Photodynamic inactivation of recombinant bioluminescent Escherichia coli by cationic porphyrins under artificial and solar irradiation

  • Eliana Alves
  • Carla M. B. Carvalho
  • João P. C. Tomé
  • Maria A. F. Faustino
  • Maria G. P. M. S. Neves
  • Augusto C. Tomé
  • José A. S. Cavaleiro
  • Ângela Cunha
  • Sónia Mendo
  • Adelaide Almeida
Original Paper

Abstract

A faster and simpler method to monitor the photoinactivation process of Escherichia coli involving the use of recombinant bioluminescent bacteria is described here. Escherichia coli cells were transformed with luxCDABE genes from the marine bioluminescent bacterium Vibrio fischeri and the recombinant bioluminescent indicator strain was used to assess, in real time, the effect of three cationic meso-substituted porphyrin derivatives on their metabolic activity, under artificial (40 W m−2) and solar irradiation (≈620 W m−2). The photoinactivation of bioluminescent E. coli is effective (>4 log bioluminescence decrease) with the three porphyrins used, the tricationic porphyrin Tri-Py+-Me-PF being the most efficient compound. The photoinactivation process is efficient both with solar and artificial light, for the three porphyrins tested. The results show that bioluminescence analysis is an efficient and sensitive approach being, in addition, more affordable, faster, cheaper and much less laborious than conventional methods. This approach can be used as a screening method for bacterial photoinactivation studies in vitro and also for the monitoring of the efficiency of novel photosensitizer molecules. As far as we know, this is the first study involving the use of bioluminescent bacteria to monitor the antibacterial activity of porphyrins under environmental conditions.

Keywords

Cationic porphyrins Photodynamic antimicrobial therapy Bioluminescence Escherichia coli Solar irradiation 

Notes

Acknowledgments

We are grateful to Professor James Slock (King’s College, EUA) for kindly providing E.coli strain with plasmid pHK555 and also plasmid pHK724. Thanks are due to the University of Aveiro, Fundação para a Ciência e a Tecnologia (FCT) and FEDER for funding the Organic Chemistry Research Unit (QOPNA) and the project POCI/CTM/58183/2004. To CESAM (Centro de Estudos do Ambiente e do Mar) for funding the Microbiology Research Group. C. M. B. Carvalho and J. P. Tomé are also grateful to FCT for their grants.

References

  1. 1.
    Jemli M, Alouini Z, Sabbahi S, Gueddari M (2002) Destruction of fecal bacteria in wastewater by three photosensitizers. J Environ Monit 4(4):511–516PubMedCrossRefGoogle Scholar
  2. 2.
    Alouini Z, Jemli M (2001) Destruction of helminth eggs by photosensitized porphyrin. J Environ Monit 3(5):548–551PubMedCrossRefGoogle Scholar
  3. 3.
    Bonnett R, Krysteva MA, Lalov IG, Artarsky SV (2006) Water disinfection using photosensitizers immobilized on chitosan. Water Res 40(6):1269–1275PubMedCrossRefGoogle Scholar
  4. 4.
    Wainwright M (1998) Photodynamic antimicrobial chemotherapy (PACT). J Antimicrob Chemother 42(1):13–28PubMedCrossRefGoogle Scholar
  5. 5.
    DeRosa M, Crutchley R (2002) Photosensitized singlet oxygen and its applications. Coord Chem Rev (233–234):351–371Google Scholar
  6. 6.
    Carvalho CMB, Gomes ATPC, Fernandes SCD, Prata ACB, Almeida MA, Cunha MA, Tomé JPC, Faustino MAF, Neves MGPMS, Tomé AC, Cavaleiro JAS, Lin Z, Rainho JP, Rocha J (2007) Photoinactivation of bacteria in wastewater by porphyrins: bacterial ß-galactosidase activity and leucine-uptake as methods to monitor the process. J Photochem Photobiol B 88(2–3):112–118PubMedCrossRefGoogle Scholar
  7. 7.
    Jiménez-Hernández ME, Manjón F, Garcia-Fresnadillo D, Orellana G (2005) Solar water disinfection by singlet oxygen photogenerated with polymer-supported Ru(II) sensitizers. Solar Energy 80:1382–1387CrossRefGoogle Scholar
  8. 8.
    Jori G, Fabris C, Soncin M, Ferro S, Coppellotti O, Dei D, Fantetti L, Chiti G, Roncucci G (2006) Photodynamic therapy in the treatment of microbial infections: basic principles and perspective applications. Lasers Surg Med 38(5):468–481PubMedCrossRefGoogle Scholar
  9. 9.
    Banfi S, Caruso E, Buccafurni L, Battini V, Zazzaron S, Barbieri P, Orlandi V (2006) Antibacterial activity of tetraaryl-porphyrin photosensitizers: an in vitro study on Gram negative and Gram positive bacteria. J Photochem Photobiol B 85(1):28–38PubMedCrossRefGoogle Scholar
  10. 10.
    Caminos DA, Spesia MB, Durantini EN (2005) Photodynamic inactivation of Escherichia coli by novel meso-substituted porphyrins by 4-(3-N, N, N-trimethylammoniumpropoxy)phenyl and 4-(trifluoromethyl)phenyl groups. Photochem Photobiol Sci 5(1):56–65PubMedCrossRefGoogle Scholar
  11. 11.
    Lazzeri D, Rovera M, Pascual L, Durantini EN (2004) Photodynamic studies and photoinactivation of Escherichia coli using meso-substituted cationic porphyrin derivatives with asymmetric charge distribution. Photochem Photobiol 80(2):286–293PubMedCrossRefGoogle Scholar
  12. 12.
    Merchat M, Spikes JD, Bertoloni G, Jori G (1996) Studies on the mechanism of bacteria photosensitization by meso-substituted cationic porphyrins. J Photochem Photobiol B 35(3):149–157PubMedCrossRefGoogle Scholar
  13. 13.
    Merchat M, Bertolini G, Giacomini P, Villanueva A, Jori G (1996) Meso-substituted cationic porphyrins as efficient photosensitizers of gram-positive and gram-negative bacteria. J Photochem Photobiol B 32(3):153–157PubMedCrossRefGoogle Scholar
  14. 14.
    Ashkenazi H, Nitzan Y, Gal D (2003) Photodynamic effects of antioxidant substituted porphyrin photosensitizers on Gram-positive and -negative bacteria. Photochem Photobiol 77(2):186–191PubMedCrossRefGoogle Scholar
  15. 15.
    Milanesio M, Alvarez M, Silber J, Rivarola V, Durantini E (2003) Photodynamic activity of monocationic and non-charged methoxyphenylporphyrin derivatives in homogeneous and biological media. Photochem Photobiol Sci 2(9):926–933PubMedCrossRefGoogle Scholar
  16. 16.
    Demidova T, Hamblin M (2005) Effect of cell-photosensitizer binding and cell density on microbial photoinactivation. Antimicrob Agents Chemother 49(6):2329–2335PubMedCrossRefGoogle Scholar
  17. 17.
    Vesterlund S, Paltta J, Laukova A, Karp M, Ouwehand AC (2004) Rapid screening method for the detection of antimicrobial substances. J Microbiol Methods 57(1):23–31PubMedCrossRefGoogle Scholar
  18. 18.
    Hamblin MR, O’Donnell DA, Murthy N, Contag CH, Hasan T (2002) Rapid control of wound infections by targeted photodynamic therapy monitored by in vivo bioluminescence imaging. Photochem Photobiol 75(1):51–57PubMedCrossRefGoogle Scholar
  19. 19.
    Francis KP, Yu J, Bellinger-Kawahara C, Joh D, Hawkinson MJ, Xiao G, Purchio TF, Caparon MG, Lipsitch M, Contag PR (2001) Visualizing pneumococcal infections in the lungs of live mice using bioluminescent Streptococcus pneumoniae transformed with a novel gram-positive lux transposon. Infect Immun 69(5):3350–3358PubMedCrossRefGoogle Scholar
  20. 20.
    Rocchetta HL, Boylan CJ, Foley JW, Iversen PW, LeTourneau DL, McMillian CL, Contag PR, Jenkins DE, Parr TR Jr (2001) Validation of a noninvasive, real-time imaging technology using bioluminescent Escherichia coli in the neutropenic mouse thigh model of Infection. Antimicrob Agents Chemother 45(1):129–137PubMedCrossRefGoogle Scholar
  21. 21.
    Meighen EA (1993) Bacterial bioluminescence: organization, regulation, and application of the lux genes. FASEB J 7(11):1016–1022PubMedGoogle Scholar
  22. 22.
    Rodriguez A, Nabi I, Meighen E (1985) ATP turnover by the fatty acid reductase complex of Photobacterium phosphoreum. Can J Biochem Cell Biol (63):1106–1111Google Scholar
  23. 23.
    Meighen EA (1991) Molecular biology of bacterial bioluminescence. Microbiol Mol Biol Rev 55(1):123–142Google Scholar
  24. 24.
    Meighen EA (1994) Genetics of bacterial bioluminescence. Ann Rev Genet 28(1):117–139PubMedCrossRefGoogle Scholar
  25. 25.
    Contag CH, Jenkins D, Contag PR, Negrin RS (2000) Use of reporter genes for optical measurements of neoplastic disease in vivo. Neoplasia 2:41–52PubMedCrossRefGoogle Scholar
  26. 26.
    Demidova T, Gad F, Zahra T, Francis K, Hamblin M (2005) Monitoring photodynamic therapy of localized infections by bioluminescence imaging of genetically engineered bacteria. J Photochem Photobiol B 81(1):25CrossRefGoogle Scholar
  27. 27.
    Doyle TC, Nawotka KA, Kawahara CB, Francis KP, Contag PR (2006) Visualizing fungal infections in living mice using bioluminescent pathogenic Candida albicans strains transformed with the firefly luciferase gene. Microb Pathog 40(2):82–90PubMedCrossRefGoogle Scholar
  28. 28.
    Jawhara S, Mordon S (2004) In vivo imaging of bioluminescent Escherichia coli in a cutaneous wound infection model for evaluation of an antibiotic therapy. Antimicrob Agents Chemother 48(9):3436–3441PubMedCrossRefGoogle Scholar
  29. 29.
    Burlage RS, Sayler GS, Larimer F (1990) Monitoring of naphthalene catabolism by bioluminescence with nah-lux transcriptional fusions. J Bacteriol 172(9):4749–4757PubMedGoogle Scholar
  30. 30.
    Grande R, Pietro SD, Campli ED, Bartolomeo SD, Filareto B, Cellini L (2007) Bio-toxicological assays to test water and sediment quality. J Environ Sci Health A 42(1):33–38Google Scholar
  31. 31.
    Johnson B (2005) Microtox® acute toxicity test. Small-scale freshwater toxicity investigations, Springer Netherlands, pp 69–105Google Scholar
  32. 32.
    Ptitsyn LR, Horneck G, Komova O, Kozubek S, Krasavin EA, Bonev M, Rettberg P (1997) A biosensor for environmental genotoxin screening based on an SOS lux assay in recombinant Escherichia coli cells. Appl Environ Microbiol 63(11):4377–4384PubMedGoogle Scholar
  33. 33.
    Verschaeve L, Van Gompel J, Thilemans L, Regniers L, Vanparys P, van der Lelie D (1999) VITOTOX® bacterial genotoxicity and toxicity test for the rapid screening of chemicals. Environ Mol Mutagen 33(3):240–248PubMedCrossRefGoogle Scholar
  34. 34.
    Maoz A, Mayr R, Bresolin G, Neuhaus K, Francis KP, Scherer S (2002) Sensitive in situ monitoring of a recombinant bioluminescent Yersinia enterocolitica reporter mutant in real time on camembert cheese. Appl Environ Microbiol 68(11):5737–5740PubMedCrossRefGoogle Scholar
  35. 35.
    Kadurugamuwa JL, Sin L, Albert E, Yu J, Francis K, DeBoer M, Rubin M, Bellinger-Kawahara C, Parr TR Jr, Contag PR (2003) Direct Continuous Method for Monitoring Biofilm Infection in a Mouse Model. Infect Immun 71(2):882–890PubMedCrossRefGoogle Scholar
  36. 36.
    Contag C, Contag P, Mullins J, Spilman S, Stevenson D, Benaron D (1995) Photonic detection of bacterial pathogens in living hosts. Mol Microbiol 4(18):593–603CrossRefGoogle Scholar
  37. 37.
    Francis KP, Joh D, Bellinger-Kawahara C, Hawkinson MJ, Purchio TF, Contag PR (2000) Monitoring bioluminescent Staphylococcus aureus infections in living mice using a novel luxABCDE construct. Infect Immun 68(6):3594–3600PubMedCrossRefGoogle Scholar
  38. 38.
    Salisbury Salisbury V, Pfoestl A, Wiesinger-Mayr H, Lewis R, Bowker KE, MacGowan AP (1999) Use of a clinical Escherichia coli isolate expressing lux genes to study the antimicrobial pharmacodynamics of moxifloxacin. J Antimicrob Chemother 43(6):829–832CrossRefGoogle Scholar
  39. 39.
    Marincs F (2000) On-line monitoring of growth of Escherichia coli in batch cultures by bioluminescence. Appl Microbiol Biotechnol 53(5):536–541PubMedCrossRefGoogle Scholar
  40. 40.
    Beard S, Salisbury V, Lewis R, Sharpe J, MacGowan A (2002) Expression of lux genes in a clinical isolate of Streptococcus pneumoniae: using bioluminescence to monitor gemifloxacin activity. Antimicrob Agents Chemother 46(2):538–542PubMedCrossRefGoogle Scholar
  41. 41.
    Jawhara S, Mordon S (2006) Monitoring of bactericidal action of laser by in vivo imaging of bioluminescent E. coli in a cutaneous wound infection. Lasers Med Sci 21(3):153–159PubMedCrossRefGoogle Scholar
  42. 42.
    Simon L, Fremaux C, Cenatiempo Y, Berjeaud JM (2001) Luminescent method for the detection of antibacterial activities. Appl Microbiol Biotechnol V57(5):757–763CrossRefGoogle Scholar
  43. 43.
    Spesia MB, Lazzeri D, Pascual L, Rovera M, Durantini EN (2005) Photoinactivation of Escherichia coli using porphyrin derivatives with different number of cationic charges. FEMS Immunol Med Microbiol 44(3):289–295PubMedCrossRefGoogle Scholar
  44. 44.
    Alves E, Almeida A, Cunha Â, Carvalho C, Faustino M, Tomé J, Neves M, Tomé A, Cavaleiro J (2007) Effect of the porphyrin charge in the inactivation of enteric bacteria. Faro, Portugal pp 195Google Scholar
  45. 45.
    Sirish M, Chertkov V, Schneider H (2002) Porphyrin-based peptide receptors: synthesis and NMR analysis. Chem Eur J 8(5):1181–1188CrossRefGoogle Scholar
  46. 46.
    Tomé JPC, Neves MGPMS, Tomé AC, Cavaleiro JAS, Soncin M, Magaraggia M, Ferro S, Jori G (2004) Synthesis and antibacterial activity of new poly-S-lysine-porphyrin conjugates. J Med Chem 47(26):6649–6652PubMedCrossRefGoogle Scholar
  47. 47.
    Kaplan HB, Greenberg EP (1987) Overproduction and Purification of the luxR Gene Product: Transcriptional Activator of the Vibrio fischeri Luminescence System. PNAS 84(19):6639–6643PubMedCrossRefGoogle Scholar
  48. 48.
    Slock J, VanRiet D, Kolibachuk D, Greenberg EP (1990) Critical regions of the Vibrio fischeri luxR protein defined by mutational analysis. J Bacteriol 172(7):3974–3979PubMedGoogle Scholar
  49. 49.
    Sambrook J, Fritsch E, Maniatis T (1989) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, New YorkGoogle Scholar
  50. 50.
    Hill P, Rees C, Winson M, Stewart G (1993) The application of lux genes. Biotechnol Appl Biochem (17):3–14Google Scholar
  51. 51.
    Maisch T, Szeimies R, Jori G, Abels C (2004) Antibacterial photodynamic therapy in dermatology. Photochem Photobiol Sci (3):907–917Google Scholar
  52. 52.
    Komerik N, Nakanishi H, MacRobert AJ, Henderson B, Speight P, Wilson M (2003) In vivo killing of Porphyromonas gingivalis by toluidine blue-mediated photosensitization in an animal model. Antimicrob Agents Chemother 47(3):932–940PubMedCrossRefGoogle Scholar
  53. 53.
    Costa L, Alves E, Carvalho C, Tomé J, Faustino M, Neves M, Tomé A, Cavaleiro J, Cunha Â, Almeida A (2008) Sewage bacteriophage photoinactivation by cationic porphyrins: a study of charge effect. Photochem Photobiol Sci 7:415–422PubMedCrossRefGoogle Scholar

Copyright information

© Society for Industrial Microbiology 2008

Authors and Affiliations

  • Eliana Alves
    • 1
    • 2
  • Carla M. B. Carvalho
    • 1
    • 3
  • João P. C. Tomé
    • 3
  • Maria A. F. Faustino
    • 3
  • Maria G. P. M. S. Neves
    • 3
  • Augusto C. Tomé
    • 3
  • José A. S. Cavaleiro
    • 3
  • Ângela Cunha
    • 1
  • Sónia Mendo
    • 1
  • Adelaide Almeida
    • 1
  1. 1.Department of Biology, CESAMUniversity of AveiroAveiroPortugal
  2. 2.School of Health TechnologyPorto Polytechnic InstitutePortoPortugal
  3. 3.Department of Chemistry, QOPNAUniversity of AveiroAveiroPortugal

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