Photodynamic inactivation of Escherichia coli immobilized on agar surfaces by a tricationic porphyrin Daniel A. Caminos and Edgardo N. Durantini * Departamento de Quı ´mica, Universidad Nacional de Rı ´o Cuarto, Rı ´o Cuarto, Agencia Postal Nro. 3, X5804BYA Rı ´o Cuarto, Co ´rdoba, Argentina Received 29 December 2005; revised 24 January 2006; accepted 24 January 2006 Available online 14 February 2006 Abstract—Thephotodynamicactivity of5,10,15-tris[4-(3-N,N,N-trimethylammoniumpropoxy)phenyl]-20-(4-trifluoromethylphe nyl)porphyrin iodide (A 3 B 3+ ) has been studied in vitro on a typical Gram-negative bacterium Escherichia coli immobilized on a surfaces. The results obtained for the tricationic A 3 B 3+ porphyrin were compared with those of 5,10,15,20-tetra(4-N,N,N-trimethy- lammoniumphenyl)porphyrin p-tosylate (TTAP 4+ ), which is a standard active sensitizer established to eradicate E. coli in cellular suspension. The photobleaching of these porphyrins in solution was evaluated by decay in absorbance and in fluorescence cases, a higher photostability was found for A 3 B 3+ than for TTAP 4+ . Photodynamic inactivation capacities of these sensitizers were analyzed in E. coli cells immobilized on agar surfaces. Small colonies were treated with different amount of sensitizer (0–1 and irradiated with visible light for 3 h. The light source used was either a projector or midday sun. The A 3 B 3+ porphyrin produced a growth delay of E. coli colonies on agar surfaces. Similar result was obtained irradiating only one isolated colony through cal fiber. Under these conditions, A 3 B 3+ porphyrin shows a high activity to inactivate localized bacterial cells. The higher photod namic activity of A 3 B 3+ was confirmed by mechanical spreading of the colonies before treatment. This procedure produces co inactivation of E. coli cells on the agar surface. Therefore, tricationic A 3 B 3+ porphyrin is an interesting sensitizer with potential applications in photodynamic inactivation of bacteria growing as localized foci of infection. 2006 Elsevier Ltd. All rights reserved. 1. Introduction New approaches to the treatment of bacterial infections have been developed because of the changing patterns of infectious disease and the emergence of bacterial strains resistanto currentantibiotics. 1 In this way, bacterial photodynamic inactivation (PDI) represents an interest- ing alternativeto inactivatemicroorganisms. 2,3 This methodology is based on the administration of a photo- sensitizer, which is preferentially accumulated in the microbialcells.The subsequent irradiation with visible light, in the presence of oxygen, specifically produces cell damages that inactivate the microorganisms. Two oxi- dativemechanismsare considered to beprincipally implicated in the photodamage of cells.In the type I photochemical reaction,the photosensitizer interacts with a biomolecule to produce free radicals, while in the type II mechanism,singlet molecularoxygen, O 2 ( 1 D g ), is produced asthe main species responsible for cell inactivation. 4,5 It was discovered that in general Gram-positive bacteria are efficiently photoinactivated by a variety of porphy- rins,whereas Gram-negative bacteria are usually resis- tant to the action of anionic or neutral agents. 6–9 The resistance of Gram-negative bacteria to the action of photoactivated sensitizers has been ascribed to the pres- ence of highly organized outer membrane, which hinders the interaction of the photosensitizer with the cytoplas- mic membrane and intercepts the photogenerated reac- tive species. 7,10,11 Alternativelycationic porphyrins have been shown to photoinduce direct inactivation of Gram-negative bacteria without the presence of an addi- tional permeabilization agent. 6–10,12 The positive charg- es on the photosensitizer molecule appear to promote a tight electrostatic interaction with negatively charged sites at the outer surface of the Gram-negative bacteria, increasing the efficiency of the photoinactivation pro- cesses. 3 Besides efficacy, PDI has shown other benefits. First, the sensitizers used are highly selective, that is, 0968-0896/$ - see front matter 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.bmc.2006.01.058 Keywords: Cationic porphyrin; Photosensitizer; Bacteria;Escherichia coli; Photodynamic inactivation. * Correspondingauthor. Tel.: +54 358 4676157;fax: +54 358 4676233; e-mail: edurantini@exa.unrc.edu.ar Bioorganic & Medicinal Chemistry 14 (2006) 4253–4259