Photodynamic Treatment: a Novel Method for Sanitation of Food Handling and Food Processing Surfaces

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Journal of Food Protection, Vol. 72, No. 5, 2009, Pages 1020–1024 Copyright ᮊ, International Association for Food Protection

Photodynamic Treatment: A Novel Method for Sanitation of Food Handling and Food Processing Surfaces

LUBOV Y. BROVKO,1* ANN MEYER,1 ARVINDER S. TIWANA,1 WEI CHEN,2 HAN LIU,2 CARLOS D. M. FILIPE,2 AND MANSEL W. GRIFFITHS1

1Canadian Research Institute for Food Safety, University of Guelph, Guelph, Ontario, Canada N1G 2W1; and 2Department of Chemical Engineering, McMaster University, Hamilton, Ontario, Canada L8S 4L7

MS 08-507: Received 6 October 2008/Accepted 14 December 2008 Downloaded from http://meridian.allenpress.com/jfp/article-pdf/72/5/1020/1677894/0362-028x-72_5_1020.pdf by guest on 01 October 2021

ABSTRACT

The photodynamic bactericidal effect of the photoactive dyes acriflavine neutral, rose bengal, phloxine B, and malachite green (oxalate salt) at concentrations of 5 to 5,000 ␮g/ml against two gram-negative strains (Escherichia coli LJH 128 and Salmonella Typhimurium C1058), two gram-positive strains (Bacillus sp. C578 and Listeria monocytogenes LJH 375), and yeast (Saccharomyces cerevisiae C1172) was investigated. Incubation of the investigated bacteria with acriflavine neutral under illumination resulted in a significant reduction in cell numbers compared with dark incubation. Rose bengal caused a significant killing effect for bacteria incubated both in the dark and under illumination. Malachite green was active against gram-positive bacteria under illumination and did not affect gram-negative bacteria or yeasts. Incubation with phloxine B resulted in a significant decline in cell numbers for gram-positive bacteria, both in the dark and under illumination; gram-negative bacteria and yeasts were unaffected. Conjugation of rose bengal and phloxine B with poly(vinyl amine) resulted in an enhanced bactericidal effect during both dark and light incubation. This was explained by electrostatic interaction of the polymer with the cell surface, which resulted in closer contact of the photoactive dye and cell. No killing effect was observed for yeasts incubated with dye conjugates. Filter paper treated with dye–poly(vinyl amine) conjugates showed high photodynamic bactericidal activity against the bacterial strains, but not against the yeasts. The extent of bacterial killing depended on the nature and concentration of the dye conjugate and the type of microorganism. The presented data suggest that a photodynamic approach for constructing ‘‘self-decontaminating’’ materials has potential.

Numerous outbreaks of foodborne illness have been the need for novel cleaning and sanitation strategies has attributed to postprocess contamination of product due to been recognized, especially to combat biofilms. inadequate sanitation of food contact surfaces (8). Recent The bactericidal effect of visible light illumination on research has indicated that pathogens can acquire resistance bacteria treated with nontoxic photosensitizers has been to commonly used sanitizers and, as a result of such ad- shown (13, 17, 18). Their efficacy depended on both cell aptation, cross-resistance to antibiotics has been observed type and the nature of the photosensitizer used. The method (4, 12). Emergence of multiantibiotic resistant pathogens is relies on illumination of microorganisms treated with nontoxic a risk to animal health and to the safety of food products. photosensitizers by low-power visible (red, blue, or white) The problem of removing bacteria from food processing light. Interaction of light with photosensitizers produces surfaces is compounded by the fact that microorganisms highly active, short-lived free radicals that are able to de- growing in a biofilm secrete extracellular polymeric sub- stroy cell components in close vicinity of the dye. This stances, which can remain attached to the cell in a capsular results, in some cases, in a 5- to 7-log cycle reduction in form or, alternatively, be released as a slime in which the bacterial counts, indicating the promise of the approach. cells form a complex multicellular structure (6, 12, 20). Development of resistance to antimicrobial photodynamic Bacteria in biofilms are more resistant to sanitizers. Deter- treatment has not been reported, which makes this approach gents can be formulated to remove particular types of soils, more attractive for investigation. As visible light can pen- for example, proteinaceous, fatty, carbohydrate or mineral etrate thick layers, antimicrobial photodynamic treatment soils, rather than to remove microorganisms. Gibson et al. may also provide a method for eracidating biofilms in situ (9) reported that detergents did not significantly improve (18). the removal of attached gram-positive and gram-negative Clinical applications of antimicrobial photodynamic organisms from food contact surfaces. Many commonly treatment are being investigated, including treatment of used enzymatic cleaners also fail to reduce the viable bac- wound infections and burns, infections in body cavities, terial load or remove the bacterial extracellular polymeric such as mouth, ear, sinus, stomach, and surface infections substances from surfaces (2, 16). In view of these findings, of cornea and skin (7, 10, 19). Currently, photoantimicrobials are used for the disinfection of blood products, e.g., * Author for correspondence. Tel: 519-824-4120, Ext 58301; Fax: 519- the Marco Blueflex system (19, 21). 763-0952; E-mail: [email protected]. In a preliminary investigation (3), we had shown that J. Food Prot., Vol. 72, No. 5 PHOTODYNAMIC SURFACE SANITATION 1021

TABLE 1. Physicochemical and photochemical properties of the photoactive dyes Molecular Absorption/emission Molar extinction Dye mass (Da) wavelength (nm) (1) coefﬁcient (MϪ1 cmϪ1) Current applications

Acriflavin neutral 665.8 436/520 3.6 ϫ 104 Biological stain, topical antiseptic, and treatment of bacte- (euflavine) rial, fungal, and parasitic infection in fish Rose bengal 1,017.6 525,540/550–600 7.28 ϫ 104 Biological stain, eye drops to assess the damage of con- junctiva and corneal cells, and treatment of certain can- cers Malachite green 927.02 629/NAa 15.0 ϫ 104 Dye for silk, leather and paper, biological stain, topical antiseptic, and treatment for parasitic, fungal, and bacterial infections in fish Phloxine B 829.64 524/600 10.1 ϫ 104 Colorant for food, cosmetics and drugs, biological stain, disinfection and detoxication of waste water, toxicant Downloaded from http://meridian.allenpress.com/jfp/article-pdf/72/5/1020/1677894/0362-028x-72_5_1020.pdf by guest on 01 October 2021 for fruit fly, bacteriocidal agent in plants

a NA, not available.

photoactive dyes such as toluedine blue, and tetramethyl The PVAm (degree of hydrolysis of 97% and viscosity average rosamine and its derivatives possess high photodynamic molecular mass of 150 kDa; BASF, Mississauga, Ontario, Canada) killing activity against both gram-positive and gram-nega- was further hydrolyzed with 5% NaOH at 75ЊC for 48 h, under tive pathogens present as planktonic cells and in biofilms, nitrogen purge, which was followed by thorough dialysis in water as well as against bacterial spores and viruses. The objec- and freeze-drying (5). One hundred milligrams of hydrolyzed PVAm was dissolved in 4 ml of water (pH 5.5), and 150 mg of tive of this study was to assess the degree of inactivation the dye was added to the solution. Conjugation was started by of microorganisms as a result of photodynamic treatment addition of 100 mg of 1-ethyl-3-(3-dimethylaminopropyl)-carbo- with common photoactive dyes that are compatable with diimide hydrochloride (EDC; Sigma-Aldrich, Canada). The mix- food industry requirements. The second objective was to ture was stirred for 24 h in the dark and at room temperature. The explore the possibility of designing self-decontaminating sample was then dialyzed (molecular weight cutoff of 3,500; materials on the basis of the dyes that would show a strong Spectra/Pro 3 Dialysis Membrane, Spectra Laboratories, Rancho bacteriocidal effect on illumination with incident light. Dominguez, CA) against deionized water for 1 week to separate uncoupled dye and excess EDC from the PVAm. The concentra- MATERIALS AND METHODS tion of labeled PVAm in these samples was determined from the Bacterial cultures and cultivation. The microorganisms absorbance at 540 and 550 nm for PVAm-PhB and PVAm-RB, used in the study were obtained from the culture collection of the respectively. The final concentration of PVAm-PhB was 0.46 mg/ Canadian Research Institute for Food Safety (University of ml, and 0.48 mg/ml for PVAm-RB, with dye contents of 9.2 and Guelph, Ontario) and comprised two gram-negative strains (Esch- 9.6%, respectively. This corresponded to dye concentrations of 42 ␮ ␮ erichia coli LJH 128 and Salmonella Typhimurium C1058), two g/ml for PhB and 46 g/ml for RB. gram-positive strains (Bacillus sp. C578 and Listeria monocyto- Photoinactivation in broth. Photoinactivation was per- genes LJH 375), and yeast (Saccharomyces cerevisiae C1172). All formed in a 96-well microtiter plate by adding 150 ␮l of bacterial microorganisms were cultivated at 30 or 37ЊC, with shaking (120 suspension (105 to 106 CFU/ml) in PBS and 150 ␮l of the dye rpm; Incubator Shaker, New Brunswick, Scientific, Edison, NY) solution to each well. The plate was then illuminated for 30 min for 24 h by using Luria-Bertani broth (Difco, Becton Dickinson, by white light under the Illumatool illuminator (Montreal Biotech, Sparks, MD) for E. coli and Salmonella, brain heart infusion broth Inc., Montreal, Quebec, Canada). The light source (halogen lamp) (Difco, Becton Dickinson) for L. monocytogenes and Bacillus sp., was placed at a distance of 6.5 cm from the surface and provided and yeast broth (Difco, Becton Dickinson) for S. cerevisiae. Enu- uniform illumination with an average intensity of 0.45 Ϯ 0.04 meration of bacterial cells was performed by using the spread- mW/cm2. An identical plate was kept in the dark and used as a plate method in which serial dilutions of bacterial suspension were control. After 30 min of incubation, the bacterial population in inoculated onto the plate with the respective agar-containing me- each well was enumerated by the spread-plate method as described dia. Plates were incubated for 24 h at 30 or 37ЊC, and the number above. of formed colonies was counted. The number of cells in the initial samples was calculated taking into consideration the dilution fac- Preparation of self-decontaminating paper. Pieces of filter tor and was presented as CFU per milliliter. paper (Whatman No. 4) were soaked in the solutions of dye con- Photoactive dyes. The photactive dyes used were acriflavine jugate at initial concentrations 9.2 to 9.6% (wt/vol) for PhB and neutral, rose bengal (RB), phloxine B (PhB), and malachite green RB, as well as in 10-fold and 100-fold dilutions of the dyes. Sam- (oxalate salt) (all obtained from Sigma-Aldrich Canada, Ltd., Oak- ples of treated paper were air dried under sterile conditions. Paper ville, Ontario, Canada). Physicochemical and photochemical prop- treated with a solution of PVAm was used as a control. erties of these dyes are presented in Table 1. The dyes were pre- Photoinactivation on the surface of paper. pared in phosphate-buffered saline (PBS) to obtain concentrations Photoinactiva- ␮ of 5, 50, 500, and 5,000 ␮g/ml and kept in the dark. tion was performed by placing 100 l of bacterial suspension in PBS (ϳ103 CFU/ml) onto the surface of the dye-treated filter pa- Conjugates of RB and PhB with poly(vinyl amine) per (ϳ2cm2), which was then placed in a petri dish. This is the (PVAm). The conjugates were prepared by the following method. maximum volume of bacterial suspension that can be totally ab- 1022 BROVKO ET AL. J. Food Prot., Vol. 72, No. 5

TABLE 2. Killing effect of photodynamic treatment observed for microorganisms in suspension Mean log reduction in count after 30 min of treatment with the dye

E. coli Salmonella Typhimurium Bacillus sp. L. monocytogenes S. cerevisiae Concn Dye (␮g/ml) Dark Light Dark Light Dark Light Dark Light Dark Light

Acriflavin 5 Ϫ0.09 1.13a 0.004 0.55 0.14 Ͼ6a 0.05 0.01 neutral 50 Ϫ0.4 Ͼ5a 0.27 Ͼ5a 0.24 Ͼ6a —b 1.5 500 Ͼ6 Ͼ6 0.09 Ͼ5a 0.47 2.2 — Ͼ5 Rose bengal 5 0.05 0.2 0.05 2.0 0.17 Ͼ5a Ͼ6 Ͼ6 Ϫ0.02 1.01 50 0.2 Ͼ6a Ϫ0.32 Ͼ6a Ͼ5 Ͼ5 Ͼ6 Ͼ6 Ϫ0.48 Ͼ6a 500 Ϫ0.3 Ͼ6a 0.35 Ͼ6a Ͼ5 Ͼ5 Ͼ6 Ͼ6 1.5 Ͼ6a Malachite 5 Ϫ0.08 0.09 Ϫ0.15 Ϫ0.01 Ϫ0.17 Ϫ0.09 0.04 0.04 Ϫ0.06 0.21 green 50 Ϫ0.04 0.05 Ϫ0.06 0.02 Ϫ0.15 Ϫ0.02 0.09 Ͼ6a 0.36 0.39 Downloaded from http://meridian.allenpress.com/jfp/article-pdf/72/5/1020/1677894/0362-028x-72_5_1020.pdf by guest on 01 October 2021 500 Ϫ0.06 0.69 Ϫ0.14 2.39a 0.04 Ͼ5a Ϫ0.01 1.93a Ϫ0.13 0.68 Phloxine B 50 Ϫ0.5 0.16 Ϫ0.35 Ϫ0.21 1.5 Ͼ6a 0.63 Ͼ6a Ϫ0.58 0.33 500 Ϫ0.6 0.45 Ϫ0.05 0.93 Ͼ5 Ͼ6 2.23 Ͼ6a Ϫ0.44 1.14 5,000 Ϫ0.52 Ϫ0.05 Ϫ0.17 0.06 Ͼ6 Ͼ6 Ͼ6 Ͼ6 0.10 1.15 a Killing effect of the dye on illumination with white light significantly higher than on dark incubation (P Ͻ 0.05). b —, no data. sorbed by the paper. The sample was illuminated for 30 min under and PhB were active against gram-positive bacteria and did the Illumatool, as descibed earlier. An identical dish was kept in not produce significant photokilling effects for gram-nega- the dark and was used as a control. After 30 min, 10 ml of molten tive strains or yeast. The dark toxicity of malachite green Њ nutrient soft agar (45 C) was added to both dishes. The third and was low, but PhB inactivated gram-positive bacteria incu- fourth identical dishes that contained nontreated paper were pro- bated in the dark as well as on light incubation. cessed the same way and were used as controls. Bacteria were enumerated in all four dishes by counting the formed colonies In order to incorporate photodynamic dyes onto sur- after incubation at 37ЊC for 24 h. faces, conjugates of RB and PhB with PVAm were obtained by using the method descibed above. The choice of poly- Statistical analysis. All experiments were performed twice mer for conjugation was based on the fact that PVAm car- with three replicates. Microbiological data were transformed to ries a net positive charge and can attach almost irreversibly log values, and log reductions were calculated by subtracting from to a negatively charged surfaces. Other available negatively pretreatment levels and expressed as means. Comparison of mul- tiple means were made by using anaylsis of variance, with the charged surfaces are based on cellulosic materials, includ- limit of statistical significance set at P Ͻ 0.05. ing paper. Currently, PVAm is used as a strengthening agent in the paper-making process and, thus, techniques for the RESULTS incorporation of this polymer onto surface have already Photoinactivation of microorganisms in suspension been developed (11). by the photoactive dyes and their conjugates with poly- Photodynamic killing of microorganisms with dye con- mers. The photokilling effect of all dyes was tested against jugates was tested by using cell suspensions. Incubation of the microorganisms in suspension. The results are presented microorganisms with PVAm conjugated with RB and PhB in Table 2. Acriflavine neutral was shown to possess high resulted in significant reductions in cell numbers (Table 3). photokilling activity against all the investigated microor- For all tested bacterial strains, the treatment with dye con- ganisms. At a concentration of 50 ␮g/ml, the observed log jugates produced a significant killing effect both under dark reduction in cells numbers was significantly higher after incubation and on illumination. The killing effect here was incubation under light compared with dark incubation. RB, much more pronounced than it was for comparable con- though also active against all investigated micoorganisms, centrations of dyes in solution. This phenomenon may be inactivated the two gram-positive bacteria both during in- due to the positive charge of the polymer, which interacted cubation under light and in the dark. Both malachite green with the negatively charged surface of microorganisms,

TABLE 3. Killing effect of dye–poly(vinyl amine) conjugates Mean log reduction in count after 30 min of treatment with the dye conjugate

E. coli Salmonella Typhimurium Bacillus sp. L. monocytogenes S. cerevisiae

Conjugatea Dark Light Dark Light Dark Light Dark Light Dark Light

Poly(vinyl amine)–rose bengal Ͼ6 Ͼ6 2.04 2.31 Ͼ4 Ͼ4 Ͼ5 Ͼ5 0.36 0.08 Poly(vinyl amine)–phloxine B Ͼ6 Ͼ6 2.89 1.33 Ͼ4 Ͼ4 Ͼ5 Ͼ5 0.17 Ϫ0.46 a Rose bengal and phloxine B at concentrations of 4.6 and 4.8% (wt/vol), respectively. J. Food Prot., Vol. 72, No. 5 PHOTODYNAMIC SURFACE SANITATION 1023

TABLE 4. Killing effect of paper treated with photoactive dyes conjugated with poly(vinyl amine) Mean log reduction of number of cells after 30 min of incubation

E. coli Salmonella Typhimurium Bacillus sp. L. monocytogenes

Relative dilution of dye conjugatea Dark Light Dark Light Dark Light Dark Light

Poly(vinyl amine)–rose bengal 0 Ϫ0.11 0.13 Ͻ0.01 Ͻ0.01 0.4 Ͻ0.01 0.36 Ϫ0.1 0.01 0.05 0.17 Ͼ1 Ͼ1 1.4 Ͼ2 Ϫ0.01 0.23 0.1 0.13 0.1 0.85 Ͼ1 Ͼ2 Ͼ2 0.57 0.12 1 0.53 Ͼ2b 0.37 Ͼ1b 1.45 Ͼ2 0.38 0.7 Poly(vinyl amine)–phloxine B Ϫ Ͻ Ͻ Ϫ Ϫ 0 0.11 0.13 0.01 0.48 0.01 0.01 0.16 0.09 Downloaded from http://meridian.allenpress.com/jfp/article-pdf/72/5/1020/1677894/0362-028x-72_5_1020.pdf by guest on 01 October 2021 0.01 0.19 0.25 Ͻ0.01 Ͼ2b Ͻ0.01 Ͼ2b Ϫ0.09 Ϫ0.07 0.1 0.83 0.57 Ͻ0.01 Ͼ2b Ͻ0.01 Ͼ2b Ϫ0.12 Ϫ0.09 1 1.27 0.64 Ͼ2 Ͼ2 0.87 Ͼ2 Ϫ0.15 Ͻ0.01 a The dilutions of conjugates used for paper treatment is presented in relative units, starting with concentration of 9.2 and 9.6% for poly(vinyl amine)–rose bengal and poly(vinyl amine)–phloxine B, respectively. b Killing effect of the dye on illumination with white light is significantly higher than the killing effect with dark incubation (P Ͻ 0.05). thus increasing the effective concentration of the dye in the dark (Fig. 1-2) and nontreated paper (Fig. 1-1). A signifi- vicinity of the cell. However, there was no significant kill- cant photokilling effect (90 to 99%) was observed for all ing effect observed for yeast cells. It is known that the bacterial strains on the papers treated with the original con- surface charge of S. cerevisiae is only slightly negative at centration of the RB-PVAm conjugate. For yeast cells, the physiological pH; however, at the ionic strength of nutrient effect was less, with only 80% of S. cerevisieae cells being medium, yeasts were shown to be electrically neutral at all killed after 30 min of incubation on RB-PVAm–treated pa- pH values (15). This may result in poor interaction of cells per. Treatment of paper with the 10- and 100-fold dilutions with the positively charged polymer, and explain the ab- of the original conjugate solution resulted in significantly sence of a photokilling effect. lower killing efficiency. Photoinactivation of microorganisms on the surface PhB-PVAm–treated paper produced a similar killing Listeria Bacil- of paper treated with photoactive dye conjugates. This effect under light for the gram-positive and lus E. coli processes resulted in a significant reduction in both gram- strains, but cells were slightly more resistant to positive (L. monocytogenes) and gram-negative (Salmonel- the photodynamic treatment, and S. cereviseae was not af- la Typhimurium) bacterial counts after 30 min of incuba- fected by PhB-PVAm–treated paper. tion on the paper treated with the PhB-PVAm conjugate DISCUSSION under illumination compared with incubation in dark and incubation on nontreated paper. The observed log reduc- In our previous work (3, 13), it was observed that cer- tions in count are presented in Table 4 for E. coli, L. mon- tain dyes are very effective bacteriocidal agents on illumi- ocytogenes, Bacillus sp., and S. cereviseae. The data for nation with white light. Both planktonic cells and cells in Salmonella were not calculated, as the used strain had high biofilms, as well as bacterial spores and viruses (bacterio- motility in semisolid agar and did not form distinct colo- phages) were eliminated as a result of the photodynamic nies. However, visual examination of plates containing pa- treatment. However, to develop a surface photosanitation per contaminated with Salmonella showed a substantial re- technique compatable with the requirements of the food duction in bacterial growth for the samples containing treat- industry, more basic research was needed to choose the ed paper incubated under illumination (Fig. 1-3) as com- most appropriate dye with activity against a wide variety pared with the samples containing treated paper kept in the of different food-related microorganisms in different forms,

FIGURE 1. Images of the plates showing the effect of paper treated with PhB-PVAm conjugate on bacterial growth. Paper was inoculated with 103 CFU of Salmonella Typhimurium per plate. 1, Control (nontreated paper); 2, treated paper incubated for 30 min in the dark; 3, treated paper incubated for 30 min under illumination. 1024 BROVKO ET AL. J. Food Prot., Vol. 72, No. 5 while at the same time, not interfering with food quality. REFERENCES The choice of photoactive dyes in the present study was 1. Anonymous. Fluorochrome absorption emission wavelengths. Avail- based on their availablity, potential photodynamic activity able at: http://www.sciencegateway.org/resources/fae1.htm. Ac- based on their photochemical properties, and the lowest cessed 1 October 2008. possible toxicity. All of the used dyes are readily available, 2. Augustin, M., T. Ali-Vehmas, and F. Atroshi. 2004. 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