofilms through embedding in a polymeric matrix.

Lipopolysaccharides

Surface molecules, part of the outer membrane of the bacteria with a Gram-negative cell wall.

Respiratory burst

Production of superoxide upon the activation of polymorphonuclear leukocytes with rapid consumption of oxygen.

Degranulation

The liberation of the granular content of antimicrobial agents during the activation of polymorphonuclear leukocytes.

Pf bacteriophages

Temperate filamentous phages that can initiate a chronic infection cycle, during which virions are continuously extruded from the bacterial surface without cell lysis. They can be activated under various conditions such as antibiotic treatment or biofilm growth.

Rhamnolipids

Virulence factors with biosurfactant activity produced by Pseudomonas aeruginosa.

Persister cells

Non-dividing cells that can resume growth and cause relapse of the infection when antibiotic treatment is terminated.

Bacteriophage

Often referred to as simply phages. Naturally occurring viruses that infect bacteria. Lytic phages replicate inside their hosts and release new bacteriophages able to infect more bacteria. Temperate phages incorporate their genetic material into the bacterial chromosome and can be activated under special conditions (for example, treatment with antibiotics).

Small colony variants

(SCVs). Phenotypic variants that are slow-growing and tolerant to immune cells and antimicrobials. They strongly adhere to surfaces and can auto-aggregate.

Stringent response

A universal stress response that is induced by starvation and results in decreased cell growth to promote cell survival.

SOS response

A stress response that counteracts various types of DNA damage.

RpoS response

RpoS is a global stress response regulator induced under various stress conditions dependent on the RpoS alternative sigma factor and a central regulator of many stationary-phase inducible genes. This response enables cells to become more resistant not only to the stress that they first encounter but also to other stress-inducing treatments.

Membrane vesicles

Small (20–400 nm in diameter) lipid bilayer-enclosed particles released from Gram-negative bacteria. They contain both cytoplasmic and periplasmic components. Membrane vesicles from Gram-positive bacteria have also been described.

Nitrosative stress

Stress response to reactive nitrogen species such as nitric oxide (NO−) and its derivatives (nitrous acid (HNO2), peroxinitrite (ONOO–), alkylperoxynitrate (ROONO).

CRISPR–Cas

An immunity mechanism in archaea and bacteria that confers resistance to foreign genetic elements, such as phages and plasmids, and represents an acquired form of immunity.

Fitness cost

The consequence of mutation-induced resistance on the bacterial growth rate.

Clinical resistance breakpoint

Concentrations of antimicrobial that are used to distinguish strains with a high likelihood of treatment success and those in which treatment is more likely to fail.

Antimicrobial resistance genes

(ARGs). Examples of ARGs transmitted by horizontal gene transfer. Conjugation: plasmid-encoded extended spectrum β-lactamases (ESBLs), carbapenemases, plasmid-encoded quinolone resistance genes (qnr), usually on multi-drug resistance plasmids, between different Gram-negative species such as Escherichia coli and Klebsiella pneumoniae, vancomycin-resistance genes between Enterococcus faecalis and Staphylococcus aureus. Transduction: tetracycline and penicillin resistance between S. aureus strains. Natural transformation: resistance to metronidazole in Helicobacter pylori.

Integrases

Enzymes required for the site-specific recombination of integrons. Integrons are genetic elements that facilitate the acquisition and reassembly of gene cassettes encoding products with a variety of functions, including drug resistance.

Pharmacokinetics and pharmacodynamics

Parameters that describe the time-dependent concentration of the antibiotic in the host (pharmacokinetics) and its effect at the infection site (pharmacodynamics).

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Ciofu, O., Moser, C., Jensen, P.Ø. et al. Tolerance and resistance of microbial biofilms. Nat Rev Microbiol 20, 621–635 (2022). https://doi.org/10.1038/s41579-022-00682-4

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