Lasers in Medical Science

, Volume 34, Issue 6, pp 1159–1165 | Cite as

Sub-lethal antimicrobial photodynamic inactivation: an in vitro study on quorum sensing-controlled gene expression of Pseudomonas aeruginosa biofilm formation

  • Saghar Hendiani
  • Majid Pornour
  • Nasim KashefEmail author
Original Article


During antimicrobial photodynamic inactivation (APDI) in the treatment of an infection, it is likely that microorganisms would be exposed to sub-lethal doses of APDI (sAPDI). Although sAPDI cannot kill microorganisms, it can significantly affect microbial virulence. In this study, we evaluated the effect of sAPDI using methylene blue (MB) on the expression of genes belonging to two quorum sensing (QS) operons (rhl and las systems) and two genes necessary for biofilm formation (pelF and pslA) under QS control in Pseudomonas aeruginosa. Biofilm formation ability of P. aeruginosa ATCC 27853 exposed to sAPDI (MB at 0.012 mM and light dose of 23 J/cm2) was evaluated using triphenyl tetrazolium chloride (TTC) assay and scanning electron microscopy (SEM). The effect of sAPDI on expression of rhlI, rhlR, lasI, lasR, pelF, and pslA were also evaluated by quantitative real-time polymerase chain reaction. Quantitative assay (TTC) results and morphological observations (SEM) indicated that a single sAPDI treatment resulted in a significant decrease in biofilm formation ability of P. aeruginosa ATCC 27853 compared to their non-treated controls (P = 0.012). These results were consistent with the expression of genes belonging to rhl and las systems and pelF and pslA genes. The results suggested that the transcriptional decreases caused by MB-sAPDI did lead to phenotypic changes.


Pseudomonas aeruginosa Biofilm formation Quorum sensing Antimicrobial photodynamic inactivation Oxidative stress 



This study was supported by the College of Science, University of Tehran.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.


  1. 1.
    Taylor PW, Stapleton PD, Paul Luzio J (2002) New ways to treat bacterial infections. Drug Discov Today 7(21):1086–1091CrossRefPubMedGoogle Scholar
  2. 2.
    Wainwright M (1998) Photodynamic antimicrobial chemotherapy (PACT). J Antimicrob Chemother 42:13–28CrossRefGoogle Scholar
  3. 3.
    Huang L, Xuan Y, Koide Y, Zhiyentayev T, Tanaka M, Hamblin MR (2012) Type I and type II mechanisms of antimicrobial photodynamic therapy: an in vitro study on gram-negative and gram-positive bacteria. Lasers Surg Med 44:490–499CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Caminos DA, Spesia MB, Pons P, Durantini EN (2008) Mechanisms of Escherichia coli photodynamic inactivation by an amphiphilic tricationic porphyrin and 5,10,15,20-tetra(4-N,N,Ntrimethylammoniumphenyl) porphyrin. Photochem Photobiol Sci 7:1071–1078CrossRefPubMedGoogle Scholar
  5. 5.
    Niederhoffer EC, Naranjo CM, Bradley KL (1990) Control of Escherichia coli superoxide dismutase (sodA and sodB) genes by the ferric uptake regulation (fur) locus. J Bacteriol 172(4):1930–1938CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Ziegelhoffer EC, Donohue TJ (2009) Bacterial responses to photo-oxidative stress. Nat Rev Microbiol 7(12):856–863CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Boles BR, Singh PK (2008) Endogenous oxidative stress produces diversity and adaptability in biofilm communities. Proc Natl Acad Sci U S A 105(34):12503–12508CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Anderson SM, Krinsky NI (1973) Protective action of carotenoid pigments against photodynamic damage to liposomes. Photochem Photobiol 18(5):403–408CrossRefPubMedGoogle Scholar
  9. 9.
    Orlandi VT, Bolognese F, Chiodaroli L, Tolker-Nielsen T, Barbieri P (2015) Pigments influence the tolerance of Pseudomonas aeruginosa PAO1 to photodynamically induced oxidative stress. Microbiology 161(12):2298–2309CrossRefPubMedGoogle Scholar
  10. 10.
    Gad F, Zahra T, Hasan T, Hamblin MR (2004) Effects of growth phase and extracellular slime on photodynamic inactivation of gram-positive pathogenic bacteria. Antimicrob Agents Chemother 48(6):2173–2178CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Tegos GP, Hamblin MR (2006) Phenothiazinium antimicrobial photosensitizers are substrates of bacterial multidrug resistance pumps. Antimicrob Agents Chemother 50(1):196–203CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Nakonieczna J, Michta E, Rybicka M, Grinholc M, Gwizdek-Wiśniewska A, Bielawski K (2010) Superoxide dismutase is upregulated in Staphylococcus aureus following protoporphyrin-mediated photodynamic inactivation and does not directly influence the response to photodynamic treatment. BMC Microbiol 10(1):323CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    St Denis TG, Huang L, Dai T, Hamblin MR (2011) Analysis of the bacterial heat shock response to photodynamic therapy-mediated oxi- dative stress. Photochem Photobiol 87:707–713CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Jayaseelan S, Ramaswamy D, Dharmaraj S (2014) Pyocyanin: production, applications, challenges and new insights. World J Microbiol Biotechnol 30(4):1159–1168CrossRefGoogle Scholar
  15. 15.
    Donlan RM (2002) Biofilms: microbial life on surfaces. Emerg Infect Dis 8(9):881CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Watnick P, Kolter R (2000) Biofilm, city of microbes. J Bacteriol 182(10):2675–2679CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Al-Wrafy F, Brzozowska E, Górska S, Gamian A (2017) Pathogenic factors of Pseudomonas aeruginosa-the role of biofilm in pathogenicity and as a target for phage therapy. Adv Hyg Exp Med Hig Med Dosw 71:78–91Google Scholar
  18. 18.
    Colvin KM, Irie Y, Tart CS, Urbano R, Whitney JC, Ryder C et al (2012) The Pel and Psl polysaccharides provide Pseudomonas aeruginosa structural redundancy within the biofilm matrix. Environ Microbiol 14(8):1913–1928CrossRefPubMedGoogle Scholar
  19. 19.
    Lam J, Chan R, Lam K, Costerton JW (1980) Production of mucoid microcolonies by Pseudomonas aeruginosa within infected lungs in cystic fibrosis. Infect Immun 28:546–556PubMedPubMedCentralGoogle Scholar
  20. 20.
    Friedman L, Kolter R (2004) Genes involved in matrix formation in Pseudomonas aeruginosa PA14 biofilms. Mol Microbiol 51(3):675–690CrossRefPubMedGoogle Scholar
  21. 21.
    Swartjes JJTM, Das T, Sharifi S et al (2013) A functional DNasei coating to prevent adhesion of bacteria and the formation of biofilm. Adv Funct Mater 23(22):2843–2849CrossRefGoogle Scholar
  22. 22.
    Smith RS, Iglewski BH (2003) P aeruginosa quorum-sensing systems and virulence. Curr Opin Microbiol 6:56–60CrossRefPubMedGoogle Scholar
  23. 23.
    Passador L, Passador L, Cook JM, Gambello MJ, Rust L, Iglewski BH (1993) Expression of Pseudomonas aeruginosa virulence genes requires cell-to-cell communication. Science 260:1127–1130CrossRefPubMedGoogle Scholar
  24. 24.
    Latifi A, Winson MK, Foglino M, Bycroft BW, Stewart GS, Lazdunski A, Williams P (1995) Multiple homologues of LuxR and LuxI control expression of virulence determinants and secondary metabolites through quorum sensing in Pseudomonas aeruginosa PAO1. Mol Microbiol 17:333–343CrossRefPubMedGoogle Scholar
  25. 25.
    Wei Q, Ma LZ (2013) Biofilm matrix and its regulation in Pseudomonas aeruginosa. Int J Mol Sci 14(10):20983–21005CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Sakuragi YKR (2007) Quorum-sensing regulation of the biofilm matrix genes (pel) of Pseudomonas aeruginosa. J Bacteriol 189(14):5383–5386CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Kashef N, Hamblin MR (2017) Can microbial cells develop resistance to oxidative stress in antimicrobial photodynamic inactivation? Drug Resist Updat 31:31–42CrossRefPubMedPubMedCentralGoogle Scholar
  28. 28.
    Moody CS, Hassan HM (1982) Mutagenicity of oxygen free radicals. Proc Natl Acad Sci 79(9):2855–2859CrossRefPubMedGoogle Scholar
  29. 29.
    Kashef N, Akbarizare M, Kamrava SK (2013) Effect of sub-lethal photodynamic inactivation on the antibiotic susceptibility and biofilm formation of clinical Staphylococcus aureus isolates. Photodiagn Photodyn Ther 10(4):368–373CrossRefGoogle Scholar
  30. 30.
    Orlandi VT, Bolognese F, Martegani E, Cantaluppi V, Medana C, Barbieri P (2017) Response to photo-oxidative stress of Pseudomonas aeruginosa PAO1 mutants impaired in different functions. Microbiology 163(11):1557–1567CrossRefPubMedGoogle Scholar
  31. 31.
    Sabaeifard P, Abdi-Ali A, Soudi MR, Dinarvand R (2014) Optimization of tetrazolium salt assay for Pseudomonas aeruginosa biofilm using microtiter plate method. J Microbiol Methods 105:134–140CrossRefPubMedGoogle Scholar
  32. 32.
    Hendiani S, Abdi-Ali A, Mohammadi P, Kharrazi SH (2015) Synthesis of silver nanoparticles and its synergistic effects in combination with imipenem and two biocides against biofilm producing Acinetobacter baumannii. Nanomed J 2:291–298Google Scholar
  33. 33.
    Schmittgen TD, Livak KJ (2008) Analyzing real-time PCR data by the comparative CT method. Nat Protoc 3(6):1101–1108CrossRefGoogle Scholar
  34. 34.
    Boyce JM, Pittet D (2002) Guideline for hand hygiene in health-caresettings. Recommendations of the healthcare infection controlpractices advisory committee and the HIPAC/SHEA/APIC/IDSAhand hygiene task force. Am J Infect Control 30:1–46CrossRefGoogle Scholar
  35. 35.
    Franklin MJ, Nivens DE, Weadge JT, Howello PL (2011) Biosynthesis of the Pseudomonas aeruginosa extracellular polysaccharides, alginate, Pel, and Psl. Front Microbiol 2:167CrossRefPubMedPubMedCentralGoogle Scholar
  36. 36.
    Schembri MA, Hjerrild L, Gjermansen M, Klemm P (2003) Differential expression of the Escherichia coli autoaggregation factor antigen 43. J Bacteriol 185(7):2236–2242CrossRefPubMedPubMedCentralGoogle Scholar
  37. 37.
    Wen ZT, Suntharaligham P, Cvitkovitch DG, Burne RA (2005) Trigger factor in Streptococcus mutans is involved in stress tolerance, competence development, and biofilm formation. Infect Immun 73(1):219–225CrossRefPubMedPubMedCentralGoogle Scholar
  38. 38.
    Palma M, DeLuca D, Worgall S, Quadri LEN (2004) Transcriptome analysis of the response of Pseudomonas aeruginosa to hydrogen peroxide. J Bacteriol 186(1):248–252CrossRefPubMedPubMedCentralGoogle Scholar
  39. 39.
    Brown SM, Howell ML, Vasil ML, Anderson AJ, Hassett DJ (1995) Cloning and characterization of the katB gene of Pseudomonas aeruginosa encoding a hydrogen peroxide-inducible catalase: purification of KatB, cellular localization, and demonstration that it is essential for optimal resistance to hydrogen peroxide. J Bacteriol 177(22):6536–6544CrossRefPubMedPubMedCentralGoogle Scholar
  40. 40.
    Ochsner UA, Vasil ML, Alsabbagh E, Parvatiyar K, Hassett DJ (2000) Role of the Pseudomonas aeruginosa oxyR-recG operon in oxidative stress defense and DNA repair: OxyR-dependent regulation of katB-ankB, ahpB, andahpC-ahpF. J Bacteriol 182(16):4533–4544CrossRefPubMedPubMedCentralGoogle Scholar
  41. 41.
    Mathee K, Ciofu O, Sternberg C, Lindum PW, Campbell J, I A, Jensen P et al (1999) Mucoid conversion of Pseudomonas aeruginos by hydrogen peroxide: a mechanism for virulence activation in the cystic fibrosis lung. Microbiology 145(6):1349–1357CrossRefPubMedGoogle Scholar
  42. 42.
    Gambino M, Cappitelli F (2016) Mini-review: biofilm responses to oxidative stress. Biofouling 32(2):167–178CrossRefPubMedGoogle Scholar
  43. 43.
    Pogliano J, Lynch AS, Belin D, Lin EC, Beckwith J (1997) Regulation of Escherichia coli cell envelope proteins involved in protein folding and degradation by the Cpx two-component system. Genes Dev 11(9):1169–1182CrossRefPubMedGoogle Scholar
  44. 44.
    Zhao X, Drlica K (2014) Reactive oxygen species and the bacterial response to lethal stress. Curr Opin Microbiol 21:1–6CrossRefGoogle Scholar
  45. 45.
    Dorsey-Oresto A, Lu T, Mosel M, Wang X, Salz T, Drlica K, Zhao X (2013) YihE kinase is a central regulator of programmed cell death in bacteria. Cell Rep 3(2):528–537CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer-Verlag London Ltd., part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of Microbiology, School of Biology, College of ScienceUniversity of TehranTehranIran
  2. 2.Department of Photo Healing and Regeneration, Medical Laser Research Center, Yara InstituteAcademic Center for Education, Culture and Research (ACECR)TehranIran

Personalised recommendations