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Volume 162, Issue 5

The phenotypic evolution of populations changes in the presence of subinhibitory concentrations of ciprofloxacin Free

Abstract

Ciprofloxacin is a widely used antibiotic, in the class of quinolones, for treatment of infections. The immediate response of to subinhibitory concentrations of ciprofloxacin has been investigated previously. However, the long-term phenotypic adaptation, which identifies the fitted phenotypes that have been selected during evolution with subinhibitory concentrations of ciprofloxacin, has not been studied. We chose an experimental evolution approach to investigate how exposure to subinhibitory concentrations of ciprofloxacin changes the evolution of populations compared to unexposed populations. Three replicate populations of PAO1 and its hypermutable mutant Δ were cultured aerobically for approximately 940 generations by daily passages in LB medium with and without subinhibitory concentration of ciprofloxacin and aliquots of the bacterial populations were regularly sampled and kept at  − 80 °C for further investigations. We investigate here phenotypic changes between the ancestor (50 colonies) and evolved populations (120 colonies/strain). Decreased protease activity and swimming motility, higher levels of quorum-sensing signal molecules and occurrence of mutator subpopulations were observed in the ciprofloxacin-exposed populations compared to the ancestor and control populations. Transcriptomic analysis showed downregulation of the type III secretion system in evolved populations compared to the ancestor population and upregulation of denitrification genes in ciprofloxacin-evolved populations. In conclusion, the presence of antibiotics at subinhibitory concentration in the environment affects bacterial evolution and further studies are needed to obtain insight into the dynamics of the phenotypes and the mechanisms involved.

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© 2016 The Authors
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2016-05-01
2024-04-25
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References

  1. Andersson D. I., Hughes D. 2014; Microbiological effects of sublethal levels of antibiotics. Nat Rev Microbiol 12:465–478 [View Article][PubMed]
     [Google Scholar]
  2. Becher A., Schweizer H. P. 2000; Integration-proficient Pseudomonas aeruginosa vectors for isolation of single-copy chromosomal lacZ and lux gene fusions. Biotechniques 29:948–950, 952[PubMed]
     [Google Scholar]
  3. Bjarnsholt T., Jensen P. O., Jakobsen T. H., Phipps R., Nielsen A. K., Rybtke M. T., Tolker-Nielsen T., Givskov M., Høiby N., Ciofu O., Scandinavian Cystic Fibrosis Study Consortium. 2010; Quorum sensing and virulence of Pseudomonas aeruginosa during lung infection of cystic fibrosis patients. PLoS One 5:e10115 [View Article][PubMed]
     [Google Scholar]
  4. Blázquez J., Couce A., Rodríguez-Beltrán J., Rodríguez-Rojas A. 2012; Antimicrobials as promoters of genetic variation. Curr Opin Microbiol 15:561–569 [View Article][PubMed]
     [Google Scholar]
  5. Brazas M. D., Hancock R. E. 2005; Ciprofloxacin induction of a susceptibility determinant in Pseudomonas aeruginosa . Antimicrob Agents Chemother 49:3222–3227 [View Article][PubMed]
     [Google Scholar]
  6. Brazas M. D., Breidenstein E. B., Overhage J., Hancock R. E. 2007; Role of lon, an ATP-dependent protease homolog, in resistance of Pseudomonas aeruginosa to ciprofloxacin. Antimicrob Agents Chemother 51:4276–4283 [View Article][PubMed]
     [Google Scholar]
  7. Brochmann R. P., Toft A., Ciofu O., Briales A., Kolpen M., Hempel C., Bjarnsholt T., Høiby N., Jensen P. O. 2014; Bactericidal effect of colistin on planktonic Pseudomonas aeruginosa is independent of hydroxyl radical formation. Int J Antimicrob Agents 43:140–147 [View Article][PubMed]
     [Google Scholar]
  8. Ciofu O., Mandsberg L. F., Bjarnsholt T., Wassermann T., Høiby N. 2010; Genetic adaptation of Pseudomonas aeruginosa during chronic lung infection of patients with cystic fibrosis: strong and weak mutators with heterogeneous genetic backgrounds emerge in mucA and/or lasR mutants. Microbiology 156:1108–1119 [View Article][PubMed]
     [Google Scholar]
  9. Cirz R. T., O'Neill B. M., Hammond J. A., Head S. R., Romesberg F. E. 2006; Defining the Pseudomonas aeruginosa SOS response and its role in the global response to the antibiotic ciprofloxacin. J Bacteriol 188:7101–7110 [View Article][PubMed]
     [Google Scholar]
  10. Dwyer D. J., Kohanski M. A., Collins J. J. 2009; Role of reactive oxygen species in antibiotic action and resistance. Curr Opin Microbiol 12:482–489 [View Article][PubMed]
     [Google Scholar]
  11. Edgar R., Domrachev M., Lash A. E. 2002; Gene Expression Omnibus: NCBI gene expression and hybridization array data repository. Nucleic Acids Res 30:207–210 [View Article][PubMed]
     [Google Scholar]
  12. Fàbrega A., Madurga S., Giralt E., Vila J. 2009; Mechanism of action of and resistance to quinolones. Microb Biotechnol 2:40–61 [View Article][PubMed]
     [Google Scholar]
  13. Gambello M. J., Iglewski B. H. 1991; Cloning and characterization of the Pseudomonas aeruginosa lasR gene, a transcriptional activator of elastase expression. J Bacteriol 173:3000–3009[PubMed]
     [Google Scholar]
  14. Gocke E. 1991; Mechanism of quinolone mutagenicity in bacteria. Mutat Res 248:135–143 [View Article][PubMed]
     [Google Scholar]
  15. Gustafsson I., Sjölund M., Torell E., Johannesson M., Engstrand L., Cars O., Andersson D. I. 2003; Bacteria with increased mutation frequency and antibiotic resistance are enriched in the commensal flora of patients with high antibiotic usage. J Antimicrob Chemother 52:645–650 [View Article][PubMed]
     [Google Scholar]
  16. Ha D. G., Kuchma S. L., O'Toole G. A. 2014; Plate-based assay for swimming motility in Pseudomonas aeruginosa . Methods Mol Biol 1149:59–65 [View Article][PubMed]
     [Google Scholar]
  17. Hall B. M., Ma C. X., Liang P., Singh K. K. 2009; Fluctuation analysis CalculatOR: a web tool for the determination of mutation rate using Luria-Delbruck fluctuation analysis. Bioinformatics 25:1564–1565 [View Article][PubMed]
     [Google Scholar]
  18. Hansen S. K., Rau M. H., Johansen H. K., Ciofu O., Jelsbak L., Yang L., Folkesson A., Jarmer H. O., Aanæs K., other authors. 2012; Evolution and diversification of Pseudomonas aeruginosa in the paranasal sinuses of cystic fibrosis children have implications for chronic lung infection. ISME J 6:31–45 [View Article][PubMed]
     [Google Scholar]
  19. Hassett D. J., Ma J. F., Elkins J. G., McDermott T. R., Ochsner U. A., West S. E., Huang C. T., Fredericks J., Burnett S., other authors. 1999; Quorum sensing in Pseudomonas aeruginosa controls expression of catalase and superoxide dismutase genes and mediates biofilm susceptibility to hydrogen peroxide. Mol Microbiol 34:1082–1093 [View Article][PubMed]
     [Google Scholar]
  20. Hauser A. R. 2009; The type III secretion system of Pseudomonas aeruginosa: infection by injection. Nat Rev Microbiol 7:654–665 [View Article][PubMed]
     [Google Scholar]
  21. Herrero M., de Lorenzo V., Timmis K. N. 1990; Transposon vectors containing non-antibiotic resistance selection markers for cloning and stable chromosomal insertion of foreign genes in gram-negative bacteria. J Bacteriol 172:6557–6567[PubMed]
     [Google Scholar]
  22. Hoang T. T., Kutchma A. J., Becher A., Schweizer H. P. 2000; Integration-proficient plasmids for Pseudomonas aeruginosa: site-specific integration and use for engineering of reporter and expression strains. Plasmid 43:59–72 [View Article][PubMed]
     [Google Scholar]
  23. Høiby N., Jarløv J. O., Kemp M., Tvede M., Bangsborg J. M., Kjerulf A., Pers C., Hansen H. 1997; Excretion of ciprofloxacin in sweat and multiresistant Staphylococcus epidermidis . Lancet 349:167–169 [View Article][PubMed]
     [Google Scholar]
  24. Holloway B. W. 1955; Genetic recombination in Pseudomonas aeruginosa . J Gen Microbiol 13:572–581[PubMed]
     [Google Scholar]
  25. Hong C. S., Shitashiro M., Kuroda A., Ikeda T., Takiguchi N., Ohtake H., Kato J. 2004; Chemotaxis proteins and transducers for aerotaxis in Pseudomonas aeruginosa . FEMS Microbiol Lett 231:247–252 [View Article][PubMed]
     [Google Scholar]
  26. Hurley M., Smyth A. 2012; Fluoroquinolones in the treatment of bronchopulmonary disease in cystic fibrosis. Ther Adv Respir Dis 6:363–373 [View Article][PubMed]
     [Google Scholar]
  27. Jansen G., Crummenerl L. L., Gilbert F., Mohr T., Pfefferkorn R., Thänert R., Rosenstiel P., Schulenburg H. 2015; Evolutionary transition from pathogenicity to commensalism: global regulator mutations mediate fitness gains through virulence attenuation. Mol Biol Evol 32:2883–2896 [View Article][PubMed]
     [Google Scholar]
  28. Jørgensen K. M., Wassermann T., Jensen P. O., Hengzuang W., Molin S., Høiby N., Ciofu O. 2013; Sublethal ciprofloxacin treatment leads to rapid development of high-level ciprofloxacin resistance during long-term experimental evolution of Pseudomonas aeruginosa . Antimicrob Agents Chemother 57:4215–4221 [View Article][PubMed]
     [Google Scholar]
  29. Jørgensen K. M., Wassermann T., Johansen H. K., Christiansen L. E., Molin S., Høiby N., Ciofu O. 2015; Diversity of metabolic profiles of cystic fibrosis Pseudomonas aeruginosa during the early stages of lung infection. Microbiology 161:1447–1462 [View Article][PubMed]
     [Google Scholar]
  30. Jyet J., Ramphal R. 2008; Flagella and pili of Pseudomonas aeruginosa . In Pseudomonas: Model Organism, Pathogen and Cell Factory pp 85–108 Edited by Rehm B. H. A. Oxford: Wiley-Blackwell; [View Article]
     [Google Scholar]
  31. Kessler E., Safrin M. 2014; Elastinolytic and proteolytic enzymes. Methods Mol Biol 1149:135–169 [View Article][PubMed]
     [Google Scholar]
  32. Kohanski M. A., DePristo M. A., Collins J. J. 2010; Sublethal antibiotic treatment leads to multidrug resistance via radical-induced mutagenesis. Mol Cell 37:311–320 [View Article][PubMed]
     [Google Scholar]
  33. Köhler T., Perron G. G., Buckling A., van Delden C. 2010; Quorum sensing inhibition selects for virulence and cooperation in Pseudomonas aeruginosa . PLoS Pathog 6:e1000883 [View Article][PubMed]
     [Google Scholar]
  34. Linares J. F., Gustafsson I., Baquero F., Martinez J. L. 2006; Antibiotics as intermicrobial signaling agents instead of weapons. Proc Natl Acad Sci U S A 103:19484–19489 [View Article][PubMed]
     [Google Scholar]
  35. Line L., Alhede M., Kolpen M., Kühl M., Ciofu O., Bjarnsholt T., Moser C., Toyofuku M., Nomura N., other authors. 2014; 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[PubMed] [CrossRef]
     [Google Scholar]
  36. Mandsberg L. F., Ciofu O., Kirkby N., Christiansen L. E., Poulsen H. E., Høiby N. 2009; Antibiotic resistance in Pseudomonas aeruginosa strains with increased mutation frequency due to inactivation of the DNA oxidative repair system. Antimicrob Agents Chemother 53:2483–2491 [View Article][PubMed]
     [Google Scholar]
  37. Markussen T., Marvig R. L., Gómez-Lozano M., Aanæs K., Burleigh A. E., Høiby N., Johansen H. K., Molin S., Jelsbak L. 2014; Environmental heterogeneity drives within-host diversification and evolution of Pseudomonas aeruginosa . MBio 5:e01592–e01514 [View Article][PubMed]
     [Google Scholar]
  38. Masuda N., Gotoh N., Ishii C., Sakagawa E., Ohya S., Nishino T. 1999; Interplay between chromosomal beta-lactamase and the MexAB-OprM efflux system in intrinsic resistance to beta-lactams in Pseudomonas aeruginosa . Antimicrob Agents Chemother 43:400–402[PubMed]
     [Google Scholar]
  39. Morita Y., Kimura N., Mima T., Mizushima T., Tsuchiya T. 2001; Roles of MexXY- and MexAB-multidrug efflux pumps in intrinsic multidrug resistance of Pseudomonas aeruginosa PAO1. J Gen Appl Microbiol 47:27–32 [View Article][PubMed]
     [Google Scholar]
  40. Mulet X., Moyá B., Juan C., Macià M. D., Pérez J. L., Blázquez J., Oliver A. 2011; Antagonistic interactions of Pseudomonas aeruginosa antibiotic resistance mechanisms in planktonic but not biofilm growth. Antimicrob Agents Chemother 55:4560–4568 [View Article][PubMed]
     [Google Scholar]
  41. Nair C. G., Chao C., Ryall B., Williams H. D. 2013; Sub-lethal concentrations of antibiotics increase mutation frequency in the cystic fibrosis pathogen Pseudomonas aeruginosa . Lett Appl Microbiol 56:149–154 [View Article][PubMed]
     [Google Scholar]
  42. Newman J. R., Fuqua C. 1999; Broad-host-range expression vectors that carry the l-arabinose-inducible Escherichia coli araBAD promoter and the araC regulator. Gene 227:197–203 [View Article][PubMed]
     [Google Scholar]
  43. Olivares J., Álvarez-Ortega C., Martinez J. L. 2014; Metabolic compensation of fitness costs associated with overexpression of the multidrug efflux pump MexEF-OprN in Pseudomonas aeruginosa . Antimicrob Agents Chemother 58:3904–3913 [View Article][PubMed]
     [Google Scholar]
  44. Palmer K. L., Mashburn L. M., Singh P. K., Whiteley M. 2005; Cystic fibrosis sputum supports growth and cues key aspects of Pseudomonas aeruginosa physiology. J Bacteriol 187:5267–5277 [View Article][PubMed]
     [Google Scholar]
  45. Poole K. 2011; Pseudomonas aeruginosa: resistance to the max. Front Microbiol 2:65[PubMed] [CrossRef]
     [Google Scholar]
  46. Rainey P. B., Buckling A., Kassen R., Travisano M. 2000; The emergence and maintenance of diversity: insights from experimental bacterial populations. Trends Ecol Evol 15:243–247 [View Article][PubMed]
     [Google Scholar]
  47. Rakhimova E., Munder A., Wiehlmann L., Bredenbruch F., Tümmler B. 2008; Fitness of isogenic colony morphology variants of Pseudomonas aeruginosa in murine airway infection. PLoS One 3:e1685 [View Article][PubMed]
     [Google Scholar]
  48. Schuster M., Greenberg E. P. 2006; A network of networks: quorum-sensing gene regulation in Pseudomonas aeruginosa . Int J Med Microbiol 296:73–81 [View Article][PubMed]
     [Google Scholar]
  49. Skindersoe M. E., Alhede M., Phipps R., Yang L., Jensen P. O., Rasmussen T. B., Bjarnsholt T., Tolker-Nielsen T., Høiby N., Givskov M. 2008; Effects of antibiotics on quorum sensing in Pseudomonas aeruginosa . Antimicrob Agents Chemother 52:3648–3663 [View Article][PubMed]
     [Google Scholar]
  50. Stephenson K., Hoch J. A. 2002; Two-component and phosphorelay signal-transduction systems as therapeutic targets. Curr Opin Pharmacol 2:507–512 [View Article][PubMed]
     [Google Scholar]
  51. Taguchi K., Fukutomi H., Kuroda A., Kato J., Ohtake H. 1997; Genetic identification of chemotactic transducers for amino acids in Pseudomonas aeruginosa . Microbiology 143:3223–3229 [View Article][PubMed]
     [Google Scholar]
  52. Tanimoto K., Tomita H., Fujimoto S., Okuzumi K., Ike Y. 2008; Fluoroquinolone enhances the mutation frequency for meropenem-selected carbapenem resistance in Pseudomonas aeruginosa, but use of the high-potency drug doripenem inhibits mutant formation. Antimicrob Agents Chemother 52:3795–3800 [View Article][PubMed]
     [Google Scholar]
  53. Toder D. S., Gambello M. J., Iglewski B. H. 1991; Pseudomonas aeruginosa LasA: a second elastase under the transcriptional control of lasR . Mol Microbiol 5:2003–2010 [View Article][PubMed]
     [Google Scholar]
  54. Torres-Barceló C., Kojadinovic M., Moxon R., MacLean R. C. 2015; The SOS response increases bacterial fitness, but not evolvability, under a sublethal dose of antibiotic. Proc Biol Sci 282:20150885 [View Article][PubMed]
     [Google Scholar]
  55. Wong A., Kassen R. 2011; Parallel evolution and local differentiation in quinolone resistance in Pseudomonas aeruginosa . Microbiology 157:937–944 [View Article][PubMed]
     [Google Scholar]
  56. Wong A., Rodrigue N., Kassen R. 2012; Genomics of adaptation during experimental evolution of the opportunistic pathogen Pseudomonas aeruginosa . PLoS Genet 8:e1002928 [View Article][PubMed]
     [Google Scholar]
  57. Zumft W. G. 1997; Cell biology and molecular basis of denitrification. Microbiol Mol Biol Rev 61:533–616[PubMed]
     [Google Scholar]
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The phenotypic evolution of Pseudomonas aeruginosa populations changes in the presence of subinhibitory concentrations of ciprofloxacin
Microbiology 162, 865 (2016); https://doi.org/10.1099/mic.0.000273
/content/journal/micro/10.1099/mic.0.000273
/content/journal/micro/10.1099/mic.0.000273
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