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1. CESAM Centre for Environmentaland Marine Studies www.casam.ua.pt Influence of external bacterial structures on the efficiency of photodynamic inactivation by a cationic porphyrin M.A. Pereira,a M.A.F. Faustino,b J.P.C. Tomé,b M.G.P.M.S. Neves,b A.C. Tomé,b J.A.S. Cavaleiro,b Â. Cunhaa and A. Almeidaa aDepartment of Biology & CESAM, University of Aveiro, 3810-193 Aveiro, Portugal bDepartment of Chemistry & QOPNA, University of Aveiro, 3810-193 Aveiro, Portugal • Introduction • Antimicrobial photodynamic inactivation (PDI) has recently been used to inactivate efficiently microorganisms. The PDI procedure uses a photosensitizer (PS) in the presence of light and molecular oxygen leading to the production of reactive oxygen species (ROS) (e.g. singlet oxygen and free radicals) that destroy those microorganisms.1-2 The main targets of PDI are the external bacterial structures, cytoplasmic membrane and cell wall.3 • The aim of this study was to evaluate how the external bacterial structures influence the PDI efficiency. To reach this objective, 8 bacterial species with different external structures (4 Gram-negative and 4 Gram-positive bacteria) were selected and a cationic porphyrin was used as photosensitizer. • Material andMethods • Photosensitizers • Thecationicderivative 5,10,15,20-tetrakis(1-methylpyridinium-4-yl)porphyrin tetra-iodide(Tetra-Py+-Me) wasusedat a concentrationof 5.0 µM • Bacterial strainsandgrowthconditions • Gram-negative: • EscherichiacoliATCC 13706, Aeromonassalmonocida,4 • Aeromonashydrophilastrain ATTC 7966 andStaphylococcusaureus5 • grownovernight in TSB at37ºC • Gram-positive: • Rhodopirellulasp. strainLF26grownfor 48 h in 607 DSMZ mediumat25ºC. • Deinococcus radiodurans7grownfor 72 h in 607 DSMZ mediumat30ºC. • Deinococcus geothermalis8andTrueperaradiovictrix9grownfor 48 h in • 607 DSMZ mediumat50ºC • Photoinactivationassays • Bacterial suspension: concentration~108CFU mL-1incubatedwiththe photosensitizer in thedark, 15 min, 25ºC • Irradiationconditions: 40 W.m-2white light (380-700nm) understirring (100 rpm) • Irradiation time: 270 min (samples werecollectedafter 0, 15, 30, 60, 90, 180 and 270 min of light exposure) • Light Control: bacterial suspensionexposedto irradiationconditions • DarkControl: bacterial suspensionincubatedwith 5 µM PS in thesamecondition as thetests, butprotectedfromlight • Threeindependentassayswithtworeplicateswereconducted for eachbacteria • Quantification: pour plating in solid medium and determination of CFU mL-1 • Cellularuptake of porphyrin • Bacterial suspension: concentration~108cellsmL-1incubatedwiththe photosensitizer in thedark, 15 min, 25ºC • Centrifugation: at 17.000 g for 15 min to remove the unbound Tetra-Py+-Me • Digestion: pellets resuspended in 1 mL of 0.1 M NaOH-1% sodium dodecyl sulfate and incubated at room temperature for at least 24 h • Fluorimetry: samples were excited at 423 nm, fluorescence emission of the PS was monitored in the 600–800 nm range • Results • Photoinactivationassays Gram-negative bacteria Gram-positive bacteria Variation of viability of E. coli, A. salmonocida, A. hydrophilaand Rhodopirellulasp. after exposure to 5.0 µM of Tetra-Py+-Me and irradiated with white light (380-700 nm) with an irradiance of 40 W m-2 during 270 min Variation of viability of S. aureus, D. radiodurans, D. geothermalisand T. radiovictrixafter exposure to 5.0 µM of Tetra-Py+-Me and irradiated with white light (380-700 nm) with an irradiance of 40 W m-2 during 270 min • Cellularuptake of porphyrin • Discussion • The Gram-positive bacteria were more easily inactivated than Gram-negative strains, and this is even true for T. radiovictrix, D. geothermalisand D. radiodurans, which have a complex multi-layered cell wall, composed by 5 layers. • The results support the theory that the outer cell structures are major bacterial targets for PDI. • The chemical composition of the external structures has a stronger effect on PDI efficiency than complexity and number of layers of the external coating; • Lipids seem to be an important target of PDI. • These findings reinforce the importance of the chemical composition of bacteria external structures in order to understand the cellular and molecular basis of antimicrobial PDI. This is still an unexplored field that deserves further research. Gram-negative Gram-positive Uptake of Tetra-Py+-Me by Gram-positive and Gram-negative bacteria after 15 min of dark incubation at 25°C in the presence of 5.0 µM of Tetra-Py+-Me under stirring (~100 rpm) • References • L. Costa, E. Alves, C.M.B. Carvalho, J.P.C. Tomé, M.A.F. Faustino, M.G.P.M.F. Neves, A.C. Tomé, J.A.S. Cavaleiro, Â. Cunha and A. Almeida, Sewage bacteriophage photoinactivation by cationic porphyrins: a study of charge effect, Photochem. Photobiol. Sci., 2008, 7, 415-422. • E. Alves, L. Costa, C. Carvalho, J.P.C. Tomé, M.A.F. Faustino, M. G.P.M.S. Neves, A.C. Tomé, J.A.S. Cavaleiro, A. Cunha and A. Almeida, Charge effect on the photoinactivation of Gram-negative and Gram-positive bacteria by cationic meso-substituted porphyrins, BMC Microbiol., 2009, 9, 70-83. • M.R. Hamblin and T. Hasan, Photodynamic therapy: a new antimicrobial approach to infectious disease?, Photochem. Photobiol. Sci., 2004, 3, 436-450. • C. Pereira, Y.J. Silva, A.L. Santos, Â. Cunha, N.C.M. Gomes and A. Almeida, Bacteriophages with potential for inactivation of fish pathogenic bacteria: survival, host specificity and effect on bacterial community structure, Marine drugs, 2011, 9(11), 2236-55. • O.M. Lage, J. Bondoso and F. Viana, Isolation and characterisation of Planctomycetes from the sediments of a fish farm wastewater treatment tank, Arch. Microbiol., 2012, 194, 879-885. • C.V. Romão, E.P. Mitchell and S. McSweeney, The crystal structure of DeinococcusradioduransDps protein (DR2263) reveals the presence of a novel metal centre in the N-terminus, J. Biol. Inorg. Chem., 2006, 11, 891-902. • A.C. Ferreira, M.F. Nobre, F.A. Rainey, M.T. Silva, R. Wait, J. Burghardt, A.P. Chung and M.S. da Costa, Deinococcusgeothermalis sp. nov. and Deinococcusmurrayi sp. nov., two extremely radiation-resistant and slightly thermophilic species from hot springs, Int J SystBacteriol., 1997, 47(4), 939-947. • T.N. Demidova and M.R. Hamblin, Photodynamic inactivation of Bacillus spores, mediated by phenothiazinium dyes, Applied and Environmental Microbiology, 2005, 71(11), 6918-6925. • L. Albuquerque, C. Simões, M.F. Nobre, N.M. Pino, J.R. Battista, M.T. Silva, F.A. Rainey and M.S. Da Costa, Trueperaradiovictrix gen. nov., sp. nov., a new radiation resistant species and the proposal of Trueperaceae fam. nov. FEMS, MicrobiolLett 2005, 247, 161-169. Acknowledgements Thanks are due to Fundação para a Ciência e a Tecnologia (FCT, Portugal), European Union, QREN, FEDER and COMPETE for funding the CESAM (Pest-C/MAR/LA0017/2013) and the QOPNA research unit (project PEst-C/QUI/UI0062/2013; FCOMP-01-0124-FEDER-037296).