Application of mathematical models for bacterial inactivation curves using Hypericin-based photosensitization.

AIMS: The aim of this study was to evaluate which of the three mathematical models (Weibull, Geeraerd and Logistic) makes the best approximation for Hypericin-based photosensitization bacterial inactivation. METHODS AND RESULTS: The inactivation of common food-borne pathogens Listeria monocytogenes and Bacillus cereus has been analysed. Three models, Weibull, Logistic and Geeraerd, have been used for different treatment conditions (10(-7 ) mol l(-1) and 10(-8)  mol l(-1) Hypericin concentrations and 2 and 60 min incubation time). The Logistic method showed better correspondence (estimated R(2)  ≥ 0·98, RMSE ≤ 0·41), than the Weibull (estimated R(2)  ≥ 0·91, RMSE ≤ 0·90) and the Geeraerd model (R(2)  ≥ 0·83, RMSE ≤ 1·27). After evaluation of best fitting model for the dependence of bacterial population reduction on irradiation time, the parameters describing treatment efficiency have been calculated. CONCLUSIONS: Our results indicate that the Logistic model described in the best way the inactivation of B. cereus and L. monocytogenes by Hypericin-based photosensitization. SIGNIFICANCE AND IMPACT OF THE STUDY: This study identified a best model for describing bacterial inactivation by hypericin-based photosensitization, which provides a way to evaluate the efficacy of different treatment conditions for this novel decontamination technique.

Effective inactivation of food pathogens Listeria monocytogenes and Salmonella enterica by combined treatment of hypericin-based photosensitization and high power pulsed light.

AIMS: The aim of this study was to evaluate the inactivation efficiency of Listeria monocytogenes ATC(L3) C 7644 and Salmonella enterica serovar Typhimurium strain DS88 by combined treatment of hypericin (Hyp)-based photosensitization and high power pulsed light (HPPL). METHODS AND RESULTS: Cells were incubated with Hyp (1 × 10(-5) or 1 × 10(-7) mol l(-1)) in PBS and illuminated with a light λ = 585 nm. For the combined treatment, bacteria were, after photosensitization, exposed to 350 pulses of HPPL (UV light dose = 0·023 J cm(-2)). Fluorescence measurements were performed to evaluate optimal time for cell-Hyp interaction. Results indicate that Hyp tends to bind both Listeria and Salmonella. After photosensitization treatment, Listeria population was reduced 7 log, whereas Salmonella was inactivated just 1 log. Electron photomicrograps of Salmonella and Listeria confirmed that photosensitization induced total collapse of the Listeria cell wall, but not that of Salmonella. After combined photosensitization-HPPL treatment, the population of Listeria was diminished by 7 log and Salmonella by 6·7 log. CONCLUSIONS: Listeria can be effectively inactivated by Hyp-based photosensitization (7 log), whereas Salmonella is more resistant to photosensitization and can be inactivated just by 1 log in vitro. Combined treatment of photosensitization and pulsed light inactivates effectively (6·7-7 log) both the Gram-positive and the more resistant to photosensitization Gram-negative bacteria. SIGNIFICANCE AND IMPACT OF THE STUDY: A new approach to combat Gram-positive and Gram-negative bacteria is proposed, combining photosensitization with high power pulsed light.

Novel approach to the microbial decontamination of strawberries: chlorophyllin-based photosensitization.

AIMS: This study is focused on the possibility to control microbial contamination of strawberries by chlorophyllin (Na-Chl)-based photosensitization. Moreover, photosensitization-induced effects on key quality attributes of treated strawberries was evaluated. METHODS AND RESULTS: Strawberries were inoculated with Listeria monocytogenes ATC(L3) C 7644, soaked in 1mmol l(-1) Na-Chl for 5 min and illuminated for 30 min with visible light (λ = 400 nm, energy density 12 mW cm(-2) ). Results indicated that the decontamination of strawberries using photosensitization was 98% compared to control sample. Naturally occurring yeasts/microfungi and mesophiles were inhibited by 86 and 97%, respectively. The shelf life of treated strawberries was extended by 2 days. The total antioxidant activity of treated strawberries increased by 19%. No impact on the amount of phenols, anthocyanins or surface colour was detected. CONCLUSIONS: Photosensitization may be an effective, nonthermal and environmentally friendly microbial decontamination technique which expands the shelf life of strawberries without any negative impact on antioxidant activity, and phenols, anthocyanins or colour formation. SIGNIFICANCE AND IMPACT OF THE STUDY: Experimental data support the idea that Na-Chl-based photosensitization can be a useful tool for the future development of nonthermal food preservation technology.

Inactivation of Bacillus cereus by Na-chlorophyllin-based photosensitization on the surface of packaging.

AIMS: This study was focused on the possibility to inactivate food-borne pathogen Bacillus cereus by Na-chlorophyllin (Na-Chl)-based photosensitization in vitro and after attachment to the surface of packaging material. METHODS AND RESULTS: Bacillus cereus in vitro or attached to the packaging was incubated with Na-Chl (7·5×10(-8) to 7·5×10(-5) mol l(-1) ) for 2-60min in phosphate buffer saline. Photosensitization was performed by illuminating cells under a light with a λ of 400nm and an energy density of 20mW cm(-2) . The illumination time varied 0-5min and subsequently the total energy dose was 0-6J cm(-2) . The results show that B. cereus vegetative cells in vitro or attached to the surface of packaging after incubation with 7·5×10(-7) mol l(-1) Na-Chl and following illumination were inactivated by 7log. The photoinactivation of B. cereus spores in vitro by 4log required higher (7·5×10(-6) mol l(-1) ) Na-Chl concentration. Decontamination of packaging material from attached spores by photosensitization reached 5log at 7·5×10(-5) mol l(-1) Na-Chl concentration. Comparative analysis of different packaging decontamination treatments indicates that washing with water can diminish pathogen population on the surface by <1log, 100ppm Na-hypochlorite reduces the pathogens about 1·7log and 200ppm Na-hypochlorite by 2·2log. Meanwhile, Na-Chl-based photosensitization reduces bacteria on the surface by 4·2 orders of magnitude. CONCLUSIONS: Food-borne pathogen B. cereus could be effectively inactivated (7log) by Na-Chl-based photosensitization in vitro and on the surface of packaging material. Spores are more resistant than vegetative cells to photosensitization-based inactivation. Comparison of different surface decontamination treatments indicates that Na-Chl-based photosensitization is much more effective antibacterial tool than washing with water or 200ppm Na-hypochlorite. SIGNIFICANCE AND IMPACT OF THE STUDY: Our data support the idea that Na-Chl-based photosensitization has great potential for future application as an environment-friendly, nonthermal surface decontamination technique.

Inactivation of food pathogen Bacillus cereus by photosensitization in vitro and on the surface of packaging material.

AIMS: The study was focused on the possibility to inactivate food pathogen Bacillus cereus by 5-aminolevulinic acid (ALA) - based photosensitization in vitro and after adhesion on the surface of packaging material. METHODS AND RESULTS: Bacillus cereus was incubated with ALA (3-7.5 mmol l(-1)) for 5-60 min in different environment (PBS, packaging material and wheat grains) and afterwards illuminated with visible light. The light source used for illumination emitted light at lambda = 400 nm with energy density at the position of the cells, 20 mW cm(-2). The illumination time varied from 0 to 20 min, and subsequently a total energy dose was between 0 and 24 J cm(-2). The obtained results indicate that B. cereus after the incubation with 3-7.5 mmol l(-1) ALA produces suitable amounts of endogenous photosensitizers. Following illumination, micro-organism inactivated even by 6.3 log. The inactivation of B. cereus after adhesion on the surface of food packaging by photosensitization reached 4 log. It is important to note that spores of B. cereus were susceptible to this treatment as well; 3.7-log inactivation in vitro and 2.7-log inactivation on the surface of packaging material were achieved at certain experimental conditions. CONCLUSIONS: Vegetative cells and spores of Gram-positive food pathogen B. cereus were effectively inactivated by ALA-based photosensitization in vitro. Moreover, the significant inactivation of B. cereus adhered on the surface of packaging material was observed. It was shown that photosensitization-based inactivation of B. cereus depended on the total light dose (illumination time) as well as on the amount of endogenous porphyrins (initial ALA concentration, time of incubation with ALA). SIGNIFICANCE AND IMPACT OF THE STUDY: Our previous data, as well as the one obtained in this study, support the idea that photosensitization with its high selectivity, antimicrobial efficiency and nonthermal nature could serve in the future for the development of completely safe, nonthermal surface decontamination and food preservation techniques.

Modelling the photosensitization-based inactivation of Bacillus cereus.

AIMS: To study and to develop a model for the photo-destruction of the foodborne pathogen Bacillus cereus, initially treated with a precursor of endogenous photosensitizers (5-aminolevulinic acid, ALA). MATERIALS AND METHODS: The cells were incubated in the presence of ALA (3 or 7.5 mmol l(-1)) for incubation times ranging from 2 to 60 min, inoculated onto the surface of LB Agar plates and submitted to light irradiation. The Weibull model was used to describe the survival curves of B. cereus. Quadratic equations were used to describe the effects of ALA concentration and incubation time on the Weibull model parameters. RESULTS: ALA-based photosensitization proved to be an effective tool for inactivation of B. cereus. The decrease in viable counts observed after 20 min of irradiation, ranged from 4 to 6 log CFU g(-1). CONCLUSIONS: The developed model proved to be a parsimonious and robust solution to describe the observed data. SIGNIFICANCE AND IMPACT OF THE STUDY: The study demonstrates the effectiveness of photosensitization on B. cereus on agar plates. The model developed may be useful to optimize inactivation treatments by photosensitization.

Prospects of photosensitization in control of pathogenic and harmful micro-organisms.

Photosensitization is a treatment involving the interaction of the two nontoxic factors, photoactive compound and visible light, which in the presence of oxygen results in the selective destruction of the target cell. Different micro-organisms, such as multidrug-resistant bacteria, yeasts, microfungi and viruses, are susceptible to this treatment. Therefore, a photosensitization phenomenon might open a new avenue for the development of nonthermal, effective and ecologically friendly antimicrobial technology, which might be applied for food safety.

Novel approach to control Salmonella enterica by modern biophotonic technology: photosensitization.

AIMS: Salmonellosis is one of the most common foodborne diseases in the world. The aim of this study was to evaluate the antibacterial efficiency of 5-aminolevulinic acid (ALA) based photosensitization against one of food pathogens Salmonella enterica. METHODS AND RESULTS: Salmonella enterica was incubated with ALA (7.5 mmol l(-1)) for 1-4 h and afterwards illuminated with visible light. The light source used for illumination of S. enterica emitted light lambda = 400 nm with energy density 20 mW cm(-2). The illumination time varied from 0 to 20 min and subsequently a total energy dose reached 0-24 J cm(-2). The data obtained indicate that S. enterica is able to produce endogenous photosensitizer PpIX when incubated with ALA. Remarkable inactivation of micro-organisms can be achieved (6 log) after photosensitization. It is obvious that photosensitization-based inactivation of S. enterica depends on illumination as well as incubation with ALA time. CONCLUSION: ALA-based photosensitization can be an effective tool against multi-drug resistant Gram-negative bacteria S. enterica serovar Typhimurium. SIGNIFICANCE AND IMPACT OF THE STUDY: Experimental data and mathematical evaluations support the idea that ALA-based photosensitization can be a useful tool for the development of nonthermal food preservation technology in future.

Advanced high-power pulsed light device to decontaminate food from pathogens: effects on Salmonella typhimurium viability in vitro.

AIMS: The aim of this study was to construct an advanced high-power pulsed light device for decontamination of food matrix and to evaluate its antibacterial efficiency. Key parameters of constructed device-emitted light spectrum, pulse duration, pulse power density, frequency of pulses, dependence of emitted spectrum on input voltage, irradiation homogenicity, possible thermal effects as well as antimicrobial efficiency were evaluated. METHODS AND RESULTS: Antimicrobial efficiency of high-power pulsed light technique was demonstrated and evaluated by two independent methods - spread plate and Miles-Misra method. Viability of Salmonella typhimurium as function of a given light dose (number of pulses) and pulse frequency was examined. According to the data obtained, viability of Salmonella typhimurium reduced by 7 log order after 100 light pulses with power density 133 W cm(-2). In addition, data indicate, that the pulse frequency did not influence the outcome of pathogen inactivation in the region 1-5 Hz. Moreover, no hyperthermic effect was detected during irradiation even after 500 pulses on all shelves with different distance from light source and subsequently different pulse power density (0-252 W cm(-2)). CONCLUSION: Newly constructed high-power pulsed light technique is effective nonthermal tool for inactivation of Salmonella typhimurium even by 7 log order in vitro. SIGNIFICANCE AND IMPACT OF THE STUDY: Novel advanced high-power pulsed light device can be a useful tool for development of nonthermal food decontamination technologies.

Evaluation of protoporphyrin IX production, phototoxicity and cell death pathway induced by hexylester of 5-aminolevulinic acid in Reh and HPB-ALL cells.

Production of protoporphyrin IX (PpIX) in human B-cell leukemia cell line (Reh) and T-cell lymphoma cell line (HPB-ALL) was studied by flow cytometry after incubation with 5-aminolevulinic acid (ALA) or its hexylester in vitro. Cell survival and cell death pathway were also investigated in these two cell lines by cell growth curves, flow cytometry, and electron microscopy after ALA hexylester-mediated photodynamic therapy. Both ALA and its hexylester could induce PpIX production in the two cell lines, but ALA hexylester was about 100 times more efficient than ALA. Reh cells appear to be more sensitive than HPB-ALL cells to ALA hexylester-mediated phototoxicity. Apoptosis was the major cell death pathway of Reh cells, while necrosis played a major role in the case of HPB-ALL cells.

On the combination of photodynamic therapy with ionizing radiation.

Ehrlich ascites carcinoma growth and cell damage have been examined after photodynamic therapy (PDT), radiotherapy (RT) and combined treatment. Haematoporphyrin dimethyl ether (HPde) is used as a photosensitizer for PDT and tested as a radiosensitizer for RT. For PDT a non-coherent light source (370 < lambda < 680 nm) equipped with filters is used. gamma-Irradiation consists of 60Co irradiation at a dose of 2 Gy. Both PDT and RT induce a significant delay and inhibition in tumour growth (33 and 38%, respectively). Nevertheless cell damage after these treatments is different: after PDT the cell membrane integrity is damaged and no serious chromosomal aberrations are observed; whereas after gamma-irradiation there is no cell membrane integrity damage, but more significant DNA injuries are observed. It seems evident that HPde is able to act as a photosensitizer as well as a radiosensitizer. Combining PDT and RT produces an additive effect, not dependent on the sequence in which the two treatments are given, when a 1 h time window is used.

Combined treatment of ionizing radiation and photosensitization by 5-aminolevulinic acid-induced protoporphyrin IX.

The response of human colon adenocarcinoma cells of the line WiDr to the combined treatment of ionizing radiation and photosensitization by 5-aminolevulinic acid-induced protoporphyrin IX was assessed by a colony-forming assay. A dose of X rays inactivating approximately 50% of the cells was used. Seventy to 85% of the cells accumulated in S and G2 + M phase 12-24 h after such a treatment as measured by flow cytometry, while the distribution of cells in the phases of the cell cycle approached that of untreated cells 48 h after X-ray treatment. Cellular photosensitization was developed by endogenous synthesis of protoporphyrin IX (PPIX) from the precursor 5-aminolevulinic acid (5-ALA). This was performed by treating the cells with 1 mM 5-ALA for 4 h in a serum-free medium. The endogenous synthesis of PPIX increased with time after the cells had been subcultured, i.e. the ability of the cells to synthesize PPIX increased 1.5-2-fold within 48 h of incubation. This was not due to effects of trypsin on the cells. Photochemotherapy with 5-ALA was given 0-48 h after X rays. The combined cytotoxic effect was analyzed by an isobologram after correction of the survival curves for microcolony formation and differences in intracellular concentration of PPIX. The results indicate that 5-ALA PCT given 0-4 h after X rays acts slightly antagonistically while 5-ALA PCT given 12-48 h after X rays acts slightly synergistically.