International Journal of Food Microbiology 191 (2014) 129–134
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International Journal of Food Microbiology journal homepage: www.elsevier.com/locate/ijfoodmicro
Inactivation of Escherichia coli O157:H7 in biofilm on food-contact surfaces by sequential treatments of aqueous chlorine dioxide and drying Jihyun Bang a, Ayoung Hong a, Hoikyung Kim b, Larry R. Beuchat c, Min Suk Rhee a, Younghoon Kim d, Jee-Hoon Ryu a,⁎ a
Department of Biotechnology, Korea University, Anam-dong, Sungbuk-ku, Seoul 136-701, Republic of Korea Division of Human Environmental Sciences, Wonkwang University, Shinyong-dong, Iksan, Jeonbuk 570-749, Republic of Korea Center for Food Safety and Department of Food Science and Technology, University of Georgia, 1109 Experiment Street, Griffin, GA 30223-2797, USA d BK 21 Plus Graduate Program, Department of Animal Science and Institute Agricultural Science and Technology, Chonbuk National University, Jeonju 561-756, Republic of Korea b c
a r t i c l e
i n f o
Article history: Received 14 July 2014 Received in revised form 11 September 2014 Accepted 14 September 2014 Available online 19 September 2014 Keywords: Food-contact surface Escherichia coli O157:H7 Biofilm Chlorine dioxide Drying
a b s t r a c t We investigated the efficacy of sequential treatments of aqueous chlorine and chlorine dioxide and drying in killing Escherichia coli O157:H7 in biofilms formed on stainless steel, glass, plastic, and wooden surfaces. Cells attached to and formed a biofilm on wooden surfaces at significantly (P ≤ 0.05) higher levels compared with other surface types. The lethal activities of sodium hypochlorite (NaOCl) and aqueous chlorine dioxide (ClO2) against E. coli O157:H7 in a biofilm on various food-contact surfaces were compared. Chlorine dioxide generally showed greater lethal activity than NaOCl against E. coli O157:H7 in a biofilm on the same type of surface. The resistance of E. coli O157:H7 to both sanitizers increased in the order of wood N plastic N glass N stainless steel. The synergistic lethal effects of sequential ClO2 and drying treatments on E. coli O157:H7 in a biofilm on wooden surfaces were evaluated. When wooden surfaces harboring E. coli O157:H7 biofilm were treated with ClO2 (200 μg/ml, 10 min), rinsed with water, and subsequently dried at 43% relative humidity and 22 °C, the number of E. coli O157:H7 on the surface decreased by an additional 6.4 CFU/coupon within 6 h of drying. However, when the wooden surface was treated with water or NaOCl and dried under the same conditions, the pathogen decreased by only 0.4 or 1.0 log CFU/coupon, respectively, after 12 h of drying. This indicates that ClO2 treatment of food-contact surfaces results in residual lethality to E. coli O157:H7 during the drying process. These observations will be useful when selecting an appropriate type of food-contact surfaces, determining a proper sanitizer for decontamination, and designing an effective sanitization program to eliminate E. coli O157:H7 on foodcontact surfaces in food processing, distribution, and preparation environments. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Escherichia coli O157:H7 was first recognized as an enteric pathogen in 1982 (Riley et al., 1983). The pathogen can cause serious diseases such as hemorrhagic colitis, hemolytic uremic syndrome, and thrombotic thrombocytopenic purpura in humans (Doyle, 1991; Tschäpe and Fruth, 2001). Outbreaks of E. coli O157:H7 infections are often associated with consumption of contaminated undercooked ground beef, but foods such as fresh produce have also been associated with these infections (Rodríguez et al., 2011). Since the infectious dose of E. coli O157:H7 is low (b 100 cells), cross-contamination of foods by foodcontact surfaces harboring low numbers of the pathogen can potentially lead to outbreaks (Sheen and Hwang, 2010; Wilks et al., 2005). A biofilm can be defined as a bacterial community that has attached to and grown on abiotic or biological surfaces (Costerton, 1995). E. coli O157:H7 is known to form biofilms on various types of food-contact ⁎ Corresponding author. Tel.: +82 2 3290 3409; fax: +82 2 3290 3918. E-mail address: [email protected] (J.-H. Ryu).
http://dx.doi.org/10.1016/j.ijfoodmicro.2014.09.014 0168-1605/© 2014 Elsevier B.V. All rights reserved.
surfaces, e.g., stainless steel (Dewanti and Wong, 1995; Ryu et al., 2004a), glass (Oh et al., 2007), and plastic (Dourou et al., 2011). Microbial cells in biofilms are frequently embedded in extracellular polymeric substances (EPSs), resulting in enhanced resistance to environmental stresses such as exposure to oxidizing sanitizers (Ryu and Beuchat, 2005; Vogeleer et al., 2014). EPSs are known to play an important role in various steps leading to biofilm formation including the production of a conditioning film (Allison and Sutherland, 1987), the attachment of cells (Ofek and Doyle, 1994), and the formation of threedimensional structures (Danese et al., 2000). Foodborne pathogens that have formed biofilm on food-contact surfaces increase the potential for food cross-contamination (Kim and Wei, 2012; Giaouris et al., 2014). Effective procedures for elimination of foodborne pathogens on foodcontact surfaces are therefore critical for controlling outbreaks of foodborne illnesses. Sanitizers such as sodium hypochlorite (NaOCl), chlorine dioxide (ClO2), ozone, peracetic acid, and quaternary ammonium, are used in the food industry to inactivate pathogenic microorganisms in biofilms on food-contact surfaces. Chlorine dioxide has several advantages over
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liquid chlorine sanitizers such as NaOCl. For example, ClO2 has 2.5-fold greater oxidizing power than liquid chlorine (Marriott and Gravani, 2006) and its lethal activity is stable over a wider pH range (3.0–8.0) compared to NaOCl, whose lethality is decreased at neutral pH (Huang et al., 1997). We have observed that foodborne pathogens exposed to aqueous ClO2 become more sensitive to drying (Kim et al., 2010) and dry heat (Bang et al., 2011a–c). If sequential treatments with ClO2 and drying cause synergistic lethality to E. coli O157:H7 in biofilms, then this approach could be used to kill the pathogen on food-contact surfaces. The objectives of this study were to determine the relative propensity of E. coli O157:H7 to attach to and form biofilm on various foodcontact surfaces (stainless steel, glass, plastic, and wood), compare the efficacy of NaOCl and ClO2 in terms of killing E. coli O157:H7 in a biofilm on food-contact surfaces, and confirm the synergistic lethal effects of sequential treatments of ClO2 and drying against E. coli O157:H7 in a biofilm on a wooden surface.
2. Materials and methods 2.1. Bacterial strains Three strains of E. coli O157:H7 were used; ATCC 43895 (isolated from hamburger), ATCC 43894 (isolated from human feces), and E0018 (isolated from bovine feces; a laboratory stock culture). Cryopreserved E. coli O157:H7 cells maintained at −20 °C with 15% glycerol in tryptic soy broth (TSB; BBL/Difco, Sparks, MD, USA) were activated in TSB at 37 °C for 24 h. The cultures (10 μl) were transferred to TSB (10 ml) three times at 24-h intervals and incubated at 37 °C before use in experiments.
2.2. Preparation of food-contact surfaces Stainless steel (type 304, no. 4 finish), glass (Paul Marienfeld GmbH & Co. KG, Lauda-Königshofen, Germany), plastic (polyethylene; Wonju Chemical Industries Co., Busan, Korea), and wood (pine wood; obtained from local retail shop) plates were purchased and cut to an identical size (5-cm width × 2-cm length × 1–3-mm height). Stainless steel, glass, and plastic coupons were sonicated in 15% phosphoric acid (Daejung, Siheung, Republic of Korea) at 70 °C for 20 min in an ultrasonic water bath (model JAC-1505; KODO, Hwaseong, Republic of Korea) and rinsed in distilled water. After rinsing, the coupons were sonicated in a 15% alkaline detergent solution (FS Pro-Chlor, Zep, Atlanta, GA, USA) at 70 °C for 20 min and rinsed in distilled water. Wooden coupons were vigorously scrubbed with a moist sponge with powdered precision cleaner (Alconox, Inc., White Plains, NY, USA) for approximately 30 s, followed by thorough rinsing with distilled water. Clean stainless steel, glass, and wooden coupons were dried in a laminar biosafety hood and drysterilized at 121 °C for 15 min. Plastic coupons were immersed in 70% EtOH for at least 2 h and sterilized under UV light in a laminar biosafety hood at 21 ± 2 °C overnight.
2.3. Preparation of sanitizers NaOCl solution (Reagent grade, available chlorine 10–15%; SigmaAldrich, St. Louis, MO, USA) was diluted in sterile distilled water to give concentrations of 50 and 200 μg/ml. ClO2 solution was prepared by combining 150 ml of sodium chlorite solution (10,000 μg/ml) with 3.5 ml of hydrochloric acid (1 N) followed by shaking at 150 rpm on a rotary shaker for 1 h. The ClO2 solution was diluted in sterile distilled water to give concentrations of 50 or 200 μg/ml. The concentrations of NaOCl or ClO2 were measured using a chlorine colorimeter (model Dr/ 820; Hach, Loveland, CO, USA) immediately before experiments.
2.4. Preparation of a 43% relative humidity (RH) environment Saturated potassium carbonate (Daejung) solution was prepared in distilled water. The saturated solution (0.8 ml) was deposited in 50-ml conical centrifuge tubes and caps were firmly closed. The tubes were incubated at 22 °C for at least 24 h before using in experiments.
2.5. Biofilm formation of E. coli O157:H7 on various food-contact surfaces Cell suspensions (2 ml each) of three strains of E. coli O157:H7 grown in TSB at 37 °C for 24 h were combined and centrifuged at 2600 ×g for 10 min. The supernatant was decanted and the pelleted cells were resuspended in phosphate-buffered saline (PBS; pH 7.4) to give a population of ca. 5 log CFU/ml. The cell suspension (30 ml) was deposited in a Whirl-pak® bag (207 ml; Nasco, Fort Atkinson, WI, USA) containing a sterile stainless steel, glass, plastic, or wooden coupon and incubated at 4 °C for 24 h to facilitate cell attachment. Coupons were removed from the suspension with sterile forceps and washed in 500 ml of sterile distilled water by gently moving in a circular motion for 5 s. The washed coupons were separately deposited in Whirl-pak® bags containing 30 ml of M9 medium (pH 7.2; Biosesang, Inc., Bundang, Kyung-gi, Republic of Korea) and incubated at 25 °C for up to 5 days to enable biofilm formation. After 1, 2, 3, 4, and 5 days, the coupons were washed in sterile distilled water (500 ml) by gently moving in a circular motion for 5 s and transferred to 50-ml conical centrifuge tubes containing 30 ml of 0.1% peptone water (PW) and 3 g of sterile glass beads (425–600 μm diameter; Sigma-Aldrich). The mixtures were vortexed at maximum speed for 1 min. After vortexing, undiluted suspensions (0.1 ml in duplicate) and suspensions (0.1 ml in duplicate) serially diluted in 0.1% PW were surface-plated on tryptic soy agar (TSA; BBL/Difco) and incubated at 37 °C for 24 h before E. coli O157:H7 colonies were counted. The detection limit for the direct plating was 1.5 log CFU/coupon (30 CFU/coupon).
2.6. Visualization of the biofilm structures produced by E. coli O157:H7 on food-contact surfaces Biofilms of E. coli O157:H7 formed on stainless steel, glass, plastic, and wooden coupon surfaces were visualized using scanning electron microscopy (SEM). Stainless steel, glass, plastic, and wooden plates were cut to 1 cm × 1 cm (coupons). E. coli O157:H7 biofilms were formed on coupons immersed in M9 medium at 22 °C for 5 days, as described above. After biofilm formation, the coupons were rinsed in 50 ml of sterile distilled water by gently moving in a circular motion for 5 s. The rinsed coupons were transferred to 50 ml of PBS buffer (pH 7.2) containing 0.1% concanavalin A, 0.1% CaCl2 and 0.1% MgCl 2 for at least 20 min before rinsing with PBS (pH 7.4). The rinsed coupons were fixed in 1 M sodium cacodylate buffer containing 2.5% glutaraldehyde and 4% paraformaldehyde at 4 °C for at least 30 min and washed three times in 0.1 M sodium cacodylate buffer for 10 min at 21 ± 2 °C. The fixed coupons were dehydrated using a graded series of ethanol concentrations (50, 60, 70, 80, and 90, and twice with 100%) for 10 min each. Coupons with dehydrated biofilms were soaked in mixtures of ethanol and isoamyl acetate at ratios 3:1, 1:1, and 1:3 for 20 min, and finally treated twice with 100% isoamyl acetate solution for 30 min. After treatment with isoamyl acetate solution, the coupons were dried in a critical point dryer (13200-AB, SPI supplies, West Chester, PA, USA) using carbon dioxide and coated with platinum (40 nm thick) using sputter coating (Cressington 208HR, Cressington Scientific Instruments, Watford, UK). The samples were visualized using a Hitachi S-4700 scanning electron microscope (Hitachi Ltd., Tokyo, Japan).
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2.7. Lethality of NaOCl and ClO2 against E. coli O157:H7 in a biofilm formed on various food-contact surfaces Biofilms of E. coli O157:H7 were produced on stainless steel, glass, plastic, and wooden coupons in M9 medium at 22 °C for 3 days, as described above. The coupons were washed in sterile distilled water (500 ml) for 5 s and then placed in sterile centrifuge tubes (50 ml) containing 30 ml of sterile distilled water, NaOCl (50 and 200 μg/ml) solution, or ClO2 (50 and 200 μg/ml) solutions for 1, 5, 10, and 15 min. The coupons treated with sterile distilled water were transferred to 50-ml centrifuge tubes containing 30 ml of 0.1% PW and 3 g of glass beads. Coupons treated with sanitizers were transferred to centrifuge tubes (50 ml) containing 30 ml of Dey–Engley (DE) neutralizing broth (BBL/ Difco) and 3 g of glass beads to neutralize sanitizers, and vortexed for 1 min at maximum speed. Undiluted suspensions (0.25 ml in quadruplicate and 0.1 ml in duplicate) and suspensions (0.1 ml in duplicate) serially diluted in 0.1% PW were surface plated on MacConkey Sorbitol agar (MSA, BBL/Difco) and incubated at 37 °C for 24 h before counting colonies. The remaining mixtures (coupons, 0.1% PW or DE neutralizing broth, and glass beads) were combined with 100 ml of TSB and incubated at 37 °C for 24 h to enrich for E. coli O157:H7. When colonies did not form on MSA plates, the enriched suspension was streaked on MSA plates and incubated at 37 °C for 24 h. Colonies formed on MSA plates were randomly selected and tested using the E. coli O157 latex agglutination test (Oxoid, Basingstoke, UK) to confirm E. coli O157. The detection limits of E. coli O157:H7 by direct plating and enrichment were 1.5 log CFU/coupon (30 CFU/coupon) and 0.0 log CFU/coupon (1 CFU/ coupon), respectively. 2.8. Synergistic lethal effects of ClO2 and drying treatments on E. coli O157:H7 in a biofilm formed on a wooden surface Biofilms of E. coli O157:H7 were produced on wooden coupons in M9 medium at 22 °C for 3 days, as described above. Coupons were rinsed in 500 ml of distilled water for 5 s, immersed in 30 ml of sterile water, NaOCl (200 μg/ml), or ClO2 (200 μg/ml) solutions in 50-ml centrifuge tubes for 10 min, and rinsed twice in sterile distilled water (30 ml) to remove residual sanitizers. The coupons were transferred, without contact with saturated salt solution, to 50-ml centrifuge tubes in which the RH had been adjusted to 43%. The caps were firmly closed and tubes were incubated at 22 °C for 1, 3, 6, 9, and 12 h. At each sampling time, the number of viable E. coli O157:H7 on wooden coupons was determined as described above. 2.9. Statistical analysis All experiments were replicated at least three times and two coupons were examined per replicate. Data were analyzed using the general linear model of the Statistical Analysis Systems procedure (SAS 9.1; SAS Institute, Cary, NC, USA). The number of E. coli O157:H7 attached to coupons as affected by type of surface, the number in biofilm as affected by surface type and incubation period, the number in biofilm after treatment with a sanitizer as affected by type of sanitizer, type of surface, and treatment time, and the number in biofilm on wooden surfaces as affected by drying time were analyzed using Fisher's least significant difference test. Significant differences were assessed at a 95% confidence level (P ≤ 0.05). 3. Results 3.1. Biofilm formation by E. coli O157:H7 on various food-contact surfaces Fig. 1 shows the number of E. coli O157:H7 on various food-contact surfaces (stainless steel, glass, plastic, or wood) when the pathogen had attached on those surfaces and incubated in M9 medium at 22 °C for up to 5 days. We considered that a biofilm was formed when the
Fig. 1. Maturation curves of E. coli O157:H7 biofilms formed on stainless steel, glass, plastic, and wooden coupon surfaces. Cells grown in TSB at 37 °C for 24 h were collected, resuspended in PBS, and allowed to attach to the coupons at 4 °C for 24 h. The coupons with attached cells were rinsed with sterile water and immersed in M9 medium at 22 °C for up to 5 days. On the same surface type, values not noted by the same lowercase letters were significantly different (P ≤ 0.05). At the same incubation time, values not noted by the uppercase letters were significantly different (P ≤ 0.05).
population of E. coli O157:H7 on the surface increased significantly (P ≤ 0.05) during immersion in M9 medium. The numbers of attached cells of E. coli O157:H7 on stainless steel, glass, plastic, and wooden coupons at day 0 were 4.9, 4.3, 4.1, and 5.3 log CFU/coupon, respectively. When stainless steel, glass, plastic, and wooden coupons containing attached E. coli O157:H7 were immersed in M9 medium and incubated at 22 °C, the number of E. coli O157:H7 on those coupons increased significantly (P ≤ 0.05) to 8.6, 7.9, 8.6, and 9.5 log CFU/coupon, respectively, within 2 days and remained constant for an additional 3 days. Fig. 2 shows SEM images of biofilms formed by E. coli O157:H7 on stainless steel, glass, plastic, and wooden coupons in M9 medium at 22 °C for 5 days. Communities (clusters) of E. coli O157:H7 were
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Fig. 2. Scanning electron microscopy (SEM) images of a biofilm formed by E. coli O157:H7 on stainless steel, glass, plastic, and wooden coupon surfaces in M9 medium at 22 °C for 5 days.
observed on the surfaces of all coupons. Each type of surface showed different degrees of roughness in the order of wood N plastic N stainless steel N glass. The glass surface was without crevices. Stainless steel had many oblique, straight-line, narrow crevices on the surface. The plastic surface exhibited convex patterns, with a greater number of E. coli O157:H7 present at the rim of those patterns. Wood showed the roughest surface, with many pores and deep crevices in which dense E. coli O157:H7 communities had developed.
3.2. Lethality of NaOCl and ClO2 against E. coli O157:H7 in a biofilm formed on various food-contact surfaces Table 1 shows populations of E. coli O157:H7 in biofilms on stainless steel, glass, plastic, and wooden coupon surfaces after treatment with water, NaOCl (50 or 200 μg/ml), or ClO2 (50 or 200 μg/ml) for up to 15 min. Initial populations of E. coli O157:H7 in biofilms on these food-contact surfaces were 8.4–9.5 log CFU/coupon. When treated
Table 1 Populations of E. coli O157:H7 in biofilms formed on stainless steel, glass, plastic, or wooden coupons treated with water or sanitizers (50 and 200 μg/ml of NaOCl or ClO2) for 0, 1, 5, 10, and 15 min. Coupon
Sanitizer
Conc. (μg/ml)
Population ± standard deviation (log CFU/coupon)a Treatment time (min) 0
Stainless steel
Water NaOCl ClO2
Glass
Water NaOCl ClO2
Plastic
Water NaOCl ClO2
Wood
Water NaOCl ClO2
0 50 200 50 200 0 50 200 50 200 0 50 200 50 200 0 50 200 50 200
A 8.5 A 8.5 A 8.5 A 8.4 A 8.4 A 8.8 A 8.8 A 8.8 A 8.8 A 8.8 A 8.7 A 8.7 A 8.7 A 8.7 A 8.7 A 9.5 A 9.5 A 9.5 A 9.5 A 9.5
1 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.4 a 0.4 a 0.4 a 0.4 a 0.4 a 0.1 a 0.1 a 0.1 a 0.1 a 0.1 a 0.5 a 0.5 a 0.5 a 0.5 a 0.5 a 0.0 a 0.0 a 0.0 a 0.0 a 0.0 a
A 8.3 B 4.7 BC 4.3 CD 1.5 D 0.4 A 8.8 AB 7.7 B 6.9 C 5.3 D 0.9 A 8.5 AB 7.4 AB 6.8 BC 4.9 C 3.0 A 9.5 B 8.7 B 8.7 B 8.8 B 8.6
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.4 a 2.4 b 2.8 b 3.0 b 0.8 b 0.3 a 0.7 ab 0.2 b 0.1 b 1.6 b 0.5 a 0.8 b 0.7 b 3.0 b 2.6 b 0.0 a 0.2 b 0.1 b 0.4 ab 0.4 ab
5
10
15
A 8.2 ± 0.5 a B 3.4 ± 2.6 b B 2.7 ± 2.4 bc ND (0/8)b ND (0/8) A 8.7 ± 0.3 a B 7.0 ± 0.7 bc C 5.0 ± 0.3 c D 1.5 ± 0.0 c ND (0/6) A 8.6 ± 0.4 a B 6.9 ± 0.7 b B 6.0 ± 1.0 bc C 2.5 ± 1.7 bc ND (0/6) A 9.6 ± 0.1 a B 8.5 ± 0.2 b B 8.4 ± 0.1 bc B 8.5 ± 0.2 b B 8.1 ± 0.5 b
A 8.3 ± 0.5 a B 3.8 ± 2.2 b C 2.1 ± 1.2 bc ND (0/8) ND (0/8) A 8.8 ± 0.1 a B 7.0 ± 1.1 bc C 3.9 ± 0.8 d D 0.5 ± 0.9 D 0.5 ± 0.9 A 8.4 ± 0.6 a B 6.9 ± 0.9 b C 5.4 ± 0.5 c D 1.7 ± 0.4 c ND (0/6) A 9.4 ± 0.1 ab B 8.1 ± 0.3 b B 8.3 ± 0.3 bc B 8.2 ± 0.5 b B 8.0 ± 0.7 b
A 8.3 ± 0.4 a B 3.1 ± 1.9 b C 0.6 ± 0.8 c ND (0/8) ND (0/8) A 8.7 ± 0.3 a B 6.1 ± 1.3 c C 0.5 ± 0.9 e ND (0/6) ND (0/6) A 8.4 ± 0.7 a B 6.5 ± 0.7 b C 5.1 ± 0.8 c D 0.5 ± 0.9 c ND (0/6) A 9.3 ± 0.2 b AB 8.1 ± 0.7 b AB 8.0 ± 0.5 c B 7.9 ± 1.0 b B 7.8 ± 0.8 b
a Comparison of the effect of treatment: within each type of coupon, values in the same column that are not preceded by the same uppercase letter are significantly different (P ≤ 0.05). Comparison of the effect of treatment time: values in the same row that are not followed by the same lowercase letter are significantly different (P ≤ 0.05). b None detected by direct plating. Values in parentheses represent the number of samples of a total of six to eight in three to four replicate trials that were positive for E. coli O157:H7 as determined by enrichment culture.
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with water for 15 min, populations decreased by only 0.1–0.3 log CFU/ coupon, regardless of the type of coupon. When treated with NaOCl, inactivation of E. coli O157:H7 in a biofilm varied significantly (P ≤ 0.05) according to the surface type. Reduction on stainless steel and glass coupons was greater than that on plastic and wooden coupons. For example, populations of E. coli O157:H7 in a biofilm on stainless steel and glass coupons decreased significantly (P ≤ 0.05) to below the detection limit by direct plating (b 1.5 log CFU/ coupon) when treated with 200 μg/ml NaOCl for 15 min. However, the number of E. coli O157:H7 on plastic and wooden coupons remained at 5.1 and 8.0 log CFU/coupon, respectively, after treatment with 200 μg/ml NaOCl for 15 min. ClO2 treatment also caused different rates of inactivation of E. coli O157:H7 in a biofilm on the various surfaces. However, the lethality of ClO2 was generally greater than that of NaOCl. For example, E. coli O157:H7 in a biofilm on stainless steel and glass coupons was eliminated within 5 and 15 min, respectively, after treatment with ClO2 at 50 μg/ml. On plastic coupons treated with ClO2 (200 μg/ml), E. coli O157:H7 was undetectable within 5 min. However, high numbers of E. coli O157:H7 (N 7.8 log CFU/coupon) survived on wooden coupons treated with ClO2, regardless of concentration or treatment time. 3.3. Synergistic lethal effects of ClO2 and drying treatments on E. coli O157:H7 in a biofilm formed on wooden coupon surfaces Table 2 shows populations of E. coli O157:H7 on wooden coupons treated with water, NaOCl (200 μg/ml), or ClO2 (200 μg/ml) for 10 min, followed by drying at 43% RH and 22 °C for up to 12 h. The initial population of E. coli O157:H7 in a biofilm on wooden coupons was 9.5 log CFU/coupon. When the wooden coupons were treated with water, NaOCl, or ClO2 for 10 min and thoroughly rinsed twice in sterile water, the resulting E. coli O157:H7 populations on the coupons were 9.5, 8.7, and 7.6 log CFU/coupon, respectively. When coupons treated with water or NaOCl were dried at 43% RH and 22 °C for 12 h, E. coli O157:H7 was further reduced by 0.4 or 1.0 log CFU/coupon, respectively. However, when the wooden coupons treated with ClO2 were dried for 6 h, the pathogen was reduced by 6.4 log CFU/coupon. 4. Discussion The main objective of this study was to evaluate decontamination procedures effective against E. coli O157:H7 in a biofilm on foodcontact surfaces. Many studies have explored the attachment of and biofilm formation by E. coli O157:H7 a single type of food-contact surface, but differences in the degree of attachment and biofilm formation by the pathogen as affected by various types of food-contact surfaces have not been extensively studied. Moreover, the effectiveness of sanitizers against E. coli O157:H7 in biofilms on various types of surfaces has been given only modest research attention. In addition, highly effective hurdle technologies for removing E. coli O157:H7 in biofilms on food-contact surfaces have not been developed.
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We first determined the extent to which E. coli O157:H7 forms biofilms on various types of food-contact surfaces (stainless steel, glass, plastic, and wood). Attachment and biofilm formation by E. coli O157:H7 as affected by surface type was also investigated. E. coli O157:H7 formed a biofilm on all surfaces tested. Wooden surfaces retained significantly (P ≤ 0.05) higher numbers of E. coli O157:H7 than did glass or plastic surfaces; the number attached to stainless steel was not significantly different. Cells attached to wooden surfaces persisted at higher levels than cells on the other test surfaces during biofilm formation. The degree of biofilm formation by other foodborne pathogens on various surface types has been examined by others. Adetunji and Isola (2011) compared biofilm formation by Listeria monocytogenes on glass, steel, and wooden surfaces immersed in nutrient broth. Biofilm formation was strongest on wooden surfaces, followed by steel and glass. It was theorized that L. monocytogenes might attach to wooden surfaces more readily because of its higher hydrophobicity. Similarly, in our study, the higher hydrophobicity of the wooden surfaces might have enhanced the initial attachment of E. coli O157:H7 compared with attachment to stainless steel, glass, and plastic surfaces. Also, the roughness of the wooden surface likely played an important role in bacterial attachment and biofilm formation. Cells became entrapped in the crevices and pores on wooden surfaces, and would not be easily detached. In addition, the increased surface area due to the many crevices and pores likely resulted in attachment of higher numbers of E. coli O157:H7. Others have reported similar observations. Coquet et al. (2002) confirmed that a wooden surface has a higher roughness than a PVC surface using differential interferometry. They reported that Yersinia ruckeri attached more readily to a wooden than a PVC surface. Contrary to our observations, several researchers have reported that E. coli O157:H7 is not able to form a biofilm on abiotic surfaces under specific circumstances. Biofilm formation by E. coli O157:H7 on abiotic surfaces seems to be highly affected by medium composition and incubation temperature. Uhlich et al. (2014) reported that the biofilm formation on polystyrene by some E. coli O157:H7 strains is enhanced in T-medium compared to YESCA broth at the same incubation temperature. They also reported that biofilm formation by E. coli O157:H7 is suppressed at 37 °C compared to 25 or 30 °C. Ryu et al. (2004a) compared the biofilm formation of E. coli O157:H7 on stainless steel coupons immersed in different types of media. They showed that the biofilm formed in M9 medium at 12 or 22 °C, but not in diluted TSB (10%) or lettuce juice broth. They concluded that nutrient availability is a major factor affecting the biofilm formation by E. coli O157:H7. In another series of studies, we investigated the lethality of NaOCl and ClO2 against E. coli O157:H7 in a biofilm formed on stainless steel, glass, plastic, and wooden surfaces. Generally, ClO2 exhibited greater efficacy than NaOCl in terms of killing E. coli O157:H7 in a biofilm on the same type of surface, with an exception of wooden surfaces. It was observed that the efficacy of each sanitizer was significantly affected by surface type. Wooden surfaces were the most difficult to sanitize, followed by plastic, glass, and stainless steel surfaces. Higher resistance
Table 2 Populations of E. coli O157:H7 in a biofilm formed on wooden surfaces treated with water or sanitizer (200 μg/ml of NaOCl or ClO2) and dried at 22 °C and 43% RH for up to 12 h. Treatment
Water NaOCl ClO2
Population ± standard deviation (log CFU/coupon)a Treatment time of sanitizer
Dried at 22 °C and 43% RH for 12 h
0h
10 min
1h
3h
6h
9h
12 h
A 9.5 ± 0.1 a A 9.5 ± 0.1 a A 9.5 ± 0.1 a
A 9.5 ± 0.1 ab B 8.7 ± 0.3 b C 7.6 ± 0.1 b
A 9.4 ± 0.1 abc B 8.3 ± 0.3 bc C 5.6 ± 0.3 c
A 9.3 ± 0.1 abcd A 8.3 ± 0.4 bc B 3.3 ± 1.7 d
A 9.2 ± 0.1 bcd A 7.8 ± 0.3 c B 1.2 ± 2.0 e
A 9.2 ± 0.1 cd B 8.0 ± 0.5 bc ND (0/6)b
A 9.1 ± 0.3 d B 7.7 ± 0.8 c ND (3/6)
a Comparison of the effect of treatment: values in the same column that are not preceded by the same uppercase letter are significantly different (P ≤ 0.05). Comparison of the effect of treatment time: values in the same row that are not followed by the same lowercase letter are significantly different (P ≤ 0.05). b None detected by direct plating. Values in parentheses represent the number of samples of a total of six analyzed in three replicate trials that were positive for E. coli O157:H7 as determined by enrichment.
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of E. coli O157:H7 against NaOCl and ClO2 on wooden surfaces is attributed in part to the surface structure. Limited penetration of sanitizers into the crevices and pores on the wooden surface resulted in protection of E. coli O157:H7 against exposure to sanitizers. Others have reported that it is more difficult to eliminate microorganisms on wooden surfaces, compared to other types of surfaces, by treating with sanitizers. Friedrich et al. (2009) explored the sanitizing ability of ClO2 against spores of Alicyclobacillus spp. Treatment of stainless steel, rubber conveyer belts, and wooden surfaces containing 4.4, 3.8, and 4.0 log spores/coupon, respectively, with ClO2 (50 μg/ml) for 15 min caused reductions of 4.4, 2.9, and 0.3 log spores/coupon, respectively. These researchers demonstrated that the spores were absorbed into the wooden surface and thus were protected from the exposure to ClO2. Lastly, we evaluated the synergistic lethal effects of sequential treatment of E. coli O157:H7 in a biofilm on wooden surfaces with aqueous ClO2 and drying. E. coli O157:H7 that had been exposed to ClO2 were reduced substantially during drying. Based on these observations, aqueous ClO2, compared to NaOCl, is recommended for the decontamination of E. coli O157:H7 from the food-contact surfaces, especially when the drying procedures follow treatment. Other studies have shown that treatment of E. coli O157:H7 with aqueous ClO2 increases sensitivity to other stresses such as drying or dry heat. Kim et al. (2010) reported that treatment of radish seeds with ClO2 followed by drying reduced E. coli O157:H7 more rapidly than treatment with NaOCl followed by drying. Bang et al. (2011a–c) showed synergism between ClO2 and other stresses such as drying or dry heat in inactivating E. coli O157: H7 on radish seeds. Kreske et al. (2006) reported that planktonic Bacillus cereus spores treated with aqueous ClO2 were more sensitive to wet heat compared with spores treated with NaOCl at the same concentration. Reasons underlying the enhanced reduction of E. coli O157:H7 treated with ClO2 followed by drying have not been elucidated. One possibility is that cells treated with ClO2 are sublethally injured and become more sensitive to the additional stresses. Another possibility would be the residual, lethal effects of ClO2. If ClO2 is absorbed by wood or in the EPS matrix of the biofilm and it is not removed during rinsing with sterile water, its lethality could persist during subsequent drying treatment. Further studies are required to explore the mechanism of the synergistic lethal effects of ClO2 and drying against E. coli O157:H7. In this study, we used curli-deficient strains of E. coli O157:H7. However, the influence of curli production by E. coli O157:H7 on the biofilm formation on various food-contact surfaces and its resistance to sanitizers should be further investigated. Curli production by E. coli O157:H7 has been known to play an important role in cell adhesion and biofilm formation (Matheus-Guimarães et al., 2014; Ryu et al., 2004b) and may enhance the resistance of the pathogen significantly against oxidizing sanitizers (Ryu et al., 2004b). The synergistic effects between ClO2 and drying on curli-producing E. coli O157:H7 which have formed a biofilm on wooden surfaces should be determined. Acknowledgments This work was supported by the Korea Food and Drug Administration (No. 13162MFDS045). We thank the Institute of Control Agents for Microorganisms at Korea University for providing resources and facilities. References Adetunji, V.O., Isola, T.O., 2011. Crystal violet binding assay for assessment of biofilm formation by Listeria monocytogenes and Listeria spp on wood, steel and glass surfaces. Glob. Vet. 6, 6–10. Allison, D.G., Sutherland, I.W., 1987. The role of exopolysaccharides in adhesion of freshwater bacteria. J. Gen. Microbiol. 133, 1319–1327.
Bang, J., Kim, H., Kim, H., Beuchat, L.R., Ryu, J.-H., 2011a. Combined effects of chlorine dioxide, drying, and dry heat treatments in inactivating microorganisms on radish seeds. Food Microbiol. 28, 114–118. Bang, J., Kim, H., Kim, H., Beuchat, L.R., Ryu, J.-H., 2011b. Inactivation of Escherichia coli O157:H7 on radish seeds by sequential treatments with chlorine dioxide, drying, and dry heat without loss of seed viability. Appl. Environ. Microbiol. 77, 6680–6686. Bang, J., Kim, H., Kim, H., Beuchat, L.R., Kim, Y., Ryu, J.-H., 2011c. Reduction of Escherichia coli O157:H7 on radish seeds by sequential application of aqueous chlorine dioxide and dry-heat treatment. Lett. Appl. Microbiol. 53, 424–429. Coquet, L., Cosette, P., Junter, G.-A., Beucher, E., Saiter, J.-M., Jouenne, T., 2002. Adhesion of Yersinia ruckeri to fish farm materials: influence of cell and material surface properties. Colloids Surf. B: Biointerfaces 26, 373–378. Costerton, J.W., 1995. Overview of microbial biofilms. J. Ind. Microbiol. 15, 137–140. Danese, P.N., Pratt, L.A., Kolter, R., 2000. Exopolysaccharide production is required for development of Escherichia coli K-12 biofilm architecture. J. Bacteriol. 182, 3593–3596. Dewanti, R., Wong, A.C., 1995. Influence of culture conditions on biofilm formation by Escherichia coli O157:H7. Int. J. Food Microbiol. 26, 147–164. Dourou, D., Beauchamp, C.S., Yoon, Y., Geornaras, I., Belk, K.E., Smith, G.C., Nychas, G.J., Sofos, J.N., 2011. Attachment and biofilm formation by Escherichia coli O157:H7 at different temperatures, on various food-contact surfaces encountered in beef processing. Int. J. Food Microbiol. 149, 262–268. Doyle, M.P., 1991. Escherichia coli O157:H7 and its significance in foods. Int. J. Food Microbiol. 12, 289–301. Friedrich, L.M., Goodrich-Schneider, R., Parish, M.E., Danyluk, M.D., 2009. Mitigation of Alicyclobacillus spp. spores on food contact surfaces with aqueous chlorine dioxide and hypochlorite. Food Microbiol. 26, 936–941. Giaouris, E., Heir, E., Hébraud, M., Chorianopoulos, N., Langsrud, S., Møretrø, T., Habimana, O., Desvaux, M., Renier, S., Nychas, G.J., 2014. Attachment and biofilm formation by foodborne bacteria in meat processing environments: causes, implications, role of bacterial interactions and control by alternative novel methods. Meat Sci. 97, 298–309. Huang, J., Wang, L., Ren, N., Ma, F., Juli, 1997. Disinfection effect of chlorine dioxide on bacteria in water. Water Res. 31, 607–613. Kim, S.H., Wei, C., 2012. Biofilms. In: Gómez-López, V.M. (Ed.), Decontamination of Fresh and Minimally Processed Produce. Wiley-Blackwell, Ames, lowa, pp. 59–75. Kim, H., Kim, H., Bang, J., Beuchat, L.R., Ryu, J.-H., 2010. Synergistic effect of chlorine dioxide and drying treatments for inactivating Escherichia coli O157:H7 on radish seeds. J. Food Prot. 73, 1225–1230. Kreske, A.C., Ryu, J.-H., Beuchat, L.R., 2006. Evaluation of chlorine, chlorine dioxide, and a peroxyacetic acid-based sanitizer for effectiveness in killing Bacillus cereus and Bacillus thuringiensis spores in suspensions, on the surface of stainless steel, and on apples. J. Food Prot. 69, 1892–1903. Marriott, N.G., Gravani, R.B., 2006. Principles of Food Sanitation, Fifth ed. Springer Science Business Media, Inc., New York. Matheus-Guimarães, C., Gonçalves, E.M., Cabilio Guth, B.E., 2014. Interactions of O157 and non-O157 Shiga toxin-producing Escherichia coli (STEC) recovered from bovine hide and carcass with human cells and abiotic surfaces. Foodborne Pathog. Dis. 11, 248–255. Ofek, I., Doyle, R.J., 1994. Bacterial Adhesion to Cells and Tissues. Chapman and Hall, New York, N.Y. Oh, Y.J., Jo, W., Yang, Y., Park, S., 2007. Biofilm formation and local electrostatic force characteristics of Escherichia coli O157:H7 observed by electrostatic force microscopy. Appl. Phys. Lett. 90, 143901. Riley, L.W., Remis, R.S., Helgerson, S.D., McGee, H.B., Wells, J.G., Davis, B.R., Hebert, R.J., Olcott, E.S., Johnson, L.M., Hargrett, N.T., Blake, P.A., Cohen, M.L., 1983. Hemorrhagic colitis associated with a rare Escherichia coli serotype. N. Engl. J. Med. 308, 681–685. Rodríguez, F.P., Campos, D., Ryser, E.T., Buchholz, A.L., Posada-Izquierdo, G.D., Marks, B.P., Zurera, G., Todd, E., 2011. A mathematical risk model for Escherichia coli O157:H7 cross-contamination of lettuce during processing. Food Microbiol. 28, 694–701. Ryu, J.-H., Beuchat, L.R., 2005. Biofilm formation by Escherichia coli O157:H7 on stainless steel: effect of exopolysaccharide and curli production on its resistance to chlorine. Appl. Environ. Microbiol. 71, 247–254. Ryu, J.-H., Kim, H., Beuchat, L.R., 2004a. Attachment and biofilm formation by Escherichia coli O157:H7 on stainless steel as influenced by exopolysaccharide production, nutrient availability, and temperature. J. Food Prot. 67, 2123–2131. Ryu, J.-H., Kim, H., Frank, J.F., Beuchat, L.R., 2004b. Attachment and biofilm formation on stainless steel by Escherichia coli O157:H7 as affected by curli production. Lett. Appl. Microbiol. 39, 359–362. Sheen, S., Hwang, C.-A., 2010. Mathematical modeling the cross-contamination of Escherichia coli O157:H7 on the surface of ready-to-eat meat product while slicing. Food Microbiol. 27, 37–43. Tschäpe, H., Fruth, A., 2001. Enterohemorrhagic Escherichia coli. In: Mühldorfer, I., Schäfer, K.P. (Eds.), Emerging Bacterial Pathogens vol. 8. Karger Publishing, Basel, Switzerland, pp. 1–11. Uhlich, G.A., Chen, C.Y., Cottrell, B.J., Nguyen, L.H., 2014. Growth media and temperature effects on biofilm formation by serotype O157:H7 and non-O157:H7 Shiga toxinproducing Escherichia coli. FEMS Microbiol. Lett. 354, 133–141. Vogeleer, P., Tremblay, Y.D., Mafu, A.A., Jacques, M., Harel, J., 2014. 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Contents lists available at ScienceDirect
International Journal of Food Microbiology journal homepage: www.elsevier.com/locate/ijfoodmicro
Inactivation of Escherichia coli O157:H7 in biofilm on food-contact surfaces by sequential treatments of aqueous chlorine dioxide and drying Jihyun Bang a, Ayoung Hong a, Hoikyung Kim b, Larry R. Beuchat c, Min Suk Rhee a, Younghoon Kim d, Jee-Hoon Ryu a,⁎ a
Department of Biotechnology, Korea University, Anam-dong, Sungbuk-ku, Seoul 136-701, Republic of Korea Division of Human Environmental Sciences, Wonkwang University, Shinyong-dong, Iksan, Jeonbuk 570-749, Republic of Korea Center for Food Safety and Department of Food Science and Technology, University of Georgia, 1109 Experiment Street, Griffin, GA 30223-2797, USA d BK 21 Plus Graduate Program, Department of Animal Science and Institute Agricultural Science and Technology, Chonbuk National University, Jeonju 561-756, Republic of Korea b c
a r t i c l e
i n f o
Article history: Received 14 July 2014 Received in revised form 11 September 2014 Accepted 14 September 2014 Available online 19 September 2014 Keywords: Food-contact surface Escherichia coli O157:H7 Biofilm Chlorine dioxide Drying
a b s t r a c t We investigated the efficacy of sequential treatments of aqueous chlorine and chlorine dioxide and drying in killing Escherichia coli O157:H7 in biofilms formed on stainless steel, glass, plastic, and wooden surfaces. Cells attached to and formed a biofilm on wooden surfaces at significantly (P ≤ 0.05) higher levels compared with other surface types. The lethal activities of sodium hypochlorite (NaOCl) and aqueous chlorine dioxide (ClO2) against E. coli O157:H7 in a biofilm on various food-contact surfaces were compared. Chlorine dioxide generally showed greater lethal activity than NaOCl against E. coli O157:H7 in a biofilm on the same type of surface. The resistance of E. coli O157:H7 to both sanitizers increased in the order of wood N plastic N glass N stainless steel. The synergistic lethal effects of sequential ClO2 and drying treatments on E. coli O157:H7 in a biofilm on wooden surfaces were evaluated. When wooden surfaces harboring E. coli O157:H7 biofilm were treated with ClO2 (200 μg/ml, 10 min), rinsed with water, and subsequently dried at 43% relative humidity and 22 °C, the number of E. coli O157:H7 on the surface decreased by an additional 6.4 CFU/coupon within 6 h of drying. However, when the wooden surface was treated with water or NaOCl and dried under the same conditions, the pathogen decreased by only 0.4 or 1.0 log CFU/coupon, respectively, after 12 h of drying. This indicates that ClO2 treatment of food-contact surfaces results in residual lethality to E. coli O157:H7 during the drying process. These observations will be useful when selecting an appropriate type of food-contact surfaces, determining a proper sanitizer for decontamination, and designing an effective sanitization program to eliminate E. coli O157:H7 on foodcontact surfaces in food processing, distribution, and preparation environments. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Escherichia coli O157:H7 was first recognized as an enteric pathogen in 1982 (Riley et al., 1983). The pathogen can cause serious diseases such as hemorrhagic colitis, hemolytic uremic syndrome, and thrombotic thrombocytopenic purpura in humans (Doyle, 1991; Tschäpe and Fruth, 2001). Outbreaks of E. coli O157:H7 infections are often associated with consumption of contaminated undercooked ground beef, but foods such as fresh produce have also been associated with these infections (Rodríguez et al., 2011). Since the infectious dose of E. coli O157:H7 is low (b 100 cells), cross-contamination of foods by foodcontact surfaces harboring low numbers of the pathogen can potentially lead to outbreaks (Sheen and Hwang, 2010; Wilks et al., 2005). A biofilm can be defined as a bacterial community that has attached to and grown on abiotic or biological surfaces (Costerton, 1995). E. coli O157:H7 is known to form biofilms on various types of food-contact ⁎ Corresponding author. Tel.: +82 2 3290 3409; fax: +82 2 3290 3918. E-mail address: [email protected] (J.-H. Ryu).
http://dx.doi.org/10.1016/j.ijfoodmicro.2014.09.014 0168-1605/© 2014 Elsevier B.V. All rights reserved.
surfaces, e.g., stainless steel (Dewanti and Wong, 1995; Ryu et al., 2004a), glass (Oh et al., 2007), and plastic (Dourou et al., 2011). Microbial cells in biofilms are frequently embedded in extracellular polymeric substances (EPSs), resulting in enhanced resistance to environmental stresses such as exposure to oxidizing sanitizers (Ryu and Beuchat, 2005; Vogeleer et al., 2014). EPSs are known to play an important role in various steps leading to biofilm formation including the production of a conditioning film (Allison and Sutherland, 1987), the attachment of cells (Ofek and Doyle, 1994), and the formation of threedimensional structures (Danese et al., 2000). Foodborne pathogens that have formed biofilm on food-contact surfaces increase the potential for food cross-contamination (Kim and Wei, 2012; Giaouris et al., 2014). Effective procedures for elimination of foodborne pathogens on foodcontact surfaces are therefore critical for controlling outbreaks of foodborne illnesses. Sanitizers such as sodium hypochlorite (NaOCl), chlorine dioxide (ClO2), ozone, peracetic acid, and quaternary ammonium, are used in the food industry to inactivate pathogenic microorganisms in biofilms on food-contact surfaces. Chlorine dioxide has several advantages over
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liquid chlorine sanitizers such as NaOCl. For example, ClO2 has 2.5-fold greater oxidizing power than liquid chlorine (Marriott and Gravani, 2006) and its lethal activity is stable over a wider pH range (3.0–8.0) compared to NaOCl, whose lethality is decreased at neutral pH (Huang et al., 1997). We have observed that foodborne pathogens exposed to aqueous ClO2 become more sensitive to drying (Kim et al., 2010) and dry heat (Bang et al., 2011a–c). If sequential treatments with ClO2 and drying cause synergistic lethality to E. coli O157:H7 in biofilms, then this approach could be used to kill the pathogen on food-contact surfaces. The objectives of this study were to determine the relative propensity of E. coli O157:H7 to attach to and form biofilm on various foodcontact surfaces (stainless steel, glass, plastic, and wood), compare the efficacy of NaOCl and ClO2 in terms of killing E. coli O157:H7 in a biofilm on food-contact surfaces, and confirm the synergistic lethal effects of sequential treatments of ClO2 and drying against E. coli O157:H7 in a biofilm on a wooden surface.
2. Materials and methods 2.1. Bacterial strains Three strains of E. coli O157:H7 were used; ATCC 43895 (isolated from hamburger), ATCC 43894 (isolated from human feces), and E0018 (isolated from bovine feces; a laboratory stock culture). Cryopreserved E. coli O157:H7 cells maintained at −20 °C with 15% glycerol in tryptic soy broth (TSB; BBL/Difco, Sparks, MD, USA) were activated in TSB at 37 °C for 24 h. The cultures (10 μl) were transferred to TSB (10 ml) three times at 24-h intervals and incubated at 37 °C before use in experiments.
2.2. Preparation of food-contact surfaces Stainless steel (type 304, no. 4 finish), glass (Paul Marienfeld GmbH & Co. KG, Lauda-Königshofen, Germany), plastic (polyethylene; Wonju Chemical Industries Co., Busan, Korea), and wood (pine wood; obtained from local retail shop) plates were purchased and cut to an identical size (5-cm width × 2-cm length × 1–3-mm height). Stainless steel, glass, and plastic coupons were sonicated in 15% phosphoric acid (Daejung, Siheung, Republic of Korea) at 70 °C for 20 min in an ultrasonic water bath (model JAC-1505; KODO, Hwaseong, Republic of Korea) and rinsed in distilled water. After rinsing, the coupons were sonicated in a 15% alkaline detergent solution (FS Pro-Chlor, Zep, Atlanta, GA, USA) at 70 °C for 20 min and rinsed in distilled water. Wooden coupons were vigorously scrubbed with a moist sponge with powdered precision cleaner (Alconox, Inc., White Plains, NY, USA) for approximately 30 s, followed by thorough rinsing with distilled water. Clean stainless steel, glass, and wooden coupons were dried in a laminar biosafety hood and drysterilized at 121 °C for 15 min. Plastic coupons were immersed in 70% EtOH for at least 2 h and sterilized under UV light in a laminar biosafety hood at 21 ± 2 °C overnight.
2.3. Preparation of sanitizers NaOCl solution (Reagent grade, available chlorine 10–15%; SigmaAldrich, St. Louis, MO, USA) was diluted in sterile distilled water to give concentrations of 50 and 200 μg/ml. ClO2 solution was prepared by combining 150 ml of sodium chlorite solution (10,000 μg/ml) with 3.5 ml of hydrochloric acid (1 N) followed by shaking at 150 rpm on a rotary shaker for 1 h. The ClO2 solution was diluted in sterile distilled water to give concentrations of 50 or 200 μg/ml. The concentrations of NaOCl or ClO2 were measured using a chlorine colorimeter (model Dr/ 820; Hach, Loveland, CO, USA) immediately before experiments.
2.4. Preparation of a 43% relative humidity (RH) environment Saturated potassium carbonate (Daejung) solution was prepared in distilled water. The saturated solution (0.8 ml) was deposited in 50-ml conical centrifuge tubes and caps were firmly closed. The tubes were incubated at 22 °C for at least 24 h before using in experiments.
2.5. Biofilm formation of E. coli O157:H7 on various food-contact surfaces Cell suspensions (2 ml each) of three strains of E. coli O157:H7 grown in TSB at 37 °C for 24 h were combined and centrifuged at 2600 ×g for 10 min. The supernatant was decanted and the pelleted cells were resuspended in phosphate-buffered saline (PBS; pH 7.4) to give a population of ca. 5 log CFU/ml. The cell suspension (30 ml) was deposited in a Whirl-pak® bag (207 ml; Nasco, Fort Atkinson, WI, USA) containing a sterile stainless steel, glass, plastic, or wooden coupon and incubated at 4 °C for 24 h to facilitate cell attachment. Coupons were removed from the suspension with sterile forceps and washed in 500 ml of sterile distilled water by gently moving in a circular motion for 5 s. The washed coupons were separately deposited in Whirl-pak® bags containing 30 ml of M9 medium (pH 7.2; Biosesang, Inc., Bundang, Kyung-gi, Republic of Korea) and incubated at 25 °C for up to 5 days to enable biofilm formation. After 1, 2, 3, 4, and 5 days, the coupons were washed in sterile distilled water (500 ml) by gently moving in a circular motion for 5 s and transferred to 50-ml conical centrifuge tubes containing 30 ml of 0.1% peptone water (PW) and 3 g of sterile glass beads (425–600 μm diameter; Sigma-Aldrich). The mixtures were vortexed at maximum speed for 1 min. After vortexing, undiluted suspensions (0.1 ml in duplicate) and suspensions (0.1 ml in duplicate) serially diluted in 0.1% PW were surface-plated on tryptic soy agar (TSA; BBL/Difco) and incubated at 37 °C for 24 h before E. coli O157:H7 colonies were counted. The detection limit for the direct plating was 1.5 log CFU/coupon (30 CFU/coupon).
2.6. Visualization of the biofilm structures produced by E. coli O157:H7 on food-contact surfaces Biofilms of E. coli O157:H7 formed on stainless steel, glass, plastic, and wooden coupon surfaces were visualized using scanning electron microscopy (SEM). Stainless steel, glass, plastic, and wooden plates were cut to 1 cm × 1 cm (coupons). E. coli O157:H7 biofilms were formed on coupons immersed in M9 medium at 22 °C for 5 days, as described above. After biofilm formation, the coupons were rinsed in 50 ml of sterile distilled water by gently moving in a circular motion for 5 s. The rinsed coupons were transferred to 50 ml of PBS buffer (pH 7.2) containing 0.1% concanavalin A, 0.1% CaCl2 and 0.1% MgCl 2 for at least 20 min before rinsing with PBS (pH 7.4). The rinsed coupons were fixed in 1 M sodium cacodylate buffer containing 2.5% glutaraldehyde and 4% paraformaldehyde at 4 °C for at least 30 min and washed three times in 0.1 M sodium cacodylate buffer for 10 min at 21 ± 2 °C. The fixed coupons were dehydrated using a graded series of ethanol concentrations (50, 60, 70, 80, and 90, and twice with 100%) for 10 min each. Coupons with dehydrated biofilms were soaked in mixtures of ethanol and isoamyl acetate at ratios 3:1, 1:1, and 1:3 for 20 min, and finally treated twice with 100% isoamyl acetate solution for 30 min. After treatment with isoamyl acetate solution, the coupons were dried in a critical point dryer (13200-AB, SPI supplies, West Chester, PA, USA) using carbon dioxide and coated with platinum (40 nm thick) using sputter coating (Cressington 208HR, Cressington Scientific Instruments, Watford, UK). The samples were visualized using a Hitachi S-4700 scanning electron microscope (Hitachi Ltd., Tokyo, Japan).
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2.7. Lethality of NaOCl and ClO2 against E. coli O157:H7 in a biofilm formed on various food-contact surfaces Biofilms of E. coli O157:H7 were produced on stainless steel, glass, plastic, and wooden coupons in M9 medium at 22 °C for 3 days, as described above. The coupons were washed in sterile distilled water (500 ml) for 5 s and then placed in sterile centrifuge tubes (50 ml) containing 30 ml of sterile distilled water, NaOCl (50 and 200 μg/ml) solution, or ClO2 (50 and 200 μg/ml) solutions for 1, 5, 10, and 15 min. The coupons treated with sterile distilled water were transferred to 50-ml centrifuge tubes containing 30 ml of 0.1% PW and 3 g of glass beads. Coupons treated with sanitizers were transferred to centrifuge tubes (50 ml) containing 30 ml of Dey–Engley (DE) neutralizing broth (BBL/ Difco) and 3 g of glass beads to neutralize sanitizers, and vortexed for 1 min at maximum speed. Undiluted suspensions (0.25 ml in quadruplicate and 0.1 ml in duplicate) and suspensions (0.1 ml in duplicate) serially diluted in 0.1% PW were surface plated on MacConkey Sorbitol agar (MSA, BBL/Difco) and incubated at 37 °C for 24 h before counting colonies. The remaining mixtures (coupons, 0.1% PW or DE neutralizing broth, and glass beads) were combined with 100 ml of TSB and incubated at 37 °C for 24 h to enrich for E. coli O157:H7. When colonies did not form on MSA plates, the enriched suspension was streaked on MSA plates and incubated at 37 °C for 24 h. Colonies formed on MSA plates were randomly selected and tested using the E. coli O157 latex agglutination test (Oxoid, Basingstoke, UK) to confirm E. coli O157. The detection limits of E. coli O157:H7 by direct plating and enrichment were 1.5 log CFU/coupon (30 CFU/coupon) and 0.0 log CFU/coupon (1 CFU/ coupon), respectively. 2.8. Synergistic lethal effects of ClO2 and drying treatments on E. coli O157:H7 in a biofilm formed on a wooden surface Biofilms of E. coli O157:H7 were produced on wooden coupons in M9 medium at 22 °C for 3 days, as described above. Coupons were rinsed in 500 ml of distilled water for 5 s, immersed in 30 ml of sterile water, NaOCl (200 μg/ml), or ClO2 (200 μg/ml) solutions in 50-ml centrifuge tubes for 10 min, and rinsed twice in sterile distilled water (30 ml) to remove residual sanitizers. The coupons were transferred, without contact with saturated salt solution, to 50-ml centrifuge tubes in which the RH had been adjusted to 43%. The caps were firmly closed and tubes were incubated at 22 °C for 1, 3, 6, 9, and 12 h. At each sampling time, the number of viable E. coli O157:H7 on wooden coupons was determined as described above. 2.9. Statistical analysis All experiments were replicated at least three times and two coupons were examined per replicate. Data were analyzed using the general linear model of the Statistical Analysis Systems procedure (SAS 9.1; SAS Institute, Cary, NC, USA). The number of E. coli O157:H7 attached to coupons as affected by type of surface, the number in biofilm as affected by surface type and incubation period, the number in biofilm after treatment with a sanitizer as affected by type of sanitizer, type of surface, and treatment time, and the number in biofilm on wooden surfaces as affected by drying time were analyzed using Fisher's least significant difference test. Significant differences were assessed at a 95% confidence level (P ≤ 0.05). 3. Results 3.1. Biofilm formation by E. coli O157:H7 on various food-contact surfaces Fig. 1 shows the number of E. coli O157:H7 on various food-contact surfaces (stainless steel, glass, plastic, or wood) when the pathogen had attached on those surfaces and incubated in M9 medium at 22 °C for up to 5 days. We considered that a biofilm was formed when the
Fig. 1. Maturation curves of E. coli O157:H7 biofilms formed on stainless steel, glass, plastic, and wooden coupon surfaces. Cells grown in TSB at 37 °C for 24 h were collected, resuspended in PBS, and allowed to attach to the coupons at 4 °C for 24 h. The coupons with attached cells were rinsed with sterile water and immersed in M9 medium at 22 °C for up to 5 days. On the same surface type, values not noted by the same lowercase letters were significantly different (P ≤ 0.05). At the same incubation time, values not noted by the uppercase letters were significantly different (P ≤ 0.05).
population of E. coli O157:H7 on the surface increased significantly (P ≤ 0.05) during immersion in M9 medium. The numbers of attached cells of E. coli O157:H7 on stainless steel, glass, plastic, and wooden coupons at day 0 were 4.9, 4.3, 4.1, and 5.3 log CFU/coupon, respectively. When stainless steel, glass, plastic, and wooden coupons containing attached E. coli O157:H7 were immersed in M9 medium and incubated at 22 °C, the number of E. coli O157:H7 on those coupons increased significantly (P ≤ 0.05) to 8.6, 7.9, 8.6, and 9.5 log CFU/coupon, respectively, within 2 days and remained constant for an additional 3 days. Fig. 2 shows SEM images of biofilms formed by E. coli O157:H7 on stainless steel, glass, plastic, and wooden coupons in M9 medium at 22 °C for 5 days. Communities (clusters) of E. coli O157:H7 were
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Fig. 2. Scanning electron microscopy (SEM) images of a biofilm formed by E. coli O157:H7 on stainless steel, glass, plastic, and wooden coupon surfaces in M9 medium at 22 °C for 5 days.
observed on the surfaces of all coupons. Each type of surface showed different degrees of roughness in the order of wood N plastic N stainless steel N glass. The glass surface was without crevices. Stainless steel had many oblique, straight-line, narrow crevices on the surface. The plastic surface exhibited convex patterns, with a greater number of E. coli O157:H7 present at the rim of those patterns. Wood showed the roughest surface, with many pores and deep crevices in which dense E. coli O157:H7 communities had developed.
3.2. Lethality of NaOCl and ClO2 against E. coli O157:H7 in a biofilm formed on various food-contact surfaces Table 1 shows populations of E. coli O157:H7 in biofilms on stainless steel, glass, plastic, and wooden coupon surfaces after treatment with water, NaOCl (50 or 200 μg/ml), or ClO2 (50 or 200 μg/ml) for up to 15 min. Initial populations of E. coli O157:H7 in biofilms on these food-contact surfaces were 8.4–9.5 log CFU/coupon. When treated
Table 1 Populations of E. coli O157:H7 in biofilms formed on stainless steel, glass, plastic, or wooden coupons treated with water or sanitizers (50 and 200 μg/ml of NaOCl or ClO2) for 0, 1, 5, 10, and 15 min. Coupon
Sanitizer
Conc. (μg/ml)
Population ± standard deviation (log CFU/coupon)a Treatment time (min) 0
Stainless steel
Water NaOCl ClO2
Glass
Water NaOCl ClO2
Plastic
Water NaOCl ClO2
Wood
Water NaOCl ClO2
0 50 200 50 200 0 50 200 50 200 0 50 200 50 200 0 50 200 50 200
A 8.5 A 8.5 A 8.5 A 8.4 A 8.4 A 8.8 A 8.8 A 8.8 A 8.8 A 8.8 A 8.7 A 8.7 A 8.7 A 8.7 A 8.7 A 9.5 A 9.5 A 9.5 A 9.5 A 9.5
1 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.4 a 0.4 a 0.4 a 0.4 a 0.4 a 0.1 a 0.1 a 0.1 a 0.1 a 0.1 a 0.5 a 0.5 a 0.5 a 0.5 a 0.5 a 0.0 a 0.0 a 0.0 a 0.0 a 0.0 a
A 8.3 B 4.7 BC 4.3 CD 1.5 D 0.4 A 8.8 AB 7.7 B 6.9 C 5.3 D 0.9 A 8.5 AB 7.4 AB 6.8 BC 4.9 C 3.0 A 9.5 B 8.7 B 8.7 B 8.8 B 8.6
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.4 a 2.4 b 2.8 b 3.0 b 0.8 b 0.3 a 0.7 ab 0.2 b 0.1 b 1.6 b 0.5 a 0.8 b 0.7 b 3.0 b 2.6 b 0.0 a 0.2 b 0.1 b 0.4 ab 0.4 ab
5
10
15
A 8.2 ± 0.5 a B 3.4 ± 2.6 b B 2.7 ± 2.4 bc ND (0/8)b ND (0/8) A 8.7 ± 0.3 a B 7.0 ± 0.7 bc C 5.0 ± 0.3 c D 1.5 ± 0.0 c ND (0/6) A 8.6 ± 0.4 a B 6.9 ± 0.7 b B 6.0 ± 1.0 bc C 2.5 ± 1.7 bc ND (0/6) A 9.6 ± 0.1 a B 8.5 ± 0.2 b B 8.4 ± 0.1 bc B 8.5 ± 0.2 b B 8.1 ± 0.5 b
A 8.3 ± 0.5 a B 3.8 ± 2.2 b C 2.1 ± 1.2 bc ND (0/8) ND (0/8) A 8.8 ± 0.1 a B 7.0 ± 1.1 bc C 3.9 ± 0.8 d D 0.5 ± 0.9 D 0.5 ± 0.9 A 8.4 ± 0.6 a B 6.9 ± 0.9 b C 5.4 ± 0.5 c D 1.7 ± 0.4 c ND (0/6) A 9.4 ± 0.1 ab B 8.1 ± 0.3 b B 8.3 ± 0.3 bc B 8.2 ± 0.5 b B 8.0 ± 0.7 b
A 8.3 ± 0.4 a B 3.1 ± 1.9 b C 0.6 ± 0.8 c ND (0/8) ND (0/8) A 8.7 ± 0.3 a B 6.1 ± 1.3 c C 0.5 ± 0.9 e ND (0/6) ND (0/6) A 8.4 ± 0.7 a B 6.5 ± 0.7 b C 5.1 ± 0.8 c D 0.5 ± 0.9 c ND (0/6) A 9.3 ± 0.2 b AB 8.1 ± 0.7 b AB 8.0 ± 0.5 c B 7.9 ± 1.0 b B 7.8 ± 0.8 b
a Comparison of the effect of treatment: within each type of coupon, values in the same column that are not preceded by the same uppercase letter are significantly different (P ≤ 0.05). Comparison of the effect of treatment time: values in the same row that are not followed by the same lowercase letter are significantly different (P ≤ 0.05). b None detected by direct plating. Values in parentheses represent the number of samples of a total of six to eight in three to four replicate trials that were positive for E. coli O157:H7 as determined by enrichment culture.
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with water for 15 min, populations decreased by only 0.1–0.3 log CFU/ coupon, regardless of the type of coupon. When treated with NaOCl, inactivation of E. coli O157:H7 in a biofilm varied significantly (P ≤ 0.05) according to the surface type. Reduction on stainless steel and glass coupons was greater than that on plastic and wooden coupons. For example, populations of E. coli O157:H7 in a biofilm on stainless steel and glass coupons decreased significantly (P ≤ 0.05) to below the detection limit by direct plating (b 1.5 log CFU/ coupon) when treated with 200 μg/ml NaOCl for 15 min. However, the number of E. coli O157:H7 on plastic and wooden coupons remained at 5.1 and 8.0 log CFU/coupon, respectively, after treatment with 200 μg/ml NaOCl for 15 min. ClO2 treatment also caused different rates of inactivation of E. coli O157:H7 in a biofilm on the various surfaces. However, the lethality of ClO2 was generally greater than that of NaOCl. For example, E. coli O157:H7 in a biofilm on stainless steel and glass coupons was eliminated within 5 and 15 min, respectively, after treatment with ClO2 at 50 μg/ml. On plastic coupons treated with ClO2 (200 μg/ml), E. coli O157:H7 was undetectable within 5 min. However, high numbers of E. coli O157:H7 (N 7.8 log CFU/coupon) survived on wooden coupons treated with ClO2, regardless of concentration or treatment time. 3.3. Synergistic lethal effects of ClO2 and drying treatments on E. coli O157:H7 in a biofilm formed on wooden coupon surfaces Table 2 shows populations of E. coli O157:H7 on wooden coupons treated with water, NaOCl (200 μg/ml), or ClO2 (200 μg/ml) for 10 min, followed by drying at 43% RH and 22 °C for up to 12 h. The initial population of E. coli O157:H7 in a biofilm on wooden coupons was 9.5 log CFU/coupon. When the wooden coupons were treated with water, NaOCl, or ClO2 for 10 min and thoroughly rinsed twice in sterile water, the resulting E. coli O157:H7 populations on the coupons were 9.5, 8.7, and 7.6 log CFU/coupon, respectively. When coupons treated with water or NaOCl were dried at 43% RH and 22 °C for 12 h, E. coli O157:H7 was further reduced by 0.4 or 1.0 log CFU/coupon, respectively. However, when the wooden coupons treated with ClO2 were dried for 6 h, the pathogen was reduced by 6.4 log CFU/coupon. 4. Discussion The main objective of this study was to evaluate decontamination procedures effective against E. coli O157:H7 in a biofilm on foodcontact surfaces. Many studies have explored the attachment of and biofilm formation by E. coli O157:H7 a single type of food-contact surface, but differences in the degree of attachment and biofilm formation by the pathogen as affected by various types of food-contact surfaces have not been extensively studied. Moreover, the effectiveness of sanitizers against E. coli O157:H7 in biofilms on various types of surfaces has been given only modest research attention. In addition, highly effective hurdle technologies for removing E. coli O157:H7 in biofilms on food-contact surfaces have not been developed.
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We first determined the extent to which E. coli O157:H7 forms biofilms on various types of food-contact surfaces (stainless steel, glass, plastic, and wood). Attachment and biofilm formation by E. coli O157:H7 as affected by surface type was also investigated. E. coli O157:H7 formed a biofilm on all surfaces tested. Wooden surfaces retained significantly (P ≤ 0.05) higher numbers of E. coli O157:H7 than did glass or plastic surfaces; the number attached to stainless steel was not significantly different. Cells attached to wooden surfaces persisted at higher levels than cells on the other test surfaces during biofilm formation. The degree of biofilm formation by other foodborne pathogens on various surface types has been examined by others. Adetunji and Isola (2011) compared biofilm formation by Listeria monocytogenes on glass, steel, and wooden surfaces immersed in nutrient broth. Biofilm formation was strongest on wooden surfaces, followed by steel and glass. It was theorized that L. monocytogenes might attach to wooden surfaces more readily because of its higher hydrophobicity. Similarly, in our study, the higher hydrophobicity of the wooden surfaces might have enhanced the initial attachment of E. coli O157:H7 compared with attachment to stainless steel, glass, and plastic surfaces. Also, the roughness of the wooden surface likely played an important role in bacterial attachment and biofilm formation. Cells became entrapped in the crevices and pores on wooden surfaces, and would not be easily detached. In addition, the increased surface area due to the many crevices and pores likely resulted in attachment of higher numbers of E. coli O157:H7. Others have reported similar observations. Coquet et al. (2002) confirmed that a wooden surface has a higher roughness than a PVC surface using differential interferometry. They reported that Yersinia ruckeri attached more readily to a wooden than a PVC surface. Contrary to our observations, several researchers have reported that E. coli O157:H7 is not able to form a biofilm on abiotic surfaces under specific circumstances. Biofilm formation by E. coli O157:H7 on abiotic surfaces seems to be highly affected by medium composition and incubation temperature. Uhlich et al. (2014) reported that the biofilm formation on polystyrene by some E. coli O157:H7 strains is enhanced in T-medium compared to YESCA broth at the same incubation temperature. They also reported that biofilm formation by E. coli O157:H7 is suppressed at 37 °C compared to 25 or 30 °C. Ryu et al. (2004a) compared the biofilm formation of E. coli O157:H7 on stainless steel coupons immersed in different types of media. They showed that the biofilm formed in M9 medium at 12 or 22 °C, but not in diluted TSB (10%) or lettuce juice broth. They concluded that nutrient availability is a major factor affecting the biofilm formation by E. coli O157:H7. In another series of studies, we investigated the lethality of NaOCl and ClO2 against E. coli O157:H7 in a biofilm formed on stainless steel, glass, plastic, and wooden surfaces. Generally, ClO2 exhibited greater efficacy than NaOCl in terms of killing E. coli O157:H7 in a biofilm on the same type of surface, with an exception of wooden surfaces. It was observed that the efficacy of each sanitizer was significantly affected by surface type. Wooden surfaces were the most difficult to sanitize, followed by plastic, glass, and stainless steel surfaces. Higher resistance
Table 2 Populations of E. coli O157:H7 in a biofilm formed on wooden surfaces treated with water or sanitizer (200 μg/ml of NaOCl or ClO2) and dried at 22 °C and 43% RH for up to 12 h. Treatment
Water NaOCl ClO2
Population ± standard deviation (log CFU/coupon)a Treatment time of sanitizer
Dried at 22 °C and 43% RH for 12 h
0h
10 min
1h
3h
6h
9h
12 h
A 9.5 ± 0.1 a A 9.5 ± 0.1 a A 9.5 ± 0.1 a
A 9.5 ± 0.1 ab B 8.7 ± 0.3 b C 7.6 ± 0.1 b
A 9.4 ± 0.1 abc B 8.3 ± 0.3 bc C 5.6 ± 0.3 c
A 9.3 ± 0.1 abcd A 8.3 ± 0.4 bc B 3.3 ± 1.7 d
A 9.2 ± 0.1 bcd A 7.8 ± 0.3 c B 1.2 ± 2.0 e
A 9.2 ± 0.1 cd B 8.0 ± 0.5 bc ND (0/6)b
A 9.1 ± 0.3 d B 7.7 ± 0.8 c ND (3/6)
a Comparison of the effect of treatment: values in the same column that are not preceded by the same uppercase letter are significantly different (P ≤ 0.05). Comparison of the effect of treatment time: values in the same row that are not followed by the same lowercase letter are significantly different (P ≤ 0.05). b None detected by direct plating. Values in parentheses represent the number of samples of a total of six analyzed in three replicate trials that were positive for E. coli O157:H7 as determined by enrichment.
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of E. coli O157:H7 against NaOCl and ClO2 on wooden surfaces is attributed in part to the surface structure. Limited penetration of sanitizers into the crevices and pores on the wooden surface resulted in protection of E. coli O157:H7 against exposure to sanitizers. Others have reported that it is more difficult to eliminate microorganisms on wooden surfaces, compared to other types of surfaces, by treating with sanitizers. Friedrich et al. (2009) explored the sanitizing ability of ClO2 against spores of Alicyclobacillus spp. Treatment of stainless steel, rubber conveyer belts, and wooden surfaces containing 4.4, 3.8, and 4.0 log spores/coupon, respectively, with ClO2 (50 μg/ml) for 15 min caused reductions of 4.4, 2.9, and 0.3 log spores/coupon, respectively. These researchers demonstrated that the spores were absorbed into the wooden surface and thus were protected from the exposure to ClO2. Lastly, we evaluated the synergistic lethal effects of sequential treatment of E. coli O157:H7 in a biofilm on wooden surfaces with aqueous ClO2 and drying. E. coli O157:H7 that had been exposed to ClO2 were reduced substantially during drying. Based on these observations, aqueous ClO2, compared to NaOCl, is recommended for the decontamination of E. coli O157:H7 from the food-contact surfaces, especially when the drying procedures follow treatment. Other studies have shown that treatment of E. coli O157:H7 with aqueous ClO2 increases sensitivity to other stresses such as drying or dry heat. Kim et al. (2010) reported that treatment of radish seeds with ClO2 followed by drying reduced E. coli O157:H7 more rapidly than treatment with NaOCl followed by drying. Bang et al. (2011a–c) showed synergism between ClO2 and other stresses such as drying or dry heat in inactivating E. coli O157: H7 on radish seeds. Kreske et al. (2006) reported that planktonic Bacillus cereus spores treated with aqueous ClO2 were more sensitive to wet heat compared with spores treated with NaOCl at the same concentration. Reasons underlying the enhanced reduction of E. coli O157:H7 treated with ClO2 followed by drying have not been elucidated. One possibility is that cells treated with ClO2 are sublethally injured and become more sensitive to the additional stresses. Another possibility would be the residual, lethal effects of ClO2. If ClO2 is absorbed by wood or in the EPS matrix of the biofilm and it is not removed during rinsing with sterile water, its lethality could persist during subsequent drying treatment. Further studies are required to explore the mechanism of the synergistic lethal effects of ClO2 and drying against E. coli O157:H7. In this study, we used curli-deficient strains of E. coli O157:H7. However, the influence of curli production by E. coli O157:H7 on the biofilm formation on various food-contact surfaces and its resistance to sanitizers should be further investigated. Curli production by E. coli O157:H7 has been known to play an important role in cell adhesion and biofilm formation (Matheus-Guimarães et al., 2014; Ryu et al., 2004b) and may enhance the resistance of the pathogen significantly against oxidizing sanitizers (Ryu et al., 2004b). The synergistic effects between ClO2 and drying on curli-producing E. coli O157:H7 which have formed a biofilm on wooden surfaces should be determined. Acknowledgments This work was supported by the Korea Food and Drug Administration (No. 13162MFDS045). We thank the Institute of Control Agents for Microorganisms at Korea University for providing resources and facilities. References Adetunji, V.O., Isola, T.O., 2011. Crystal violet binding assay for assessment of biofilm formation by Listeria monocytogenes and Listeria spp on wood, steel and glass surfaces. Glob. Vet. 6, 6–10. Allison, D.G., Sutherland, I.W., 1987. The role of exopolysaccharides in adhesion of freshwater bacteria. J. Gen. Microbiol. 133, 1319–1327.
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