NATALIA NORIKA YASSUNAKA

Título da Dissertação: PHOTODYNAMIC INACTIVATION MEDIATED BY ERYTHROSINE AND ITS DERIVATIVES ON FOODBORNE PATHOGENS AND SPOILAGE BACTERIA.

Orientadora: Profa. Dra. Jane Martha Graton Mikcha

Data da Defesa: 20/03/2015

RESUMO GERAL

INTRODUCTION. Microbial contamination can cause foodborne diseases and deterioration, generating important economic, social, and environmental consequences. The use of technologies to reduce or eliminate microbiological contamination in food can significantly impact the incidence of foodborne diseases and microbial spoilage. Therefore, alternative techniques are always required to ensure food quality and safety. One promising method is photodynamic inactivation (PDI). This technique involves administering a nontoxic dye or photosensitizer (PS), followed by doses of visible light of an appropriate wavelength that generates singlet oxygen and free radicals that are toxic to target cells. The successful application of PDI has also been reported for food pathogens and spoilage bacteria using different kinds of PSs and light sources. Erythrosine B (ERY) is a xanthene dye approved by the United States Food and Drug Administration (FDA) for usage in food products and it has been shown to have potent photodynamic activity against Gram-positive and Gram-negative bacteria. To improve its photodynamic properties, xanthene esters of ERY, such as erythrosine methyl ester (ERYMET) and erythrosine butyl ester (ERYBUT), were synthesized. The insertion of an alkyl substituent in the carboxylate group of ERY leads to the formation of esters that increase its hydrophobicity, thereby making the monoanionic form of ERY ester derivatives more effective than the dianionic form of ERY at interacting with lipophilic structures, such as membranes. ERY and its ester derivatives absorb light within a range that corresponds to the light emitted by green light-emitting diodes (LEDs). To our knowledge, no research has yet been reported on the effects of PDI on food pathogens and spoilage bacteria using ERYMET and ERYBUT combined with LEDs.
AIMS. The aim of the present study was to evaluate the in vitro sensitivity of pathogenic and spoilage bacteria to PDI using ERY and its ester derivatives as photosensitizers (PSs).
MATERIAL AND METHODS. Five bacterial strains were used in the experiments: One Gram-positive bacterium (Staphylococcus aureus ATCC 25923) and four Gram-negative bacteria (Aeromonas hydrophila ATCC 7966, Escherichia coli ATCC 25922, Salmonella enterica serotype Typhimurium ATCC 14028 and Pseudomonas aeruginosa ATCC 27853). ERY (1  10-3 M), ERYMET, and ERYBUT (approximately 1  10-4 M) were prepared by dissolving in phosphate-buffered saline (PBS, pH 7.4) that contained 5% dimethylsulfoxide (DMSO). The stock solutions were diluted with PBS to obtain concentrations that ranged from 1  10-4 M to 1  10-6 M. A green LED-based light source was used for the photosensitization experiments. The light doses for each dye at the respective concentrations and times were calculated based in two equations. Cytotoxicity of ERY and derivatives in VERO cells was evaluated by MTT assay. The cells were exposed to serial decimal dilutions of ERY (1  10-4 to 1  10-10 M), ERYMET (1  10-5 to 1  10-10 M), and ERYBUT (1  10-5 to 1  10-10 M) for 48 h. For photoinactivation of bacteria, one aliquot (50 μl) of bacterial suspension at 107 CFU/ml was homogenized with each compound at different concentrations (1  10-4 M to 1  10-6 M) and kept in the dark for 10 min. After incubation, each sample was illuminated with a green LED. Two controls without light exposure were also evaluated: Dark Control (DC; containing PBS + PS + DMSO + inoculum) and Solvent Control (SC; containing PBS + DMSO + inoculum). The surviving cell populations were enumerated and expressed as log colony-forming units (CFU)/ml. The morphological changes induced by ERY and its derivatives before and after PDI were examined by scanning electron microscopy (SEM). The experiments were performed in triplicate. The results were analyzed using analysis of variance (ANOVA) and the Tukey test. The statistical analysis was performed with a 5% level of significance.
RESULTS AND DISCUSSION. The cytotoxicity of ERY and its ester derivatives was evaluated in VERO cells, showing CC50 values of 55.3  10-6 M for ERY and 8.8  10-6 M for ERYMET with 48 h exposure in the dark. ERYBUT did not achieve 50% cell toxicity at the evaluated concentrations. The light dose values that were obtained for each PS concentration and irradiation time, under the same conditions, increased in the following order: ERYBUT < ERYMET < ERY. In PDI assays, ERY and its derivatives at the evaluated concentrations did not present intrinsic toxic effects on bacteria when incubated for 10 min in the dark. No significant difference was found between the DC and SC groups. S. aureus was more photosensitive than Gram-negative bacteria. Treatment with ERY at 1  10-6 M on S. aureus reached significant decrease of 4.0 log CFU/ml after 30 min (40 J/cm2) exposure. ERYMET and ERYBUT at 1  10-6 M with lower light doses (4 and 3 J/cm2) and 10 min exposure were sufficient to completely eradicate S. aureus. Among the tested Gram-negative bacteria, A. hydrophila was the least photoresistant. The photoinactivation with ERY at 1  10-5 M and 1  10-6 M on A. hydrophila exhibited higher reductions when compared with the ERY derivatives. ERYMET-mediated PDI dose-dependently decreased bacterial survival, in which higher reductions (4.4 log CFU/ml) were observed after 30 min irradiation (84 J/cm²). ERYBUT-mediated inactivation revealed significant differences in cell survival, even with lower light doses (22 J/cm² for 10 min, 43 J/cm² for 20 min, and 65 J/cm² for 30 min). The efficiency of inactivation on A. hydrophila was less pronounced when the PS concentrations decreased (1 x 10-6 M). The ERY-mediated PDI of E. coli at 1  10-5 M and 1  10-6 M with a longer exposure time (30 min) resulted in only 1.5 and 1.0 log CFU/ml reductions of cell viability, respectively. ERYMET and ERYBUT exerted limited antimicrobial effects against E. coli, in which none of the light doses at 1  10-5 M and 1  10-6 M reduced the rate of bacterial survival. The PDI of E. coli that was mediated by ERY (394 J/cm²), ERYMET (339 J/cm²), and ERYBUT (315 J/cm²) presented reductions of 3.4, 2.2, and 2.6 log CFU/ml, respectively. Under the same conditions, the PDI of S. Typhimurium with ERY and its derivatives caused a reduction of approximately 1.5 log CFU/ml. The ERY- and ERYMET-mediated photoinactivation of P. aeruginosa did not show reductions of cell viability when treated with light doses of 394 and 339 J/cm², respectively, but ERYBUT with a light dose of 315 J/cm² caused a reduction of 2.6 log CFU/ml. SEM images demonstrated that the PDI mediated by ERY and its derivatives caused alterations in the external structures of S. aureus.
CONCLUSION. Our study demonstrated that ERY, as well as ERYBUT, can be promising PSs against A. hydrophila and S. aureus, in which significant reductions of bacterial counts were observed with no toxicity in VERO cells at the evaluated concentrations. ERYMET presented this same efficacy in the PDI of S. aureus at nontoxic concentrations.
Key words: photoinactivation, erythrosine, ester derivatives, light emitting diode, bacteria.

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