Starvation, Together with the SOS Response, Mediates High Biofilm-Specific Tolerance to the Fluoroquinolone Ofloxacin
High levels of antibiotic tolerance are a hallmark of bacterial biofilms. In contrast to well-characterized inherited antibiotic resistance, molecular mechanisms leading to reversible and transient antibiotic tolerance displayed by biofilm bacteria are still poorly understood. The physiological heterogeneity of biofilms influences the formation of transient specialized subpopulations that may be more tolerant to antibiotics. In this study, we used random transposon mutagenesis to identify biofilm-specific tolerant mutants normally exhibited by subpopulations located in specialized niches of heterogeneous biofilms. Using Escherichia coli as a model organism, we demonstrated, through identification of amino acid auxotroph mutants, that starved biofilms exhibited significantly greater tolerance towards fluoroquinolone ofloxacin than their planktonic counterparts. We demonstrated that the biofilm-associated tolerance to ofloxacin was fully dependent on a functional SOS response upon starvation to both amino acids and carbon source and partially dependent on the stringent response upon leucine starvation. However, the biofilm-specific ofloxacin increased tolerance did not involve any of the SOS-induced toxin–antitoxin systems previously associated with formation of highly tolerant persisters. We further demonstrated that ofloxacin tolerance was induced as a function of biofilm age, which was dependent on the SOS response. Our results therefore show that the SOS stress response induced in heterogeneous and nutrient-deprived biofilm microenvironments is a molecular mechanism leading to biofilm-specific high tolerance to the fluoroquinolone ofloxacin.
Vyšlo v časopise:
Starvation, Together with the SOS Response, Mediates High Biofilm-Specific Tolerance to the Fluoroquinolone Ofloxacin. PLoS Genet 9(1): e32767. doi:10.1371/journal.pgen.1003144
Kategorie:
Research Article
prolekare.web.journal.doi_sk:
https://doi.org/10.1371/journal.pgen.1003144
Souhrn
High levels of antibiotic tolerance are a hallmark of bacterial biofilms. In contrast to well-characterized inherited antibiotic resistance, molecular mechanisms leading to reversible and transient antibiotic tolerance displayed by biofilm bacteria are still poorly understood. The physiological heterogeneity of biofilms influences the formation of transient specialized subpopulations that may be more tolerant to antibiotics. In this study, we used random transposon mutagenesis to identify biofilm-specific tolerant mutants normally exhibited by subpopulations located in specialized niches of heterogeneous biofilms. Using Escherichia coli as a model organism, we demonstrated, through identification of amino acid auxotroph mutants, that starved biofilms exhibited significantly greater tolerance towards fluoroquinolone ofloxacin than their planktonic counterparts. We demonstrated that the biofilm-associated tolerance to ofloxacin was fully dependent on a functional SOS response upon starvation to both amino acids and carbon source and partially dependent on the stringent response upon leucine starvation. However, the biofilm-specific ofloxacin increased tolerance did not involve any of the SOS-induced toxin–antitoxin systems previously associated with formation of highly tolerant persisters. We further demonstrated that ofloxacin tolerance was induced as a function of biofilm age, which was dependent on the SOS response. Our results therefore show that the SOS stress response induced in heterogeneous and nutrient-deprived biofilm microenvironments is a molecular mechanism leading to biofilm-specific high tolerance to the fluoroquinolone ofloxacin.
Zdroje
1. StewartP, William CostertonJ (2001) Antibiotic resistance of bacteria in biofilms. The Lancet 358: 135–138.
2. AnwarH, van BiesenT, DasguptaM, LamK, CostertonJW (1989) Interaction of biofilm bacteria with antibiotics in a novel in vitro chemostat system. Antimicrob Agents Chemother 33: 1824–1826.
3. EvansDJ, AllisonDG, BrownMR, GilbertP (1991) Susceptibility of Pseudomonas aeruginosa and Escherichia coli biofilms towards ciprofloxacin: effect of specific growth rate. J Antimicrob Chemother 27: 177–184.
4. LevinBR, RozenDE (2006) Non-inherited antibiotic resistance. Nat Rev Micro 4: 556–562.
5. LewisK (2010) Persister cells. Annu Rev Microbiol 64: 357–372.
6. GefenO, BalabanNQ (2009) The importance of being persistent: heterogeneity of bacterial populations under antibiotic stress. FEMS Microbiol Rev 33: 704–717.
7. StewartPS, FranklinMJ (2008) Physiological heterogeneity in biofilms. Nat Rev Microbiol 6: 199–210.
8. AnderlJN, ZahllerJ, RoeF, StewartPS (2003) Role of nutrient limitation and stationary-phase existence in Klebsiella pneumoniae biofilm resistance to ampicillin and ciprofloxacin. Antimicrob Agents Chemother 47: 1251–1256.
9. BolesBR, SinghPK (2008) Endogenous oxidative stress produces diversity and adaptability in biofilm communities. Proc Natl Acad Sci U S A 105: 12503–12508.
10. BolesBR, ThoendelM, SinghPK (2004) Self-generated diversity produces “insurance effects” in biofilm communities. Proc Natl Acad Sci U S A 101: 16630–16635.
11. LewisK (2007) Persister cells, dormancy and infectious disease. Nat Rev Microbiol 5: 48–56.
12. NguyenD, Joshi-DatarA, LepineF, BauerleE, OlakanmiO, et al. (2011) Active starvation responses mediate antibiotic tolerance in biofilms and nutrient-limited bacteria. Science 334: 982–986.
13. LenzAP, WilliamsonKS, PittsB, StewartPS, FranklinMJ (2008) Localized gene expression in Pseudomonas aeruginosa biofilms. Appl Environ Microbiol 74: 4463–4471.
14. Pérez-OsorioAC, WilliamsonKS, FranklinMJ (2010) Heterogeneous rpoS and rhlR mRNA levels and 16S rRNA/rDNA (rRNA gene) ratios within Pseudomonas aeruginosa biofilms, sampled by laser capture microdissection. J Bacteriol 192: 2991–3000.
15. GhigoJM (2001) Natural conjugative plasmids induce bacterial biofilm development. Nature 412: 442–445.
16. TuomanenE, CozensR, ToschW, ZakO, TomaszA (1986) 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.
17. SpoeringAL, LewisK (2001) Biofilms and planktonic cells of Pseudomonas aeruginosa have similar resistance to killing by antimicrobials. J Bacteriol 183: 6746–6751.
18. ZhaoX, MalikM, ChanN, Drlica-WagnerA, WangJY, et al. (2006) Lethal action of quinolones against a temperature-sensitive dnaB replication mutant of Escherichia coli. Antimicrob Agents Chemother 50: 362–364.
19. VegaNM, AllisonKR, KhalilAS, CollinsJJ (2012) Signaling-mediated bacterial persister formation. Nat Chem Biol 8: 431–433.
20. PooleK (2012) Stress responses as determinants of antimicrobial resistance in Gram-negative bacteria. Trends Microbiol 20: 227–234.
21. PotrykusK, CashelM (2008) (p)ppGpp: still magical? Annu Rev Microbiol 62: 35–51.
22. SrivatsanA, WangJD (2008) Control of bacterial transcription, translation and replication by (p)ppGpp. Curr Opin Microbiol 11: 100–105.
23. XiaoH, KalmanM, IkeharaK, ZemelS, GlaserG, et al. (1991) Residual guanosine 3′,5′-bispyrophosphate synthetic activity of relA null mutants can be eliminated by spoT null mutations. J Biol Chem 266: 5980–5990.
24. DorrT, LewisK, VulicM (2009) SOS response induces persistence to fluoroquinolones in Escherichia coli. PLoS Genet 5: e1000760 doi:10.1371/journal.pgen.1000760.
25. DorrT, VulicM, LewisK (2010) Ciprofloxacin Causes Persister Formation by Inducing the TisB toxin in Escherichia coli. PLoS Biol 8: e1000317 doi:10.1371/journal.pbio.1000317.
26. FungDK, ChanEW, ChinML, ChanRC (2010) Delineation of a bacterial starvation stress response network which can mediate antibiotic tolerance development. Antimicrob Agents Chemother 54: 1082–1093.
27. ButalaM, Zgur-BertokD, BusbySJ (2009) The bacterial LexA transcriptional repressor. Cell Mol Life Sci 66: 82–93.
28. BierneH, SeigneurM, EhrlichSD, MichelB (1997) uvrD mutations enhance tandem repeat deletion in the Escherichia coli chromosome via SOS induction of the RecF recombination pathway. Mol Microbiol 26: 557–567.
29. KaldaluN, MeiR, LewisK (2004) Killing by ampicillin and ofloxacin induces overlapping changes in Escherichia coli transcription profile. Antimicrob Agents Chemother 48: 890–896.
30. Van MelderenL (2010) Toxin-antitoxin systems: why so many, what for? Curr Opin Microbiol 13: 781–5.
31. PedersenK, GerdesK (1999) Multiple hok genes on the chromosome of Escherichia coli. Mol Microbiol 32: 1090–1102.
32. YamaguchiY, ParkJ-H, InouyeM (2011) Toxin-Antitoxin Systems in Bacteria and Archaea. Ann Rev Genet 45: 61–79.
33. GerdesK, MaisonneuveE (2012) Bacterial persistence and toxin-antitoxin Loci. Ann Rev Microbiol 66: 103–123.
34. ErillI, CampoyS, BarbeJ (2007) Aeons of distress: an evolutionary perspective on the bacterial SOS response. FEMS Microbiol Rev 31: 637–656.
35. JanionC, SikoraA, NowosielskaA, GrzesiukE (2002) Induction of the SOS response in starved Escherichia coli. Environ Molecular Mutagenesis 40: 129–133.
36. PetrosinoJF, GalhardoRS, MoralesLD, RosenbergSM (2009) Stress-induced beta-lactam antibiotic resistance mutation and sequences of stationary-phase mutations in the Escherichia coli chromosome. J Bacteriol 191: 5881–5889.
37. TaddeiF, MaticI, RadmanM (1995) cAMP-dependent SOS induction and mutagenesis in resting bacterial populations. Proc Natl Acad Sci U S A 92: 11736–11740.
38. AgarwalG, KapilA, KabraSK, DasBK, DwivediSN (2005) Characterization of Pseudomonas aeruginosa isolated from chronically infected children with cystic fibrosis in India. BMC Microbiol 5: 43.
39. ThomasSR, RayA, HodsonME, PittTL (2000) Increased sputum amino acid concentrations and auxotrophy of Pseudomonas aeruginosa in severe cystic fibrosis lung disease. Thorax 55: 795–797.
40. BodiniS, NunziangeliL, SantoriF (2007) Influence of amino acids on low-density Escherichia coli responses to nutrient downshifts. J Bacteriol 189: 3099–3105.
41. BeloinC, ValleJ, Latour-LambertP, FaureP, KzreminskiM, et al. (2004) Global impact of mature biofilm lifestyle on Escherichia coli K-12 gene expression. Mol Microbiol 51: 659–674.
42. GirgisHS, HarrisK, TavazoieS (2012) Large mutational target size for rapid emergence of bacterial persistence. Proc Natl Acad Sci U S A 109: 12740–12745.
43. HansenS, LewisK, VulicM (2008) Role of Global Regulators and Nucleotide Metabolism in Antibiotic Tolerance in Escherichia coli. Antimicrob Agents Chemother 52: 2718–2726.
44. BaharogluZ, MazelD (2011) Vibrio cholerae Triggers SOS and Mutagenesis in Response to a Wide Range of Antibiotics: a Route towards Multiresistance. Antimicrob Agents Chemother 55: 2438–2441.
45. BlázquezJ, Gómez-GómezJ-M, OliverA, JuanC, KapurV, et al. (2006) PBP3 inhibition elicits adaptive responses in Pseudomonas aeruginosa. Mol Microbiol 62: 84–99.
46. MaiquesE, UbedaC, CampoyS, SalvadorN, LasaI, et al. (2006) beta-lactam antibiotics induce the SOS response and horizontal transfer of virulence factors in Staphylococcus aureus. J Bacteriol 188: 2726–2729.
47. BaharogluZ, BikardD, MazelD (2010) Conjugative DNA Transfer Induces the Bacterial SOS Response and Promotes Antibiotic Resistance Development through Integron Activation. PLoS Genet 6: e1001165 doi:10.1371/journal.pgen.1001165.
48. BeaberJW, HochhutB, WaldorMK (2004) SOS response promotes horizontal dissemination of antibiotic resistance genes. Nature 427: 72–74.
49. GuerinE, CambrayG, Sanchez-AlberolaN, CampoyS, ErillI, et al. (2009) The SOS response controls integron recombination. Science 324: 1034.
50. CourcelleJ, KhodurskyA, PeterB, BrownPO, HanawaltPC (2001) Comparative gene expression profiles following UV exposure in wild-type and SOS-deficient Escherichia coli. Genetics 158: 41–64.
51. Fernandez De HenestrosaAR, OgiT, AoyagiS, ChafinD, HayesJJ, et al. (2000) Identification of additional genes belonging to the LexA regulon in Escherichia coli. Mol Microbiol 35: 1560–1572.
52. AllisonKR, BrynildsenMP, CollinsJJ (2011) Metabolite-enabled eradication of bacterial persisters by aminoglycosides. Nature 473: 216–220.
53. LuTK, CollinsJJ (2009) Engineered bacteriophage targeting gene networks as adjuvants for antibiotic therapy. Proc Natl Acad Sci U S A 106: 4629–4634.
54. SextonJZ, WigleTJ, HeQ, HughesMA, SmithGR, et al. (2010) Novel Inhibitors of E. coli RecA ATPase Activity. Curr Chem Genomics 4: 34–42.
55. WigleTJ, SextonJZ, GromovaAV, HadimaniMB, HughesMA, et al. (2009) Inhibitors of RecA Activity Discovered by High-Throughput Screening: Cell-Permeable Small Molecules Attenuate the SOS Response in Escherichia coli. J Biomol Screening 14: 1092–1101.
56. BertaniG (2004) Lysogeny at mid-twentieth century: P1, P2, and other experimental systems. J Bacteriol 186: 595–600.
57. O'TooleGA, KolterR (1998) Initiation of biofilm formation in Pseudomonas fluorescens WCS365 proceeds via multiple, convergent signalling pathways: a genetic analysis. Mol Microbiol 28: 449–461.
58. RouxA, BeloinC, GhigoJM (2005) Combined inactivation and expression strategy to study gene function under physiological conditions: application to identification of new Escherichia coli adhesins. J Bacteriol 187: 1001–1013.
59. RamageG, Vande WalleK, WickesBL, Lopez-RibotJL (2001) Standardized method for in vitro antifungal susceptibility testing of Candida albicans biofilms. Antimicrob Agents Chemother 45: 2475–2479.
60. HacekDM, DresselDC, PetersonLR (1999) Highly reproducible bactericidal activity test results by using a modified National Committee for Clinical Laboratory Standards broth macrodilution technique. J Clin Microbiol 37: 1881–1884.
61. ChiangSL, RubinEJ (2002) Construction of a mariner-based transposon for epitope-tagging and genomic targeting. Gene 296: 179–185.
62. Da ReS, GhigoJM (2006) A CsgD-independent pathway for cellulose production and biofilm formation in Escherichia coli. J Bacteriol 188: 3073–3087.
63. BabaT, AraT, HasegawaM, TakaiY, OkumuraY, et al. (2006) Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Mol Syst Biol 2: 2006 0008.
64. ChaverocheMK, GhigoJM, d'EnfertC (2000) A rapid method for efficient gene replacement in the filamentous fungus Aspergillus nidulans. Nucleic Acids Res 28: E97.
65. DerbiseA, LesicB, DacheuxD, GhigoJM, CarnielE (2003) A rapid and simple method for inactivating chromosomal genes in Yersinia. FEMS Immunol Med Microbiol 38: 113–116.
66. CherepanovPP, WackernagelW (1995) Gene disruption in Escherichia coli: TcR and KmR cassettes with the option of Flp-catalyzed excision of the antibiotic-resistance determinant. Gene 158: 9–14.
67. Miller JH (1992) A short course in bacterial genetics: a laboratory manual and handbook for Escherichia coli and related bacteria. Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press,.
68. Da ReS, Le QuereB, GhigoJM, BeloinC (2007) Tight modulation of Escherichia coli bacterial biofilm formation through controlled expression of adhesion factors. Appl Environ Microbiol 73: 3391–3403.
Štítky
Genetika Reprodukčná medicínaČlánok vyšiel v časopise
PLOS Genetics
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