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Efficacy of adding detergents to sanitizer solutions for inactivation of Escherichia coli O157:H7 on Romaine lettuce
International Journal of Food Microbiology 147 (2011) 157–161
Contents lists available at ScienceDirect
International Journal of Food Microbiology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / i j f o o d m i c r o
Review
Efficacy of adding detergents to sanitizer solutions for inactivation of Escherichia coli O157:H7 on Romaine lettuce☆ Lindsey A. Keskinen 1, Bassam A. Annous ⁎ Food Safety and Intervention Technologies Research Unit, U.S. Department of Agriculture, Agricultural Research Service, Eastern Regional Research Center, 600 East Mermaid Lane, Wyndmoor, PA 19038-8598, United States
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Article history: Received 30 September 2010 Received in revised form 22 March 2011 Accepted 5 April 2011 Available online 13 April 2011 Keywords: Escherichia coli O157:H7 Lettuce Leafy greens Sanitizer Chlorine dioxide Chlorine
a b s t r a c t Numerous Escherichia coli O157:H7 outbreaks have been linked to consumption of fresh lettuce. The development of effective and easily implemented wash treatment could reduce such incidents. The purpose of this study was to evaluate the addition of food-grade detergents to sanitizer solutions for inactivation of E. coli O157:H7 on Romaine lettuce. Freshly-cut leaves of Romaine lettuce were dip-inoculated to achieve a final cell concentration of 7.8 ± 0.2 log CFU/g, air-dried for 2 h, and stored overnight at 4 °C. Leaves were then washed for 2 min in an experimental short chain fatty acid formulation (SCFA) or in one of the following solutions with or without 0.2% dodecylbenzenesulfonic acid or 0.2% sodium 2-ethyl hexyl sulfate: 1) deionized water; 2) 100 ppm chlorine dioxide; 3) 100 ppm chlorine; and 4) 200 ppm chlorine. Following wash treatment, samples were blended in neutralizing buffer (1:3) and surface plated on the selective media CT-SMAC. The efficacy of wash treatments, with or without the detergents, in inactivating E. coli O157:H7 cells on lettuce leaves were not significantly different. The most effective wash solution was SCFA, which was capable of reducing E. coli O157:H7 populations by more than 5 log CFU/g. The rest of the wash treatments resulted in a population reduction of less than 1 log CFU/g. The effectiveness of SCFA surpasses that of other sanitizer treatments tested in this study and requires further research to optimize treatments to preserve lettuce quality. Conventional detergents did not enhance the efficacy of any of the wash treatments tested during this study. Published by Elsevier B.V.
Contents 1. 2.
Introduction . . . . . . . . . . . . . . . . . . Materials and methods . . . . . . . . . . . . . 2.1. Bacterial strain and inoculum preparation 2.2. Dip inoculation of lettuce . . . . . . . . 2.3. Preparation of treatment solutions . . . . 2.4. Sanitizing treatment of lettuce . . . . . . 2.5. Microbiological analysis . . . . . . . . . 2.6. Scanning electron microscopy . . . . . . 2.7. Statistical analysis . . . . . . . . . . . . 3. Results and discussion . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . .
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☆ Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture. ⁎ Corresponding author. Tel.: + 1 215 233 6797; fax: + 1 215 233 6406. E-mail address: [email protected] (B.A. Annous). 1 Current Address: Campbell Soup Company, 1 Campbell Place, Camden, NJ 08103. 0168-1605/$ – see front matter. Published by Elsevier B.V. doi:10.1016/j.ijfoodmicro.2011.04.002
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1. Introduction Escherichia coli O157:H7 has been implicated in recalls and outbreaks of illness linked to the consumption of raw leafy green vegetables, both in the United States and internationally (CDPH,
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1996, 2002, 2004a, 2004b, 2005, 2007a, 2007b, 2008; Hilborn et al., 1999; MMWR, 2006; Soderstrom et al., 2008). Packaged, prewashed, ready-to-eat leafy greens have been of particular concern, with recalls of prewashed baby spinach (CDPH, 2004a, 2007a; MMWR, 2006), shredded ready-to-eat Iceberg lettuce (CDPH, 2007b, 2008), pre-packaged, ready-to-eat salads containing Romaine lettuce (CDPH, 2002, 2005), and salad mixes containing Iceberg, Romaine, and other leafy greens (CDPH, 1996, 2004b; Hilborn et al., 1999). Leafy green vegetables are frequently consumed raw, so sanitizing washes are practical intervention strategies. In commercial valueadded produce processing, chlorine rinses are frequently used with concentrations varying from 50 to 200 ppm with contact times seldom exceeding 2 min (Parish et al., 2003). Studies have shown that chlorine rinses can decrease the bacterial load by b1 log CFU/g to 3.15 log CFU/g (Akbas and Olmez, 2007; Beuchat, 1999; Beuchat et al., 2004; Burnett et al., 2004; Escudero et al., 1999; Nthenge et al., 2007), depending on inoculation method, chlorine concentration, contact time, and the target bacteria tested. In some studies, the log reductions obtained with chlorine were equivalent to the water wash treatment (Beuchat, 1999; Nthenge et al., 2007). Chlorine dioxide has attracted interest as an alternative to chlorine. Rodgers et al. (2004) found that aqueous chlorine dioxide (5 ppm) was able to achieve greater than 5 log reductions of Listeria monocytogenes and E. coli O157:H7 on apples, lettuce and cantaloupe. These types of results are highly dependent on the method used to inoculate the produce—other studies have found that 5–200 ppm of aqueous chlorine dioxide were capable of reducing L. monocytogenes (Zhang and Farber, 1996) and E. coli O157:H7 (Keskinen et al., 2009) on lettuce by less than 2 log CFU/g. In a previous study, we reported that 100 and 200 ppm aqueous chlorine dioxide were significantly more effective at inactivating E. coli O157:H7 on Iceberg lettuce than 200 ppm chlorine and acidic electrolyzed water, and that 100 and 200 ppm aqueous chlorine dioxide and 200 ppm chlorine were equally as effective against E. coli O157:H7 on Romaine lettuce (Keskinen et al., 2009). Survival of bacterial cells during washing was attributed to poor contact between the bacterial cells on the lettuce surfaces and the sanitizer solution. Other factors considered to be potentially important in limiting the efficacy of washing treatments were attachment of bacteria in inaccessible sites in the damaged lettuce tissue, in stomata, and bacterial incorporation within biofilms (Keskinen et al., 2009; Annous et al., 2006, 2009). To achieve greater reductions in microbial populations on produce in general and lettuce in particular, improved methods of decontamination that overcome these limiting factors are needed. The addition of surfactant “surface active agent” to the wash solution could reduce the surface tension of the sanitizing solution and thus improve the contact between the bacterial cells and the sanitizer which could result in enhanced bacterial inactivation/ removal from the lettuce surfaces. Current reports indicate that surfactants alone are of limited use, and are not significantly more effective than water alone at removing pathogens from the surface of lettuce (Raiden et al., 2003; Takeuchi and Frank, 2000) and apples (Sapers et al., 2002), or were only capable of significantly reducing the population on surface of lettuce by 0.2 log CFU/cm2 (Hassan and Frank, 2003) and surfaces of strawberry fruit by 0.4 log CFU/g (Yu et al., 2001). The purpose of this study was to determine whether added detergents would enhance the efficacy of similar concentrations of chlorine and chlorine dioxide against E. coli O157:H7 on Romaine lettuce (Lactuca sativa L. var. longifolia). Sodium 2-ethyl hexyl sulfate and dodecylbenzenesulfonic acid are the two detergents considered in this study. Both detergents are currently approved for use as a produce rinse at a maximum of 0.2% (CFR, 2010). We also investigated the efficacy of an experimental short chain fatty acid solution against E. coli O157:H7 on Romaine lettuce.
2. Materials and methods 2.1. Bacterial strain and inoculum preparation E. coli O157:H7 SEA13B88 (human feces, apple cider-associated disease outbreak), maintained at − 80 °C in trypticase soy broth (TSB; Becton Dickinson, Sparks, MD) and 10% (v/v) glycerol, was grown for 18–24 h in TSB at 35 °C, transferred to a trypticase soy agar (TSA; Becton Dickinson) slant, and this working stock culture was stored at 4 °C for no more than 21 days. Inoculum was prepared by transferring a loopful (1 μl) of the working stock to 10 ml TSB, which was incubated in a shaking incubator for 6–8 h at 35 °C. Following incubation, 180 μl of the culture was transferred to 1.8 l of TSB, and then incubated at 35 °C in a shaking incubator for 18–24 h. The culture was then centrifuged (6740 ×g) at 4 °C for 15 min. After decanting the supernatant, the resulting pellet was resuspended in sterile deionized water and centrifuged (6740 ×g) for 15 min at 4 °C. The supernatant was decanted and the pellet was resuspended in 3.6 l of sterile deionized water. The concentration of the inoculum was determined by serially diluting the inoculum in 0.1% peptone water (PW; Becton Dickinson) and plating on TSA. 2.2. Dip inoculation of lettuce Commercially available Romaine lettuce (Lactuca sativa L. var. longifolia) was purchased at a local supermarket, and stored at 4 ± 2 °C for a maximum of 24 h before use in experiments. Damaged outer leaves were removed from each head of lettuce, the lettuce was cut into pieces approximately 4–6 cm2 and immediately submerged into the E. coli O157:H7 inoculum suspension for 5 min. Excess liquid culture on the lettuce was removed using a salad spinner (OXO Good Grips Salad Spinner, OXO International, Ltd., New York, NY) for 1 min and then the lettuce was placed into an open container and allowed to dry for 2 h at 22 ± 2 °C in a biosafety cabinet. The lettuce was then placed into plastic bags and stored for 18–24 h at 4 ± 2 °C. 2.3. Preparation of treatment solutions Chlorine solutions (500 ml) were prepared immediately before use by diluting 6.0% sodium hypochlorite (Clorox, The Clorox Company, Oakland, CA) in deionized water to achieve concentrations of 100 or 200 ppm chlorine. Concentrations were verified with chlorine concentration test strips (Advantec MHS, Inc., Dublin, CA). An aqueous chlorine dioxide stock solution was made by mixing acid and sodium chlorite dry chemicals using chlorine dioxide sachets (Tri-Nova® 2-gram solution pack, ICA Tri-Nova, LLC, Newnan, GA) in a sealed bottle of deionized water (1 l) and allowing the reaction to go to completion for at least 3 days at 22 ± 2 °C, in the absence of light. The chlorite ion concentration was then measured by titration (Titralab Model TIM840; Radiometer Analytical SAS, Villeurbanne CEDEX, France) immediately before use, and diluted with deionized water to make 500 ml solutions containing 100 ppm chlorite ion. All chlorine and chlorite solutions were used immediately following preparation and treatments were conducted in chlorine-aged glassware at 22 ± 2 °C. Detergent-containing sanitizer solutions were prepared by mixing fresh sanitizer solutions, as described above, and adding dodecylbenzenesulfonic acid (DA; Sigma-Aldrich, St. Louis, MO) or sodium 2ethyl hexyl sulfate (EHS; Stepanol EHS, Stepan Company, Northfield, IL) to obtain a concentration of 0.2% (w/v and v/v, respectively) in the wash solution. The experimental short-chain fatty acid solution (SCFA; Summerdale, Inc., Verona, WI) was prepared by diluting the full-strength solution (70% caprylic acid, 10% PE-1198 surfactant, and 20% lactic acid) supplied by the manufacturer to a 0.4% concentration (v/v) in deionized water.
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2.4. Sanitizing treatment of lettuce
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(A)
Inoculated Romaine lettuce (25 g) was stirred into 500 ml of sanitizer or sanitizer and detergent solution and held at 22 ± 2 °C for 2 min on an orbital shaker. Excess sanitizer on the lettuce was removed using a salad spinner prior to assaying for residual E. coli O157:H7 cells. 2.5. Microbiological analysis Immediately following sanitizing treatments, the Romaine lettuce samples were diluted 1:3 (w/v) in DE Neutralizing Buffer (Becton Dickinson) and blended for 1 min in a Waring commercial blender (model 51BL31, Waring Commercial, Torrington, CT). The resulting samples were then serially diluted in PW and plated on CTSMAC, consisting of Sorbitol MacConkey Agar (Remel, Lenexa, KS) supplemented with cefixime (0.05 mg/l) and potassium tellurite (2.5 mg/l) (SMAC Media Cefixime-Tellurite Supplement; Invitrogen Dynal AS, Oslo, Norway), and incubated at 35 °C for 18–24 h. To facilitate recovery of injured cells, samples were plated on TSA, which was incubated at 35 °C for 2 h, then were overlaid with CTSMAC and incubated at 35 °C for an additional 18–24 h, and were then enumerated.
(B)
2.6. Scanning electron microscopy Dip-inoculated 1–2 cm-sized samples of Romaine lettuce were stored for 18–24 h at 4 ± 2 °C, and then were fixed for scanning electron microscopy (SEM). Samples for SEM were immersed in 20 ml aliquots of 2.5% glutaraldehyde–0.1 M imidazole buffer and sealed for 12–24 h before washing in imidazole buffer and dehydrating in 50%, 80% and absolute ethanol. Samples were then critical point dried with carbon dioxide, mounted with Duco cement (ITW Performance Polymers, Riviera Beach, FL) and colloidal silver adhesive, and then were sputter-coated with a thin layer of gold. Samples were imaged using a Quanta200 environmental scanning electron microscope (FEI Co., Inc., Hillsboro, OR), operated in the high vacuum, secondary electron imaging mode.
Fig. 1. (A) SEM micrograph of Escherichia coli O157:H7 in biofilms on inoculated Romaine lettuce, stored at 4 ± 2 °C for 24 h. (B) SEM micrograph of E. coli O157:H7 in biofilms and infiltrating stomata on inoculated Romaine lettuce, stored at 4 ± 2 °C for 24 h then treated with 200 ppm chlorine for 2 min (Keskinen et al., 2009).
2.7. Statistical analysis All sanitizer experiments were replicated three times. The resulting data were analyzed using SAS software (SAS Version 8; SAS Institute, Cary, NC) with a general linear mixed effects model and analysis of variance (ANOVA) for least significant differences among the combinations of treatments (P b 0.05). Means were separated by the least significant differences (LSD) test. 3. Results and discussion In a previous study, we had observed that sanitizing solutions containing aqueous chlorine dioxide at 100 or 200 ppm was more effective than other sanitizers tested against E. coli O157:H7 on Iceberg lettuce, achieving population reductions in excess of 1 log CFU/g, while, 200 ppm chlorine or aqueous chlorine dioxide treatments were equally effective in inactivating E. coli O157:H7 on Romaine lettuce, with population reductions not significantly greater than 1 log CFU/g (Keskinen et al., 2009). Also, SEM studies shown that E. coli O157:H7cells were still present in damaged lettuce tissue, in stomata, and incorporated into mixed culture biofilms following a 2 min wash in 200 ppm chlorine (Fig. 1B; Keskinen et al., 2009). Results from this study indicated that major factors which are important in limiting the efficacy of sanitation treatments of lettuce are the attachment of pathogenic cells to inaccessible sites on the surface of lettuce and/or the incorporation of those cells within biofilms in such inaccessible sites. Therefore, the development of new technologies, capable of improving the delivery of sanitizing agents to
potential pathogens' harborage sites on lettuce is required. The addition of surfactant could reduce the surface tension of a sanitizing solution and thus enhances contact between the pathogenic cells and the sanitizers which could improve bacterial inactivation/removal. In this study, the efficacies of three solutions (100 and 200 ppm chlorine, and 100 ppm aqueous chlorine dioxide) with and without added detergents, and SCFA solution (an experimental formulation) were compared. Previous studies have been done to assess the efficacy of detergents at removing pathogens from lettuce and other produce (Hassan and Frank, 2003; Raiden et al., 2003; Takeuchi and Frank; 2000). One study has reported that Tween 85, a surfactant, was able to detach significantly more E. coli O157:H7 from Iceberg lettuce than water alone, with a difference in population between the two of 0.48 log CFU/cm2 (Hassan and Frank, 2003). In this study, the addition of detergents did not significantly change the efficacy of chlorine, aqueous chlorine dioxide, or deionized water (Table 1). This is similar to the results obtained by Raiden et al. (2003), which also showed that detergents did not significantly enhance the removal of bacterial cells from fresh produce. These results suggest that the addition of surfactants to the sanitizer solution was not able to aid in penetrating the biofilm in order to inactivate the bacterial cells. SEM studies have shown that E. coli O157:H7 cells, both individually and in clusters and mixed culture biofilms, were covering the entire surface of dip inoculated Romaine lettuce (Fig. 1A). However, the extension of treatment time beyond 2 min might prove beneficial to allow for more time for the sanitizer to penetrate the biofilm and inactivate the cells.
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Table 1 Efficacy of washing solutions on reducing Escherichia coli O157:H7 populations on artificially inoculated Romaine lettuce (Lactuca sativa L. var. longifolia) stored at 4 °C for 18–24 h. Treatment1
CTSMAC (log CFU/g) Population2
Untreated control Deionized water, pH 7.0 Deionized water + EHS, pH 7.7 Deionized water + DA, pH 8.6 Chlorine, 100 ppm, pH 8.0 Chlorine, 100 ppm + EHS, pH 9.5 Chlorine, 100 ppm + DA, pH 9.9 Chlorine, 200 ppm, pH 8.0 Chlorine, 200 ppm + EHS, pH 10.0 Chlorine, 200 ppm + DA, pH 10.1 TriNova, 100 ppm ClO-2, pH 4.1 TriNova, 100 ppm ClO-2 + EHS, pH 4.3 TriNova, 100 ppm ClO2- + DA, pH 4.7 0.4% SCFA, first treatment, pH 2.8 0.4% SCFA, second treatment, pH 2.8
Recovery (log CFU/g) Mean reduction
a
7.40 ± 0.32 6.63 ± 0.43bc 6.85 ± 0.29b 6.79 ± 0.49b 6.51 ± 0.27bc 6.46 ± 0.50bc 6.20 ± 0.65c 6.53 ± 0.32bc 6.58 ± 0.35bc 6.43 ± 0.26bc 6.63 ± 0.19bc 6.58 ± 0.37bc 6.54 ± 0.25bc 0.00 ± 0.00d 6.63 ± 0.07bc
Population2
Mean reduction
a
0.77 0.55 0.61 0.89 0.94 1.20 0.87 0.82 0.97 0.77 0.82 0.86 7.40 0.77
7.80 ± 0.14 7.32 ± 0.15b 7.21 ± 0.15bc 7.11 ± 0.24bcd 6.94 ± 0.23de 6.79 ± 0.27e 6.81 ± 0.27e 7.00 ± 0.14cde 6.95 ± 0.21de 7.02 ± 0.19cde 6.86 ± 0.12de 6.84 ± 0.33e 6.96 ± 0.25cde 0.00 ± 0.00f 6.84 ± 0.07e
0.48 0.59 0.69 0.86 1.01 0.99 0.80 0.85 0.78 0.94 0.96 0.84 7.80 0.96
1
Untreated control, n = 21; deionized water, chlorine 200 ppm, 0.4% SCFA first and second treatments, n = 3; TriNova, TriNova + EHS, TriNova + DA, deionized water + EHS, deionized water + DA, chlorine 100 ppm, chlorine 100 ppm + EHS, chlorine 100 ppm + DA, chlorine 200 ppm + EHS, and chlorine 200 ppm + DA, n = 6. Within the same column, means not followed by the same letter are significantly different (P b 0.05).
2
Therefore, further studies into the use of detergents might be needed in order to optimize this treatment in order to enhance the safety and shelf life of treated fresh and fresh-cut produce. The most effective sanitizer, SCFA, was also the most acidic sanitizer (Table 1). However, SCFA was only effective throughout one 2 min treatment. When the same solution was used again to treat inoculated Romaine lettuce, its effectiveness was not significantly (P b 0.05) different from deionized water, chlorine or chlorine dioxide (Table 1). The lack of efficacy upon the second use of the SCFA treatment seems to indicate that an active component of the sanitizer is adhering to the treated leaves upon initial contact with the treatment solution, and being removed from solution (Table 1). This component does not seem to have an effect on the pH of the treatment solution, as the pH remained the same following treatment of lettuce. It also seems that the low pH was not solely responsible for the efficacy of SCFA, since the pH remained constant throughout subsequent treatments using the same solution, although the solution was less efficacious the second time it was used (Table 1). Differences in pH between the other sanitizer/ detergent treatment combinations also do not seem to result in significant differences in E. coli O157:H7 on treated lettuce (Table 1). Similar results were previously reported by Keskinen et al. (2009), which showed that no significant reduction in microbial population using sanitizer solutions with pH between 2.6 and 8. Cut edges of inoculated lettuce have been shown to retain many viable bacteria following treatment (Hassan and Frank, 2003; Keskinen et al., 2009; Takeuchi and Frank, 2001). Lettuce treated in this study was dip-inoculated, which makes it likely that E. coli O157: H7 was present along the cut edges of the product. The significant reduction obtained by SCFA seems to indicate that cells within the cut edges of the product were inactivated, as well as the bacteria present on the intact tissue (Table 1). The sanitizer SCFA seems to be a promising treatment for the removal of bacteria from fresh produce, and is significantly more effective than chlorinated water, the current industry standard. This sanitizer was somewhat phytotoxic, and at high levels or long treatment times it may damage lettuce leaves. Therefore, further study is required in order to optimize this treatment in order to preserve the shelf-life of treated lettuce. Given the high degree of efficacy of this sanitizer, it would be interesting to investigate whether it is as effective when used to sanitize other types of produce. Acknowledgments This work was funded by USDA-CSREES grant number 200651110-03681. The authors thank Ms Gouping Bao for the scanning
electron microscopy, Mr. Joseph E. Sites for his work on the micrographs, Ms. Angela Burke for assistance with analysis of treatment solutions, and Dr. Joshua Gurtler for his assistance with the statistical analysis, and Dr. Dike Ukuku and Mr. Joseph Sites for their helpful comments on the manuscript. The authors also thank Mr. Joel Tenney of ICA Tri-Nova, LLC, and Dr. Robert Coleman of Summerdale Inc. for providing their products for evaluation.
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Parish, M.E., Beuchat, L.R., Suslow, T.V., Harris, L.J., Garrett, E.H., Farber, J.N., Busta, F.F., 2003. Methods to reduce/eliminate pathogens from fresh and fresh-cut produce. Comprehensive Reviews in Food Science and Food Safety 2, 161–173. Raiden, R.M., Sumner, S.S., Eifert, J.D., Pierson, M.D., 2003. Efficacy of detergents in removing Salmonella and Shigella spp. from the surface of fresh produce. Journal of Food Protection 66, 2210–2215. Rodgers, S.L., Cash, J.N., Siddiq, M., Ryser, E.T., 2004. A comparison of different chemical sanitizers for inactivating Escherichia coli O157:H7 and Listeria monocytogenes in solution and on apples, lettuce, strawberries, and cantaloupe. Journal of Food Protection 67, 721–731. Sapers, G.M., Miller, R.L., Annous, B.A., Burke, A.M., 2002. Improved antimicromial wash treatments for decontamination of apples. Journal of Food Science 67 (5), 1886–1891. Soderstrom, A., Osterberg, P., Lindqvist, A., Jonsson, B., Lindberg, A., Ulander, S.B., Welinder-Olsson, C., Lofdahl, S., Kaijser, B., De Jong, B., Kuhlmann-Berenzon, S., Boqvist, S., Eriksson, E., Szanto, E., Andersson, S., Allestam, G., Hedenstrom, I., Muller, L.L., Andersson, Y., 2008. A large Escherichia coli O157 outbreak in Sweden associated with locally produced lettuce. Foodborne Pathogens and Disease 5, 339–349. Takeuchi, K., Frank, J.F., 2000. Penetration of Escherichia coli O157:H7 into lettuce tissues as affected by inoculum size and temperature and the effect of chlorine treatment on cell viability. Journal of Food Protection 63, 434–440. Takeuchi, K., Frank, J.F., 2001. Direct microscopic observation of lettuce leaf decontamination with a prototype fruit and vegetable washing solution and 1% NaCl–NaHCO3. Journal of Food Protection 64, 1235–1239. Yu, K., Newman, M.C., Archbold, D.D., Hamilton-Kemp, T.R., 2001. Survival of Escherichia coli O157:H7 on strawberry fruit and reduction of the pathogen population by chemical agents. Journal of Food Protection 64, 1334–1340. Zhang, S., Farber, J.M., 1996. The effects of various disinfectants against Listeria monocytogenes on fresh-cut vegetables. Food Microbiology 13, 311–321.
Contents lists available at ScienceDirect
International Journal of Food Microbiology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / i j f o o d m i c r o
Review
Efficacy of adding detergents to sanitizer solutions for inactivation of Escherichia coli O157:H7 on Romaine lettuce☆ Lindsey A. Keskinen 1, Bassam A. Annous ⁎ Food Safety and Intervention Technologies Research Unit, U.S. Department of Agriculture, Agricultural Research Service, Eastern Regional Research Center, 600 East Mermaid Lane, Wyndmoor, PA 19038-8598, United States
a r t i c l e
i n f o
Article history: Received 30 September 2010 Received in revised form 22 March 2011 Accepted 5 April 2011 Available online 13 April 2011 Keywords: Escherichia coli O157:H7 Lettuce Leafy greens Sanitizer Chlorine dioxide Chlorine
a b s t r a c t Numerous Escherichia coli O157:H7 outbreaks have been linked to consumption of fresh lettuce. The development of effective and easily implemented wash treatment could reduce such incidents. The purpose of this study was to evaluate the addition of food-grade detergents to sanitizer solutions for inactivation of E. coli O157:H7 on Romaine lettuce. Freshly-cut leaves of Romaine lettuce were dip-inoculated to achieve a final cell concentration of 7.8 ± 0.2 log CFU/g, air-dried for 2 h, and stored overnight at 4 °C. Leaves were then washed for 2 min in an experimental short chain fatty acid formulation (SCFA) or in one of the following solutions with or without 0.2% dodecylbenzenesulfonic acid or 0.2% sodium 2-ethyl hexyl sulfate: 1) deionized water; 2) 100 ppm chlorine dioxide; 3) 100 ppm chlorine; and 4) 200 ppm chlorine. Following wash treatment, samples were blended in neutralizing buffer (1:3) and surface plated on the selective media CT-SMAC. The efficacy of wash treatments, with or without the detergents, in inactivating E. coli O157:H7 cells on lettuce leaves were not significantly different. The most effective wash solution was SCFA, which was capable of reducing E. coli O157:H7 populations by more than 5 log CFU/g. The rest of the wash treatments resulted in a population reduction of less than 1 log CFU/g. The effectiveness of SCFA surpasses that of other sanitizer treatments tested in this study and requires further research to optimize treatments to preserve lettuce quality. Conventional detergents did not enhance the efficacy of any of the wash treatments tested during this study. Published by Elsevier B.V.
Contents 1. 2.
Introduction . . . . . . . . . . . . . . . . . . Materials and methods . . . . . . . . . . . . . 2.1. Bacterial strain and inoculum preparation 2.2. Dip inoculation of lettuce . . . . . . . . 2.3. Preparation of treatment solutions . . . . 2.4. Sanitizing treatment of lettuce . . . . . . 2.5. Microbiological analysis . . . . . . . . . 2.6. Scanning electron microscopy . . . . . . 2.7. Statistical analysis . . . . . . . . . . . . 3. Results and discussion . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . .
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☆ Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture. ⁎ Corresponding author. Tel.: + 1 215 233 6797; fax: + 1 215 233 6406. E-mail address: [email protected] (B.A. Annous). 1 Current Address: Campbell Soup Company, 1 Campbell Place, Camden, NJ 08103. 0168-1605/$ – see front matter. Published by Elsevier B.V. doi:10.1016/j.ijfoodmicro.2011.04.002
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1. Introduction Escherichia coli O157:H7 has been implicated in recalls and outbreaks of illness linked to the consumption of raw leafy green vegetables, both in the United States and internationally (CDPH,
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1996, 2002, 2004a, 2004b, 2005, 2007a, 2007b, 2008; Hilborn et al., 1999; MMWR, 2006; Soderstrom et al., 2008). Packaged, prewashed, ready-to-eat leafy greens have been of particular concern, with recalls of prewashed baby spinach (CDPH, 2004a, 2007a; MMWR, 2006), shredded ready-to-eat Iceberg lettuce (CDPH, 2007b, 2008), pre-packaged, ready-to-eat salads containing Romaine lettuce (CDPH, 2002, 2005), and salad mixes containing Iceberg, Romaine, and other leafy greens (CDPH, 1996, 2004b; Hilborn et al., 1999). Leafy green vegetables are frequently consumed raw, so sanitizing washes are practical intervention strategies. In commercial valueadded produce processing, chlorine rinses are frequently used with concentrations varying from 50 to 200 ppm with contact times seldom exceeding 2 min (Parish et al., 2003). Studies have shown that chlorine rinses can decrease the bacterial load by b1 log CFU/g to 3.15 log CFU/g (Akbas and Olmez, 2007; Beuchat, 1999; Beuchat et al., 2004; Burnett et al., 2004; Escudero et al., 1999; Nthenge et al., 2007), depending on inoculation method, chlorine concentration, contact time, and the target bacteria tested. In some studies, the log reductions obtained with chlorine were equivalent to the water wash treatment (Beuchat, 1999; Nthenge et al., 2007). Chlorine dioxide has attracted interest as an alternative to chlorine. Rodgers et al. (2004) found that aqueous chlorine dioxide (5 ppm) was able to achieve greater than 5 log reductions of Listeria monocytogenes and E. coli O157:H7 on apples, lettuce and cantaloupe. These types of results are highly dependent on the method used to inoculate the produce—other studies have found that 5–200 ppm of aqueous chlorine dioxide were capable of reducing L. monocytogenes (Zhang and Farber, 1996) and E. coli O157:H7 (Keskinen et al., 2009) on lettuce by less than 2 log CFU/g. In a previous study, we reported that 100 and 200 ppm aqueous chlorine dioxide were significantly more effective at inactivating E. coli O157:H7 on Iceberg lettuce than 200 ppm chlorine and acidic electrolyzed water, and that 100 and 200 ppm aqueous chlorine dioxide and 200 ppm chlorine were equally as effective against E. coli O157:H7 on Romaine lettuce (Keskinen et al., 2009). Survival of bacterial cells during washing was attributed to poor contact between the bacterial cells on the lettuce surfaces and the sanitizer solution. Other factors considered to be potentially important in limiting the efficacy of washing treatments were attachment of bacteria in inaccessible sites in the damaged lettuce tissue, in stomata, and bacterial incorporation within biofilms (Keskinen et al., 2009; Annous et al., 2006, 2009). To achieve greater reductions in microbial populations on produce in general and lettuce in particular, improved methods of decontamination that overcome these limiting factors are needed. The addition of surfactant “surface active agent” to the wash solution could reduce the surface tension of the sanitizing solution and thus improve the contact between the bacterial cells and the sanitizer which could result in enhanced bacterial inactivation/ removal from the lettuce surfaces. Current reports indicate that surfactants alone are of limited use, and are not significantly more effective than water alone at removing pathogens from the surface of lettuce (Raiden et al., 2003; Takeuchi and Frank, 2000) and apples (Sapers et al., 2002), or were only capable of significantly reducing the population on surface of lettuce by 0.2 log CFU/cm2 (Hassan and Frank, 2003) and surfaces of strawberry fruit by 0.4 log CFU/g (Yu et al., 2001). The purpose of this study was to determine whether added detergents would enhance the efficacy of similar concentrations of chlorine and chlorine dioxide against E. coli O157:H7 on Romaine lettuce (Lactuca sativa L. var. longifolia). Sodium 2-ethyl hexyl sulfate and dodecylbenzenesulfonic acid are the two detergents considered in this study. Both detergents are currently approved for use as a produce rinse at a maximum of 0.2% (CFR, 2010). We also investigated the efficacy of an experimental short chain fatty acid solution against E. coli O157:H7 on Romaine lettuce.
2. Materials and methods 2.1. Bacterial strain and inoculum preparation E. coli O157:H7 SEA13B88 (human feces, apple cider-associated disease outbreak), maintained at − 80 °C in trypticase soy broth (TSB; Becton Dickinson, Sparks, MD) and 10% (v/v) glycerol, was grown for 18–24 h in TSB at 35 °C, transferred to a trypticase soy agar (TSA; Becton Dickinson) slant, and this working stock culture was stored at 4 °C for no more than 21 days. Inoculum was prepared by transferring a loopful (1 μl) of the working stock to 10 ml TSB, which was incubated in a shaking incubator for 6–8 h at 35 °C. Following incubation, 180 μl of the culture was transferred to 1.8 l of TSB, and then incubated at 35 °C in a shaking incubator for 18–24 h. The culture was then centrifuged (6740 ×g) at 4 °C for 15 min. After decanting the supernatant, the resulting pellet was resuspended in sterile deionized water and centrifuged (6740 ×g) for 15 min at 4 °C. The supernatant was decanted and the pellet was resuspended in 3.6 l of sterile deionized water. The concentration of the inoculum was determined by serially diluting the inoculum in 0.1% peptone water (PW; Becton Dickinson) and plating on TSA. 2.2. Dip inoculation of lettuce Commercially available Romaine lettuce (Lactuca sativa L. var. longifolia) was purchased at a local supermarket, and stored at 4 ± 2 °C for a maximum of 24 h before use in experiments. Damaged outer leaves were removed from each head of lettuce, the lettuce was cut into pieces approximately 4–6 cm2 and immediately submerged into the E. coli O157:H7 inoculum suspension for 5 min. Excess liquid culture on the lettuce was removed using a salad spinner (OXO Good Grips Salad Spinner, OXO International, Ltd., New York, NY) for 1 min and then the lettuce was placed into an open container and allowed to dry for 2 h at 22 ± 2 °C in a biosafety cabinet. The lettuce was then placed into plastic bags and stored for 18–24 h at 4 ± 2 °C. 2.3. Preparation of treatment solutions Chlorine solutions (500 ml) were prepared immediately before use by diluting 6.0% sodium hypochlorite (Clorox, The Clorox Company, Oakland, CA) in deionized water to achieve concentrations of 100 or 200 ppm chlorine. Concentrations were verified with chlorine concentration test strips (Advantec MHS, Inc., Dublin, CA). An aqueous chlorine dioxide stock solution was made by mixing acid and sodium chlorite dry chemicals using chlorine dioxide sachets (Tri-Nova® 2-gram solution pack, ICA Tri-Nova, LLC, Newnan, GA) in a sealed bottle of deionized water (1 l) and allowing the reaction to go to completion for at least 3 days at 22 ± 2 °C, in the absence of light. The chlorite ion concentration was then measured by titration (Titralab Model TIM840; Radiometer Analytical SAS, Villeurbanne CEDEX, France) immediately before use, and diluted with deionized water to make 500 ml solutions containing 100 ppm chlorite ion. All chlorine and chlorite solutions were used immediately following preparation and treatments were conducted in chlorine-aged glassware at 22 ± 2 °C. Detergent-containing sanitizer solutions were prepared by mixing fresh sanitizer solutions, as described above, and adding dodecylbenzenesulfonic acid (DA; Sigma-Aldrich, St. Louis, MO) or sodium 2ethyl hexyl sulfate (EHS; Stepanol EHS, Stepan Company, Northfield, IL) to obtain a concentration of 0.2% (w/v and v/v, respectively) in the wash solution. The experimental short-chain fatty acid solution (SCFA; Summerdale, Inc., Verona, WI) was prepared by diluting the full-strength solution (70% caprylic acid, 10% PE-1198 surfactant, and 20% lactic acid) supplied by the manufacturer to a 0.4% concentration (v/v) in deionized water.
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2.4. Sanitizing treatment of lettuce
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(A)
Inoculated Romaine lettuce (25 g) was stirred into 500 ml of sanitizer or sanitizer and detergent solution and held at 22 ± 2 °C for 2 min on an orbital shaker. Excess sanitizer on the lettuce was removed using a salad spinner prior to assaying for residual E. coli O157:H7 cells. 2.5. Microbiological analysis Immediately following sanitizing treatments, the Romaine lettuce samples were diluted 1:3 (w/v) in DE Neutralizing Buffer (Becton Dickinson) and blended for 1 min in a Waring commercial blender (model 51BL31, Waring Commercial, Torrington, CT). The resulting samples were then serially diluted in PW and plated on CTSMAC, consisting of Sorbitol MacConkey Agar (Remel, Lenexa, KS) supplemented with cefixime (0.05 mg/l) and potassium tellurite (2.5 mg/l) (SMAC Media Cefixime-Tellurite Supplement; Invitrogen Dynal AS, Oslo, Norway), and incubated at 35 °C for 18–24 h. To facilitate recovery of injured cells, samples were plated on TSA, which was incubated at 35 °C for 2 h, then were overlaid with CTSMAC and incubated at 35 °C for an additional 18–24 h, and were then enumerated.
(B)
2.6. Scanning electron microscopy Dip-inoculated 1–2 cm-sized samples of Romaine lettuce were stored for 18–24 h at 4 ± 2 °C, and then were fixed for scanning electron microscopy (SEM). Samples for SEM were immersed in 20 ml aliquots of 2.5% glutaraldehyde–0.1 M imidazole buffer and sealed for 12–24 h before washing in imidazole buffer and dehydrating in 50%, 80% and absolute ethanol. Samples were then critical point dried with carbon dioxide, mounted with Duco cement (ITW Performance Polymers, Riviera Beach, FL) and colloidal silver adhesive, and then were sputter-coated with a thin layer of gold. Samples were imaged using a Quanta200 environmental scanning electron microscope (FEI Co., Inc., Hillsboro, OR), operated in the high vacuum, secondary electron imaging mode.
Fig. 1. (A) SEM micrograph of Escherichia coli O157:H7 in biofilms on inoculated Romaine lettuce, stored at 4 ± 2 °C for 24 h. (B) SEM micrograph of E. coli O157:H7 in biofilms and infiltrating stomata on inoculated Romaine lettuce, stored at 4 ± 2 °C for 24 h then treated with 200 ppm chlorine for 2 min (Keskinen et al., 2009).
2.7. Statistical analysis All sanitizer experiments were replicated three times. The resulting data were analyzed using SAS software (SAS Version 8; SAS Institute, Cary, NC) with a general linear mixed effects model and analysis of variance (ANOVA) for least significant differences among the combinations of treatments (P b 0.05). Means were separated by the least significant differences (LSD) test. 3. Results and discussion In a previous study, we had observed that sanitizing solutions containing aqueous chlorine dioxide at 100 or 200 ppm was more effective than other sanitizers tested against E. coli O157:H7 on Iceberg lettuce, achieving population reductions in excess of 1 log CFU/g, while, 200 ppm chlorine or aqueous chlorine dioxide treatments were equally effective in inactivating E. coli O157:H7 on Romaine lettuce, with population reductions not significantly greater than 1 log CFU/g (Keskinen et al., 2009). Also, SEM studies shown that E. coli O157:H7cells were still present in damaged lettuce tissue, in stomata, and incorporated into mixed culture biofilms following a 2 min wash in 200 ppm chlorine (Fig. 1B; Keskinen et al., 2009). Results from this study indicated that major factors which are important in limiting the efficacy of sanitation treatments of lettuce are the attachment of pathogenic cells to inaccessible sites on the surface of lettuce and/or the incorporation of those cells within biofilms in such inaccessible sites. Therefore, the development of new technologies, capable of improving the delivery of sanitizing agents to
potential pathogens' harborage sites on lettuce is required. The addition of surfactant could reduce the surface tension of a sanitizing solution and thus enhances contact between the pathogenic cells and the sanitizers which could improve bacterial inactivation/removal. In this study, the efficacies of three solutions (100 and 200 ppm chlorine, and 100 ppm aqueous chlorine dioxide) with and without added detergents, and SCFA solution (an experimental formulation) were compared. Previous studies have been done to assess the efficacy of detergents at removing pathogens from lettuce and other produce (Hassan and Frank, 2003; Raiden et al., 2003; Takeuchi and Frank; 2000). One study has reported that Tween 85, a surfactant, was able to detach significantly more E. coli O157:H7 from Iceberg lettuce than water alone, with a difference in population between the two of 0.48 log CFU/cm2 (Hassan and Frank, 2003). In this study, the addition of detergents did not significantly change the efficacy of chlorine, aqueous chlorine dioxide, or deionized water (Table 1). This is similar to the results obtained by Raiden et al. (2003), which also showed that detergents did not significantly enhance the removal of bacterial cells from fresh produce. These results suggest that the addition of surfactants to the sanitizer solution was not able to aid in penetrating the biofilm in order to inactivate the bacterial cells. SEM studies have shown that E. coli O157:H7 cells, both individually and in clusters and mixed culture biofilms, were covering the entire surface of dip inoculated Romaine lettuce (Fig. 1A). However, the extension of treatment time beyond 2 min might prove beneficial to allow for more time for the sanitizer to penetrate the biofilm and inactivate the cells.
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Table 1 Efficacy of washing solutions on reducing Escherichia coli O157:H7 populations on artificially inoculated Romaine lettuce (Lactuca sativa L. var. longifolia) stored at 4 °C for 18–24 h. Treatment1
CTSMAC (log CFU/g) Population2
Untreated control Deionized water, pH 7.0 Deionized water + EHS, pH 7.7 Deionized water + DA, pH 8.6 Chlorine, 100 ppm, pH 8.0 Chlorine, 100 ppm + EHS, pH 9.5 Chlorine, 100 ppm + DA, pH 9.9 Chlorine, 200 ppm, pH 8.0 Chlorine, 200 ppm + EHS, pH 10.0 Chlorine, 200 ppm + DA, pH 10.1 TriNova, 100 ppm ClO-2, pH 4.1 TriNova, 100 ppm ClO-2 + EHS, pH 4.3 TriNova, 100 ppm ClO2- + DA, pH 4.7 0.4% SCFA, first treatment, pH 2.8 0.4% SCFA, second treatment, pH 2.8
Recovery (log CFU/g) Mean reduction
a
7.40 ± 0.32 6.63 ± 0.43bc 6.85 ± 0.29b 6.79 ± 0.49b 6.51 ± 0.27bc 6.46 ± 0.50bc 6.20 ± 0.65c 6.53 ± 0.32bc 6.58 ± 0.35bc 6.43 ± 0.26bc 6.63 ± 0.19bc 6.58 ± 0.37bc 6.54 ± 0.25bc 0.00 ± 0.00d 6.63 ± 0.07bc
Population2
Mean reduction
a
0.77 0.55 0.61 0.89 0.94 1.20 0.87 0.82 0.97 0.77 0.82 0.86 7.40 0.77
7.80 ± 0.14 7.32 ± 0.15b 7.21 ± 0.15bc 7.11 ± 0.24bcd 6.94 ± 0.23de 6.79 ± 0.27e 6.81 ± 0.27e 7.00 ± 0.14cde 6.95 ± 0.21de 7.02 ± 0.19cde 6.86 ± 0.12de 6.84 ± 0.33e 6.96 ± 0.25cde 0.00 ± 0.00f 6.84 ± 0.07e
0.48 0.59 0.69 0.86 1.01 0.99 0.80 0.85 0.78 0.94 0.96 0.84 7.80 0.96
1
Untreated control, n = 21; deionized water, chlorine 200 ppm, 0.4% SCFA first and second treatments, n = 3; TriNova, TriNova + EHS, TriNova + DA, deionized water + EHS, deionized water + DA, chlorine 100 ppm, chlorine 100 ppm + EHS, chlorine 100 ppm + DA, chlorine 200 ppm + EHS, and chlorine 200 ppm + DA, n = 6. Within the same column, means not followed by the same letter are significantly different (P b 0.05).
2
Therefore, further studies into the use of detergents might be needed in order to optimize this treatment in order to enhance the safety and shelf life of treated fresh and fresh-cut produce. The most effective sanitizer, SCFA, was also the most acidic sanitizer (Table 1). However, SCFA was only effective throughout one 2 min treatment. When the same solution was used again to treat inoculated Romaine lettuce, its effectiveness was not significantly (P b 0.05) different from deionized water, chlorine or chlorine dioxide (Table 1). The lack of efficacy upon the second use of the SCFA treatment seems to indicate that an active component of the sanitizer is adhering to the treated leaves upon initial contact with the treatment solution, and being removed from solution (Table 1). This component does not seem to have an effect on the pH of the treatment solution, as the pH remained the same following treatment of lettuce. It also seems that the low pH was not solely responsible for the efficacy of SCFA, since the pH remained constant throughout subsequent treatments using the same solution, although the solution was less efficacious the second time it was used (Table 1). Differences in pH between the other sanitizer/ detergent treatment combinations also do not seem to result in significant differences in E. coli O157:H7 on treated lettuce (Table 1). Similar results were previously reported by Keskinen et al. (2009), which showed that no significant reduction in microbial population using sanitizer solutions with pH between 2.6 and 8. Cut edges of inoculated lettuce have been shown to retain many viable bacteria following treatment (Hassan and Frank, 2003; Keskinen et al., 2009; Takeuchi and Frank, 2001). Lettuce treated in this study was dip-inoculated, which makes it likely that E. coli O157: H7 was present along the cut edges of the product. The significant reduction obtained by SCFA seems to indicate that cells within the cut edges of the product were inactivated, as well as the bacteria present on the intact tissue (Table 1). The sanitizer SCFA seems to be a promising treatment for the removal of bacteria from fresh produce, and is significantly more effective than chlorinated water, the current industry standard. This sanitizer was somewhat phytotoxic, and at high levels or long treatment times it may damage lettuce leaves. Therefore, further study is required in order to optimize this treatment in order to preserve the shelf-life of treated lettuce. Given the high degree of efficacy of this sanitizer, it would be interesting to investigate whether it is as effective when used to sanitize other types of produce. Acknowledgments This work was funded by USDA-CSREES grant number 200651110-03681. The authors thank Ms Gouping Bao for the scanning
electron microscopy, Mr. Joseph E. Sites for his work on the micrographs, Ms. Angela Burke for assistance with analysis of treatment solutions, and Dr. Joshua Gurtler for his assistance with the statistical analysis, and Dr. Dike Ukuku and Mr. Joseph Sites for their helpful comments on the manuscript. The authors also thank Mr. Joel Tenney of ICA Tri-Nova, LLC, and Dr. Robert Coleman of Summerdale Inc. for providing their products for evaluation.
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