ABSTRACT EILEEN B. SOMERS AND AMY C. LEE WONG*

Transcription

1 2218 Journal of Food Protection, Vol. 67, No. 10, 2004, Pages Copyright, International Association for Food Protection Efficacy of Two Cleaning and Sanitizing Combinations on Listeria monocytogenes Biofilms Formed at Low Temperature on a Variety of Materials in the Presence of Ready-to-Eat Meat Residue EILEEN B. SOMERS A AMY C. LEE WONG* University of Wisconsin Madison, Department of Food Microbiology and Toxicology, Food Research Institute, 1925 Willow Drive, Madison, Wisconsin 53706, USA MS : Received 19 December 2003/Accepted 16 April 2004 ABSTRACT Biofilms in the food-processing industry are a serious concern due to the potential for contamination of food products, which may lead to decreased food quality and safety. The effect of two detergent and sanitizer combinations on the inactivation of Listeria monocytogenes biofilms was studied. Combination A uses a chlorinated-alkaline, low-phosphate detergent, and dual peracid sanitizer. Combination B uses a solvated-alkaline environmental sanitation product and hypochlorite sanitizer. The survival of bacterial biofilms placed at 4 and 10 C and held for up to 5 days was also addressed. To simulate conditions found in a ready-to-eat meat-processing environment, biofilms were developed in low-nutrient conditions at 10 C (with and without meat and fat residue) on a variety of materials found in a plant setting. Included were two types of stainless steel, three materials for conveyor use, two rubber products, a wall, and floor material. Biofilms developed on all surfaces tested; numbers at day 2 ranged from 3.2 log on silicone rubber to 4.47 log CFU/cm 2 on Delrin, an acetal copolymer. Biofilm survival during storage was higher at 4 C (36.3 to 1,621%) than 10 C (4.5 to 83.2%). Small amounts of meat extract, frankfurters, or pork fat reduced biofilm formation initially; with time, the biofilm cell number and survival percentage increased. Cleaning efficacy was surface dependent and decreased with residue-soiled surfaces; biofilms developed on the brick and conveyor material were most resistant. Both detergents significantly (P 0.05) removed or inactivated biofilm bacteria. The sanitizers further reduced biofilm numbers; however, the reduction was not significant in most cases for the dual peracid. Using a benchmark efficacy of 3-log reduction, combination A was only effective on 50.0% of the samples. Combination B, at 86.1%, was more effective. Bacterial biofilms are a serious concern in the foodprocessing industry due to the potential for contamination of food products, which may lead to decreased food quality and safety (19, 39). Biofilms have gained increased interest in recent years, due in part to the emergence of Listeria monocytogenes as a foodborne pathogen. This organism can be commonly found in the environment and has been isolated from various types of food processing plants (4, 6, 26, 32, 37). One of the challenges in the control of this organism is its widespread occurrence and ability to grow at low temperatures, such as those found in ready-to-eat (RTE) meat environments. Numerous outbreaks and recalls associated with this organism in RTE products such as frankfurters and luncheon meats have made effective control of biofilms a high priority. Tailoring cleaning practices and incorporating biofilm reduction methods into hazard analysis critical control point plans are some proposed ways the industry could address this problem (31). Surfaces of equipment used for food handling and processing are recognized as sources of microbial contamination and recontamination, especially when improperly cleaned and sanitized (10). Recontamination is the primary * Author for correspondence. Tel: ; Fax: ; acwong@wisc.edu. source of L. monocytogenes in many commercially prepared RTE foods (38). Biofilm bacteria are difficult to remove, and even with cleaning practices routinely used by the food industry, they may remain and survive in the plant environment. Suihko et al. (37) used ribotyping to study the prevalence of L. monocytogenes (9%) in the meat, poultry, and seafood industry. They showed that, after normal cleaning procedures and during processing, the organism could be recovered from a variety of surfaces, conveyor belts and floor drains being the most problematic. Using pulsed-field gel electrophoresis, Miettinen et al. (26) studied the prevalence (5.1%) of L. monocytogenes in an ice cream plant. A dominant strain, which persisted in the plant for 7 years, was traced to a packaging machine, a conveyor belt, and the drain system in the freezing tunnel. A multitude of surfaces, including sponge rubber seals, peelers, slicers, dicers, hose and spray nozzles, and conveyors were reported as sources of contamination of Listeria spp. in RTE food operations that processed franks and lunch meat (38). In general, biofilm bacteria are more resistant to sanitizers than their planktonic counterparts. An effective cleaning system is essential for the control of biofilm development on surfaces in RTE processing environments. Plant sanitation regimens usually include the application of a

2 J. Food Prot., Vol. 67, No. 10 CLEANING A SANITIZING COMBINATIONS OF L. MONOCYTOGENES BIOFILMS 2219 cleaning product and water rinse, followed by a sanitizer. While chemical cleaners are designed to remove food residue from the processing environment, Dunsmore (10) considered the detergent as the most important component for controlling bacterial numbers. The majority of the research designed to study the control and inactivation of L. monocytogenes biofilms has focused on sanitizers, usually in conjunction with stainless steel surfaces (11, 33, 35). The attachment surface has been shown to have an effect on the resistance of biofilm bacteria; for example, L. monocytogenes biofilms developed on rubber surfaces were much more resistant than those on stainless steel (23, 30). An area largely unexplored is the resistance of L. monocytogenes biofilm cells to cleaning and sanitizing, especially with regard to soil from RTE foods. A 3-log reduction (99.9%) has been suggested as a target for effective inactivation of attached or biofilm bacteria (1, 12, 25, 27). Mosteller and Bishop (27) studied the effect of sanitizers on biofilms of L. monocytogenes and two other organisms developed in 2% milk on stainless steel; all sanitizers tested reduced L. monocytogenes biofilm numbers by less than 3 log. Krysinski et al. (18) showed that L. monocytogenes grown on polyester-polyurethane and polyester surfaces were more resistant to cleaning and sanitizing than when grown on stainless steel. Recently, Frank et al. (13) demonstrated that L. monocytogenes biofilms on stainless steel could be inactivated ( 5 log) using a cleaner followed by a sanitizer even when overlaid with chicken meat exudate, rendered chicken fat, or both. The purpose of this study was to examine biofilm development by L. monocytogenes on a variety of food contact and non food contact surfaces in the presence of RTE meat residues at low temperature (10 C). The survival of the biofilm cells formed on these surfaces during cold storage was followed. The efficacy of two different combinations of detergent and sanitizer on the inactivation of biofilms on these surfaces was also determined. MATERIALS A METHODS Bacterial strains. A five-strain mixture of L. monocytogenes was used for all experiments. Strains chosen were Scott A (serotype 4b, human isolate), JBL 1157 (serotype 4b, processed meat), CLIP (unknown serotype, liver pâté), F6900 (serotype 1/2a, human), and F8964 (serotype 1/2b, human). All strains were from the culture collection at the Food Research Institute and were maintained in glycerol at 80 C. Strains were inoculated individually into tryptose soy broth supplemented with 0.1% yeast extract (Difco, Becton Dickinson, Sparks, Md.), grown overnight (16 to 18 h) at 37 C, and equal amounts were pooled prior to use as an inoculum. Growth media. A low-nutrient medium, adapted from that described by Denes et al. (9), was used for biofilm studies. It contained 0.1% glucose, salts, and beef extract (EPS-BE) or yeast extract (EPS-YE) at a concentration of 125 g/ml. When needed, the medium was supplemented with RTE meat residues in the form of a commercial brand of frankfurter (all beef or all turkey), or pork back fat obtained from the Muscle Biology Laboratory at the University of Wisconsin. The meat samples were subdivided, packaged in sterile Whirl-pak bags (Nasco, Fort Atkinson, Wis.), and stored at 20 C until needed. Packaged frankfurters and pork back fat samples were tested periodically to assure there was no contaminating microflora. A 5% (wt/vol) sample was added to EPS-BE, incubated at 10 C for 5 days, and followed by plating on brain heart infusion agar (BBL, Becton Dickinson, Sparks, Md.) plates. Materials tested. A variety of food contact and non food contact materials found in food-processing environments were tested. They included two types of stainless steel, 304 with no. 4 finish (Temperature Systems, Madison, Wis.) and 316L with no. 2 finish (Wisconsin Metal, Reedsburg, Wis.); an acetal copolymer used in conveyor systems, Delrin (Adapt Plastics, Loves Park, Ill.); two conveyor belt materials, Polyester 3000 (NSW Corp., Roanoke, Va.) and TURE-2 (TPU polymer with polyester fabric reinforcement and a polyurethane surface, Mol Belting Co., Grand Rapids, Mich.); and two rubber products, food-grade silicone rubber (McMaster-Carr Supply Co., Chicago, Ill.) and Buna-N (nitrile rubber, Bardon Rubber Products, Union Grove, Wis.). All materials, unless otherwise noted, were cut into 1-cm 2 chips, washed with a hot detergent (Micro, International Products Corp, Trenton, N.J.), rinsed five times in distilled water, and autoclaved. Polyester 3000, TURE-2, and Delrin were not autoclavable and were disinfected in 95% ethanol before use. A brick material used in walls (supplied by a RTE meat processor) and a painted resin used in floors (Tufco Industrial Flooring, Gentry, Ark.) were also tested. The brick and resin were cut into by 5.41-cm (17.2 cm 2 ) and 2.5-cm 2 pieces, respectively, washed, and autoclaved before use. Biofilm formation and enumeration. Flasks containing 50 ml medium and test chips were inoculated with 100 l of the pooled L. monocytogenes mixture to achieve a final inoculum level of about 6 log CFU/ml. Unless stated otherwise, biofilms were developed at 10 C for 2 or 5 days with moderate agitation (100 rpm, Orbital shaker, LabLine Instruments, Melrose Park, Ill.). The chips were removed from the medium, rinsed twice by immersion in 20 ml of 10 mm phosphate buffered saline (PBS, ph 7.2) with agitation by manually shaking the petri dish 10 times clockwise and 10 times counterclockwise, and placed in a glass test tube containing 5 ml of PBS and glass beads. Biofilm bacteria were dislodged from the chips by placing the test tube on a vortex mixer (Vortex-Genie mixer, Scientific Industries, Bohemia, N.Y.) for 30 s. Samples were serially diluted and the bacteria enumerated by plating 100 l of the appropriate dilutions onto brain heart infusion agar plates. This method has been used routinely in our laboratory (9) and by others (3, 18, 22) and has been established as an effective recovery procedure. The plates were incubated at 32 C for up to 3 days. Colonies were counted and results are recorded as log CFU per square centimeter. The thickness of the test materials varied, which affected the total surface area of the chips. This was taken into account and the numbers given in the tables were normalized to per-square-centimeter surface area. Brick surfaces were placed in a 9 in 2 Pyrex dish with 200 ml of inoculated medium and the floor resin material was placed in a 400-ml beaker with 50 ml of inoculated medium for biofilm development. Biofilm bacteria were recovered and enumerated as above, except a 50-ml conical test tube (Fisher Scientific, Pittsburgh, Pa.) containing 10 ml of PBS and glass beads replaced the test tube. Bacterial counts were expressed as log CFU per chip. Survival of biofilm bacteria. Biofilm bacteria were tested for their ability to survive in a simulated plant environment. After removal from the medium and rinsing in PBS, two chips were

3 2220 SOMERS A WONG J. Food Prot., Vol. 67, No. 10 placed in a sterile petri dish (60 by 15 mm) and incubated at 4 or 10 C (relative humidity was 78 and 65%, respectively) for either 2 or 5 days. At the end of incubation, the surviving biofilm bacteria were enumerated as above. Survival is reported as percent of the initial biofilm bacterial numbers. Cleaning and sanitizing. The ability of biofilm bacteria to survive cleaning and sanitizing was tested. Two detergent and sanitizer combinations (gift from Ecolab, Inc., St. Paul, Minn.) were used. Chemicals were diluted and applied according to the manufacturer s recommendations. Combination A consisted of a self-foaming chlorinated-alkaline, low-phosphate detergent (2%), and a water rinse followed by a dual peracid (peroxyacetic acid and peroxyoctanoic acid) sanitizer (2,600 ppm). Combination B consisted of a solvated-alkaline environmental sanitation product (5%, applied as a thin film) and a water rinse followed by a hypochlorite sanitizer (200 ppm). Chips containing biofilms were removed from the medium, rinsed as above (except sterile tap water was used in place of PBS), placed in a petri dish (100 by 15 mm), and covered with either the alkaline detergent foam or sprayed ( 3 s) with the nonchlorinated sanitation product to achieve a thin surface layer. For immersion studies, chips were placed in a petri dish with 10 ml of alkaline detergent. After incubation at 10 C for 10 min, the chips were rinsed with agitation in tap water, removed, and immersed for 1 min at room temperature in the appropriate sanitizer. Chips were removed from the sanitizer, drained to remove residual sanitizer on the surface, and immediately added to a test tube with 5 ml of PBS and glass beads. A neutralizer was not used as, after draining, less than 50 l of liquid was left on the surface of the chip. Addition to 5 ml of PBS would result in a greater than 1: 100 dilution. To determine the effect of the mechanical processing steps of cleaning and sanitizing on biofilm removal, biofilms were exposed to tap water instead of the cleaner and sanitizer combinations. Surviving bacteria were enumerated as above. Statistical analysis. Two chips were analyzed per parameter tested per experiment and each experiment was carried out at least two times. Data were analyzed by one-way analysis of variance using Minitab Statistical Software (State College, Pa.). Comparisons were made using a significance level of P RESULTS Effect of medium and temperature. For initial evaluation of biofilm formation at 4 and 10 C, which are temperatures found in RTE meat environments, a stainless steel surface (type 304) was exposed to the five-strain cocktail of L. monocytogenes in a diluted complex medium (brain heart infusion broth diluted 1:5) or a low-nutrient medium yeast extract or EPS-BE. Planktonic and biofilm growth after 2 days of incubation at 4 C was minimal (data not shown). At 10 C, which is the temperature used for all subsequent experiments, planktonic populations ranged from 6.25 log CFU/ml in 1:5 brain heart infusion broth to 6.57 log CFU/ml in EPS-BE. Growth in yeast extract produced the highest level of bacterial biofilm (4.72 log CFU/cm 2 ). Biofilm cell numbers when grown in EPS-BE and 1:5 brain heart infusion broth were lower, 4.22 and 3.68 CFU/cm 2, respectively. The presence of beef-derived proteins in these two media may have inhibited attachment of L. monocytogenes to the stainless steel surfaces. Despite slightly lower biofilm numbers, EPS-BE was the medium chosen for TABLE 1. Formation of s on a variety of test materials and survival after storage for 2 days at 4 and 10 C Test material Delrin 304 stainless steel TURE-2 Polyester 3000 Buna N 316L stainless steel Silicone Log CFU/cm 2 (SD) a 4.47 (0.18) A a 4.39 (0.28) AB 4.18 (0.18) B 4.13 (0.28) B 3.96 (0.38) BC 3.70 (0.27) C 3.20 (0.34) D 4 C 52.5 b b % survival 10 C 43.7 b 7.2 b b,c 16.2 b,c 10.0 b,c 4.5 b,c a Within the column, values followed by the same letter are not significantly different. b Significant change (P 0.05) from level. c Significant decrease in numbers (P 0.05) from storage at 4 C. this study because it is more relevant to the RTE meat processing environment we attempted to simulate in this study. Biofilm formation and survival on a variety of materials. Biofilms developed after 2 days on all the materials tested, but the number of biofilm bacteria varied (Table 1). Biofilm numbers were highest on the acetal copolymer Delrin, followed by stainless steel type 304. Food-grade silicone rubber and stainless steel type 316L surfaces were more resistant to biofilm development. Both storage temperature and test material had an effect on survival of biofilm cells. At 4 C, bacterial numbers did not decrease significantly on five of the test materials and even increased slightly on two of them (TURE-2 and stainless steel type 316L). Biofilm cells did not survive as well when stored at 10 C; there were significant decreases in cell numbers ( 50 to 95%) on all surfaces except for TURE-2. On four of the surfaces, there was a significant difference between survival at 10 C compared with 4 C. Effect of RTE product residue on stainless steel type 304. To determine the effect of RTE product residue on biofilm formation, two types of frankfurters, containing 0 (turkey) and 45% (beef) fat, and pork back fat were added at three levels (0.5,, and % wt/vol) to EPS-BE. Presence of residue decreased the amount of biofilm formed after 2 and 5 days (Table 2). Levels of biofilm formed after 2 days in the presence of as little as 0.5% beef frankfurter or back fat residue were 0.7 log to 0.8 log CFU/cm 2 lower than when no residue was added. Increasing the residue concentration resulted in a concomitant decrease in biofilm numbers. However, the biofilm bacteria continued to grow on prolonged incubation, especially in cases where meat and fat residue were present; after 5 days, the difference in biofilm cell number between samples with and without residue addition diminished. This is most notable with pork back fat, where the difference between no added residue and 5% is only 0.46 log. Overall, a higher level of survival was observed with 5-day than s. In contrast with its effect on biofilm formation, the presence of frankfurter and fat residue had a protective effect on survival of the organism.

4 J. Food Prot., Vol. 67, No. 10 CLEANING A SANITIZING COMBINATIONS OF L. MONOCYTOGENES BIOFILMS 2221 TABLE 2. Biofilm formation for 2 and 5 days on type 304 stainless steel in the presence of metal residue and survival after storage at 4 C for 5 days Beef frankfurter, log CFU/cm 2 (SD) Turkey frankfurter, log CFU/cm 2 (SD) Pork back fat, log CFU/cm 2 (SD) % Biofilm After storage % survival Biofilm After storage % survival Biofilm After storage % survival 2 days days (0.33) 3.33 (0.21) 3.32 (0.19) 2.75 (0.61) 2.71 (0.54) a 1.25 (0.40) a 1.43 (4) a 2.14 (0.30) (0.49) b 2.52 (0.14) 2.56 (0.11) 7 (0.35) a 1.67 (0.89) 1.71 (0.51) a (0.08) 3.69 (0.24) 3.59 (0.12) 3.33 (0.26) 1.95 (0.76) a 1.52 (0.88) a 2.21 (1.64) a 2.91 (1) (0.24) 3.74 (0.43) 3.12 (0.25) 3.24 (0.63) c 3.30 (0.28) 3.07 (0.45) (0.28) 3.37 (0.46) 3.09 (0.61) 0.99 (0.49) c 2.18 (1.21) c 2.75 (1.30) (0.03) 4.23 (0.02) 4.02 (0.36) 2.31 (0.23) c 2.75 (0.09) c 4.05 (0.45) a Significant change (P 0.05) from level. b, not determined. c Significant change (P 0.05) from level. Increasing the amount of residue enhanced survival. With 5% fat, essentially all of the L. monocytogenes cells remained viable after 5 days of storage at 4 C. TABLE 3. Biofilm formation on a variety of surfaces in the presence of beef frankfurter residue and survival after storage at 4 C for 5 days Test surface % frankfurter Polyester TURE-2 0 Delrin 0 Silicone 0 Silicone 0 Log CFU/cm 2 (SD) Biofilm 4.03 (0.14) A a 3.07 (0.15) B 3.95 (0.44) A 3.20 (0.11) B 3.08 (0.31) B 4.20 (0.25) A 3.67 (0.09) AB 3.40 (0.32) B 4.21 (0.30) A 3.98 (0.28) AB 3.82 (0.10) B 4.17 (0.27) A 3.35 (0.11) B 3.96 (0.26) A 3.17 (0.21) B After storage 3.49 (0.07) b 2.76 (0.34) 1.87 (0.26) b 2.67 (0.49) 2.75 (0.45) 3.33 (0.67) b 2.84 (0.93) 3.49 (0.56) 3.67 (0.22) b 3.64 (0.10) 3.87 (0.14) % survival b (0.61) b (0.29) b (0.62) 3.20 (0.21) 4.23 (0.11) A b 3.34 (0.27) c 4.05 (0.05) B b 3.62 (0.57) 4.23 (0.26) A 3.79 (0.19) c 2.88 (0.08) B b 2.56 (0.30) (0.26) A 3.83 (0.29) c (0.27) A b 3.79 (0.25) c 20.9 a Within each test surface, values followed by the same letter are not significantly different. b Significant change (P 0.05) from level. c Significant change (P 0.05) from level. Effect of frankfurter residue on biofilm formation and survival on different surfaces. In addition to stainless steel type 304, we examined the effect of beef frankfurter residue on biofilm formation and survival on stainless steel type 316L, Polyester 3000, TURE-2, Delrin, Buna N, and food-grade silicone rubber. As with stainless steel type 304, the presence of frankfurter residue decreased biofilm formation after 2 days (Table 3). For s, the numbers increased on stainless steel type 316L and silicone rubber, and were similar to those on the control surfaces without added frankfurter residue. In contrast, there was a decrease in the numbers on Buna N developed in the presence of 1% frankfurter residue compared with the level; however, these cells were much hardier to storage conditions and almost twice as many (47.9 versus 24%) remained viable after 5 days at 4 C. In the presence of frankfurter residue, survival of biofilm cells on storage was enhanced on many of the surfaces. Efficacy of cleaner application as foam or by immersion. Cleaning with the self-foaming chlorinated-alkaline detergent used in combination A can be either by immersion in the detergent solution or by application as foam (per manufacturer s directions). The efficacy of this detergent used by immersion was tested at two application temperatures, 10 and 38 C. Residual cells were observed after detergent application at 10 C, while biofilm cells were completely inactivated or removed from both stainless steel and silicone rubber surfaces at 38 C (Table 4). Five-day biofilms were more resilient to cleaning, as residual cells were present in all except one instance. However, as foam application is more commonly used with this detergent in food processing plants, we completed our testing with this method at 10 C, a temperature commonly encountered in RTE meat plants.

5 2222 SOMERS A WONG J. Food Prot., Vol. 67, No. 10 TABLE 4. Efficacy of combination A detergent on biofilms when used as an immersion at 10 and 38 C Log CFU/cm 2 (SD) after cleaning Test surface 10 C 38 C 10 C 38 C 304 stainless With 1% frankfurter 316L stainless Buna N Silicone 1.77 (0.48) A b 0.84 (0.23) B 0.74 (0.10) B 0.85 (0.16) B 0.95 (0.48) B c 0.79 (0.23) 1.60 (0.48) AB 1.45 (0.32) B 2.07 (0.27) A 1.29 (0.63) BC 0.85 (0.23) C 2.24 (0.16) A 1.36 (0.57) B 0.85 (0.23) C 0.77 (0.14) C a All values are a significant decrease (P 0.05) from initial biofilm level. b Within the test surface, values followed by the same letter are not significantly different. c, not detectable, less than 0.69 log CFU/cm 2. Cleaning and sanitizing of biofilms on food contact materials. Results of the efficacy of cleaning and sanitizing of 2- and s formed on different surfaces with and without the presence of beef or turkey frankfurter residues are shown in Tables 5 and 6 (combination A) and Tables 7 and 8 (combination B). To determine the effect of the mechanical process of cleaning and sanitizing on biofilm removal, a set of chips were processed with sterile tap water replacing the cleaner and the sanitizer. Processing with water only reduced the number of biofilm cells by negligible amounts to just over 1.5 log CFU/cm 2 ; typical results are shown in Table 6. No apparent trend with respect to surface or meat residue soil was observed for s; the average reduction was 0.53 and 0.60 log for surfaces without and with meat residue soil, respectively. A difference between surfaces without meat residue and soiled with meat residue was observed at 5 days. Average reduction of bacterial numbers on clean surfaces was 0.38 log, slightly less than for s, and 0.81 log on soiled surfaces. Overall, significant reductions in biofilm cells were observed after cleaning with either detergent. The self-foaming chlorinated-alkaline application (combination A) reduced biofilm numbers by 0.79 to 3.53 log (83.78 to 99.97%). The nonchlorinated alkaline sanitation spray application (combination B) was slightly more effective, where an overall reduction of 1.1- to 3.5-log reduction was obtained (92.05 to 99.32%). The two conveyor belt materials, TURE-2 and Polyester 3000, consistently had higher levels of residual biofilm cells after detergent cleaning compared with the other surfaces. A further reduction in bacterial numbers was observed after sanitizer treatment. The hypochlorite sanitizer (combination B) was more effective than the dual peracid (combination A). On most of the surfaces, bacteria were not detectable after application of the hypochlorite. In most cases, the reductions after the dual peracid sanitizer were not significant compared with the numbers remaining after the detergent wash. Even without the presence of meat residue, combination A treatment of s developed on TURE-2 and Buna-N were reduced by 3 log. A 3-log reduction (99.9%) has been suggested as a target for effective inactivation of attached or biofilm bacteria. The presence of meat residue on the efficacy of cleaning and sanitizing s was test-material dependent. With combination A, the presence of beef frankfurter residue (1 and 5%) reduced the effectiveness on 304 stainless steel, polyester 3000 and had some effect on Delrin and TURE-2. Turkey frankfurter residue had the most effect on cleaning and sanitizing of TURE-2; in the presence of 5% residue, only a 1-log reduction in biofilm cells was attained (Table 6). The presence of either frankfurter residue even at 5% only had a minimal effect on combination B (Tables 7 and 8). Overall, s were not more resistant to cleaning and sanitizing than the s. The effect of pork back fat residue on the cleaning efficacy of combination A on biofilms developed on stainless steel type 304 was examined (Table 9). Cleaning with the detergent caused significant reductions in biofilm numbers at all fat levels tested. Like frankfurter residue, a further but insignificant reduction after the sanitizer treatment was obtained in most cases. The presence of the highest fat level tested (5%) appeared to have a slightly negative effect on cleaning and sanitizing efficacy on s. Cleaning and sanitizing of biofilms on non food contact materials. A brick material used in walls and a painted resin used on floors were tested. However, the supply of these two materials was limited, so extensive testing was not performed. As observed previously, the presence of turkey or beef frankfurters reduced the level of biofilm formed on both the brick and floor surfaces (Table 10). The brick material is very porous, and although significant reduction in biofilm levels was obtained after both cleaning combinations, the dual peracid sanitizer was ineffective in the presence of meat residues. Only and 90.88% of the biofilm cells were inactivated or removed in the presence of 5% beef and 5% turkey frankfurter, respectively. Although not porous, the floor material had a rough surface texture. However, we were able to obtain a significant reduction, approaching the lower limit of detection, after cleaning with combination B. Survival of biofilm bacteria developed on the brick surface was determined. Biofilm numbers remained the same after 5 days at 4 C in the presence of turkey frankfurter residue; however, in the presence of beef frankfurter or

6 J. Food Prot., Vol. 67, No. 10 CLEANING A SANITIZING COMBINATIONS OF L. MONOCYTOGENES BIOFILMS 2223 TABLE 5. Efficacy of cleaning and sanitizing (combination A) on biofilms developed on a variety of surfaces with increasing amounts of beef frankfurter Log CFU/cm 2 (SD) % frankfurter Initial After cleaning a After sanitizing % total decrease 0.5 Polyester TURE-2 0 Delrin 0 Silicone 0 Silicone (0.32) A b 3.33 (0.26) B 3.20 (0.26) BC 2.75 (0.41) C 3.79 (0.07) A 2.91 (0.27) B 4.54 (0.04) A 3.30 (0.08) B 2.99 (0.16) B 4.25 (0.28) A 3.87 (0.09) B 3.40 (0.19) B 4.55 (0.08) A 4.28 (0.22) AB 3.97 (0.06) B 3.94 (0.34) A 2.98 (0.29) B 3.85 (0.29) A 2.92 (0.10) B 1.19 (0.42) 1.35 (0.61) 0.86 (0.35) 0.85 (0.23) 0.97 (0.37) 2.87 (0.07) 2.31 (0.27) 1.16 (0.54) 3.45 (0.33) 3.08 (0.31) 1.10 (0.27) 2 (0.53) 1.83 (1) 1.10 (0.27) 2.26 (0.37) 1.37 (0.39) 1.23 (0.12) 0.93 (0.32) c,d 0.73 (0.11) 0.77 (0.14) 1.56 (0.29) d 1.20 (0.58) d 0.82 (0.28) 2.35 (0.66) d 2.70 (0.53) 1.20 (0.43) 0.79 (0.18) 0.91 (0.27) 1.20 (0.43) e (0.59) d d (0.11) D f 4.05 (0.34) D 4.25 (0.37) E 4.13 (0.34) E 5.18 (0.13) D 4.45 (0.05) D 2.83 (0.09) E 3.60 (0.04) E 4.24 (0.08) D 0.88 (0.37) 4 (0.29) 1.91 (0.79) 2.10 (0.67) 2.64 (0.58) 2.88 (0.44) 0 (0.42) 2.13 (0.21) 2.22 (0.21) (0.51) d 1.38 (0.57) d 1.13 (0.53) d d 0.74 (0.17) d (0.37) d a Within the column, all values are a significant decrease (P 0.05) from initial biofilm level. b Within the test surface at 2 days, values followed by the same letter are not significantly different. c, not detectable, less than 0.69 log CFU/cm 2. d Significant decrease from biofilm level after cleaning. e Numbers with were calculated using a lower limit of detection (0.69 log). f Within the test surface at 5 days, values followed by the same letter are not significantly different. without any residue, L. monocytogenes biofilm cells grew and the numbers increased to over 1,600 and 400%, respectively (data not shown). DISCUSSION The ability of L. monocytogenes to develop biofilms and survive on nine food contact and non food contact materials was evaluated. Unique to this study was the development of L. monocytogenes biofilms at low temperature and in the presence of meat residue, conditions encountered in RTE meat-processing environments. Bacterial biofilms developed on all surfaces tested. L. monocytogenes has been shown to attach to 17 different food-use surfaces in diluted medium at 30 C after short contact time and increase in numbers for all surfaces except polypropylene (5). We have shown that small amounts of meat and fat residue in the medium reduced biofilm formation initially but, on prolonged incubation, the cell numbers increased and, on some surfaces, exceeded the number present on unsoiled chips. Milk and milk proteins have been shown to reduce bacterial attachment to surfaces (15, 29), but studies using meat and fat are limited. Hood and Zottola (17) followed the attachment to stainless steel and growth of pathogens in Trypticase soy broth (TSB), reconstituted skim, or diluted meat juice. L. monocytogenes grew to a lower level in reconstituted skim than in TSB, while no growth occurred in diluted meat juice. Stopforth et al. (36) examined the growth of acid-adapted and nonadapted L. monocytogenes on stainless steel in fresh beef carcass decontamination washing at 15 C and the subsequent sensitivity to san-

7 2224 SOMERS A WONG J. Food Prot., Vol. 67, No. 10 TABLE 6. Efficacy of cleaning and sanitizing (combination A) on biofilms developed on a variety of surfaces with increasing amounts of turkey frankfurter Log CFU/cm 2 (SD) % frankfurter Initial After cleaning a After sanitizing % total decrease After water wash Polyester TURE (0.49) A b 2.52 (0.14) B 2.56 (0.21) B 4.33 (0.18) A 2.87 (0.32) B 2.91 (0.21) B 4.59 (0.35) A 3.05 (0.21) B 2.77 (0.27) B 3.96 (0.27) A 3.28 (0.02) B 2.87 (0.22) C 4.08 (0.22) A 3.01 (0.30) B 9 (0.46) c 1.28 (0.67) 0.77 (0.21) 1.75 (0.11) 1.15 (0.52) 1.40 (0.48) 3.19 (0.11) 2.40 (0.43) 1.80 (0.72) 2.24 (0.31) 1.64 (6) 0.98 (0.42) 0.85 (0.43) 0.81 (0.16) e 1.13 (0.63) 2.36 (0.58) e 1.67 (0.31) e 1.87 (0.59) 1.93 (0.88) 1.44 (0.63) d (0.81) 2.20 (0.11) 1.90 (0.41) 4.16 (0.21) 2.54 (0.17) 2.38 (0.16) 4.42 (0.33) 2.70 (0.32) 2.63 (0.35) 3.78 (0.12) 3.22 (0.22) 2.99 (0.14) 3.69 (0.19) 2.03 (0.43) 4.18 (0.07) D f 3.52 (0.54) DE 3.25 (0.37) E 4.03 (0.06) D 3.14 (0.49) E 3.31 (0.49) E 1.32 (0.50) 3 (0.48) 1.12 (0.41) 1.21 (0.40) 1.36 (0.57) 0.97 (0.43) 0.83 (0.28) 0.85 (0.23) 1.70 (1.15) 1.80 (0.94) (0.16) 3.06 (0.80) 3.09 (0.63) 3.95 (0.30) 3.20 (0.30) 2.83 (0.26) a Within the column, all values are a significant decrease (P 0.05) from initial biofilm level. b Within the test surface at 2 days, values followed by the same letter are not significantly different. c, not detectable, less than 0.69 log CFU/cm 2. d Numbers with were calculated using a lower limit of detection (0.69 log). e Significant decrease from biofilm level after cleaning. f Within the test surface at 5 days, values followed by the same letter are not significantly different. itizers. L. monocytogenes numbers increased for 4 days but slowly decreased after that time. The decrease was contributed to competition with the resident microbial population. Adaptation was shown not to be a factor. This study also looked at the resistance of the biofilm bacteria to two cleaner and sanitizer combinations. Combination A was a chlorinated-alkaline, low-phosphate detergent followed by a dual peracid sanitizer. Combination B was a solvated-alkaline environmental sanitation product followed by a hypochlorite sanitizer. Most studies, with the exception of those by Krysinski et al. (18) and Frank et al. (13), have only looked at the effect of sanitizers and not combinations of cleaners and sanitizers on L. monocytogenes biofilms or attached cells. A 3-log reduction (99.9%) has been suggested as a target for effective inactivation of attached or biofilm bacteria (1, 12, 25, 27). Applied to this study, we found that biofilm bacteria developed in medium alone on all food contact surfaces were more readily inactivated than those developed in the presence of frankfurter and fat residue. Surprisingly, the attachment surface was not a factor with combination B; the target 3-log reduction was achieved for all samples in medium only. Overall, Combination A was less effective (70%) and the surface did come into play. Biofilm bacteria developed on the conveyor belt material TURE-2 and Buna N were harder to clean ( 3-log decrease) than the other surfaces. Other researchers have noted the effect of the attachment surface on bacterial resistance. Ronner and Wong (30) showed that L. monocytogenes biofilms developed on rubber surfaces were much more resistant to sanitizers than those on stainless steel. Bremer et al. (8) developed L. monocytogenes and Flavobacterium ssp. biofilms on stainless steel and polyvinyl chloride conveyor material and found the concentration, exposure time, and ph of chlorine had a significant effect on biofilms developed on stainless steel but not conveyor surfaces. Krysinski et al. (18) showed, in many cases, when cleaners were followed by sanitizers, complete biofilm removal and inactivation was obtained; stainless steel was the easiest to clean followed by polyester and polyester-polyurethane. In the presence of meat residue, especially at 5%, initial bacterial biofilm levels of 3 log were often not reached on food contact surfaces. For these samples, if the number of bacteria was below the detection limit after cleaning and sanitizing, the treatment was considered to be effective and met the 3-log target level. The presence of meat and fat

8 J. Food Prot., Vol. 67, No. 10 CLEANING A SANITIZING COMBINATIONS OF L. MONOCYTOGENES BIOFILMS 2225 TABLE 7. Efficacy of cleaning and sanitizing (combination B) on biofilms developed on a variety of surfaces with increasing amounts of beef frankfurter Log CFU/cm 2 (SD) % frankfurter Initial After cleaning a After sanitizing % total decrease Polyester TURE (0.07) A b 2.74 (0.09) B 2.36 (0.09) B 3.83 (0.30) A 2.95 (0.41) B 2.66 (0.21) B 4.30 (0.13) A 2.86 (0.07) B 4.15 (0.25) A 3.39 (0.45) B 4.40 (0.03) A 3.11 (0.13) B 0.83 (0.28) 0.98 (0.40) 0.77 (0.10) 0.77 (0.14) 4 (0.57) 2.85 (0.32) 1.71 (0.25) 3.05 (0.26) 1.98 (0.48) 2.18 (0.53) 3 (0.41) c e e d (0.36) e (0.16) e e 0.73 (0.11) (0.48) C f 3.13 (0.71) D 3.52 (0.77) C 3.81 (0.29) C 3.47 (0.09) D 3.68 (0.23) C 0 (0.50) 1.50 (1.20) 1.19 (0.72) 1.46 (0.42) 0.81 (0.16) 0.81 (0.22) e a Within the column, all values are a significant decrease (P 0.05) from initial biofilm level. b Within the test surface at 2 days, values followed by the same letter (A and B) are not significantly different. c, not detectable, less than 0.69 log CFU/cm 2. d Numbers with were calculated using a lower limit of detection (0.69 log). e Significant decrease from biofilm level after cleaning. f Within the test surface at 5 days, values followed by the same letter (C and D) are not significantly different. residue decreased the efficiency of both cleaning and sanitizing regimens. However, combination B was again more effective than combination A, which was relatively ineffective. Target 3-log reductions for combination B and combination A were achieved 77 and 44% of the time, respectively, for soiled surfaces. The surface seemed to have more of an effect than the level of soil. Decreased efficacy of sanitizers in the presence of soil has been noted previously (7, 11, 27). Results of our study stress the importance of a cleaning agent in removing or inactivating biofilm bacteria. Both cleaners tested significantly reduced (P 0.05) the number of biofilm bacteria. The level of cleaner efficacy was surface dependent and, to some degree, residue dependent. For instance, bacterial numbers on some of the stainless steel samples, even in the presence of turkey residue, were below the detection limit. On the other hand, the conveyor surface (TURE-2) even without soil was difficult to clean. In the study by Krysinski et al. (18), all the cleaning agents tested were shown to reduce L. monocytogenes biofilms on stainless steel to below the detection limit but were not very effective on the polyester and polyurethane surface. They noted cleaning must precede sanitizing in order to remove and inactivate microorganisms. Frank et al. (13) tested an alkaline and a neutral cleaner on L. monocytogenes biofilms on stainless steel without and with an added overlay of chicken protein. Both cleaners exceeded his targeted 5-log reduction level. The organic load reduced the efficiency of inactivation when only sanitizers were used. The sanitizer in both of our combinations further reduced the biofilm numbers, with the hypochlorite (combination B) more effective than the peracid (combination A) sanitizer. When residual bacterial numbers after cleaning were high, significant reduction (P 0.05) after sanitizer use usually occurred, especially with the hypochlorite. Several other studies have shown hypochlorite to be the more effective sanitizer. Grönholm et al. (14) treated food-spoilage microorganisms, including L. monocytogenes, dried on stainless steel chips with and without a mixture of dairyderived soil, with four disinfectants used in the dairy and brewery industry and one cleaning agent. The cleaning agent (NaOH, ionic and cationic tensides, isopropanol, and EDTA) was effective ( 3-log reduction) on L. monocytogenes without soil but not with soil. The hypochlorite sanitizer was effective against all organisms tested without soil and on L. monocytogenes and several other pathogens with soil. The other sanitizers tested (peroxides, peracetic acid, and quaternary ammonium compounds) were not effective. Frank et al. (13) showed that, when an alkali cleaner was followed by a sanitizing product, the acidified sodium chlo-

9 2226 SOMERS A WONG J. Food Prot., Vol. 67, No. 10 TABLE 8. Efficacy of cleaning and sanitizing (combination B) on biofilms developed on a variety of surfaces with increasing amounts of turkey frankfurter Log CFU/cm 2 (SD) % frankfurter Initial After cleaning a After sanitizing % total decrease 4.21 (0.22) A b 2.80 (0.19) B 2.86 (0.11) B 0 (0.28) c d (0.26) A 2.83 (0.39) B 2.41 (0.14) C 0.77 (0.14) Polyester (0.12) A 2.99 (0.30) B 2.85 (0.32) 9 (0.21) e TURE (0.25) A 3.11 (0.07) B 3.05 (0.26) 1.97 (0.43) 0.89 (0.36) e (0.21) e (0.03) A 3.04 (0.08) B 2.18 (0.54) 1.13 (0.45) e (0.37) D f 3.26 (0.42) E 2.98 (0.04) E 0.95 (0.31) 1.10 (0.25) (0.07) D 2.93 (0.08) E 2.83 (0.19) E 0.77 (0.14) 0.91 (0.39) a Within the column, all values are a significant decrease (P 0.05) from initial biofilm level. b Within the test surface at 2 days, values followed by the same letter (A through C) are not significantly different. c, not detectable, less than 0.69 log CFU/cm 2. d Numbers with were calculated using a lower limit of detection (0.69 log). e Significant decrease from biofilm level after cleaning. f Within the test surface at 5 days, values followed by the same letter (D and E) are not significantly different. rite was more effective than the dual peracid even with soil. Contradictory results for chlorine have been reported. Aarnisola et al. (1) tested nine sanitizers and one disinfecting cleaning solution on L. monocytogenes dried on stainless steel with and without a soil layer of 2% pork with 50% fat. All agents tested were effective ( 3-log reduction) on nonsoiled samples. Five of the nine sanitizers, including peracetic acid and a quaternary ammonium compound, and the disinfectant cleaning agent (hypochlorite and tensides) were effective on soiled samples; the hypochlorite-based TABLE 9. Efficacy of cleaning and sanitizing (combination A) on biofilms developed on type 304 stainless steel with added pork back fat Log CFU/cm 2 (SD) % back fat Initial After cleaning a After sanitizing % total decrease (0.08) A b 3.69 (0.24) B 3.59 (0.12) B 3.33 (0.26) B 0.89 (0.28) 0.95 (0.38) 0.90 (0.39) 1.85 (0.36) 0.77 (0.14) c 1.33 (0.75) (0.03) A 3.99 (0.28) AB 4.23 (0.02) A 4.02 (0.36) AB 0.77 (0.15) 0 (0.24) 1.12 (0.34) 1.50 (0.92) 0.85 (0.17) 4 (0.32) 1.80 (1.27) a Within the column, all values are a significant decrease (P 0.05) from initial biofilm level. b Within the column, values followed by the same letter (A and B) are not significantly different. c, not detectable, less than 0.69 log CFU/cm 2.

10 J. Food Prot., Vol. 67, No. 10 CLEANING A SANITIZING COMBINATIONS OF L. MONOCYTOGENES BIOFILMS 2227 TABLE 10. Biofilm formation on brick and floor surfaces: efficacy of cleaning and sanitizing Log CFU/chip (SD) Frankfurter added After cleaning a After sanitizing % total decrease Combination A Brick None 5% beef 5% turkey 4.71 (0.02) A b 4.24 (0.19) B 3.88 (0.09) C 2.80 (0.12) 3.44 (0.37) 2.87 (0.12) 1.90 (0.34) c 3.66 (0.78) 2.84 (0.24) Combination B Brick Floor None 5% beef None 5% beef 4.88 (0.21) A 3.97 (0.38) B 4.94 (0.06) A 3.74 (0.23) B 3.50 (0.51) 3.34 (0.18) d 1.19 (0.35) c (0.21) c (0.50) e (0.30) e a Within the column, all values are a significant decrease (P 0.05) from initial biofilm level. b Within the same test surface, values followed by the same letter (A through C) are not significantly different. c Significantly different from biofilm level after cleaning. d, not determined. e Significant change (P 0.05) from level. disinfectant was ineffective. In an in-field trial in a salmon smokehouse, Bagge-Ravn et al. (4) compared the effects of two sanitizers; a sodium hypochlorite based foam and peroxyacetic acid based fog after surfaces were treated with a alkaline-chlorinated cleaning compound. The foam sanitation reduced microflora levels ( 10 CFU recovered) in 14 to 42% of the test sites, whereas 29 to 78% of the sites tested at this level after the fog sanitizer. Interestingly, the prevalence of Listeria ssp. was unchanged and was recovered from the residue collector, drain, one processing line, and rubber mats. Bacterial biofilms developed on the non food contact surfaces (brick and floor) in the presence of meat residue were the most resistant to cleaning regimens. Despite the rough texture of the surfaces, both cleaning products significantly reduced the biofilm population (P 0.05). Mettler and Carpentier (24) inserted test plates of flooring material into the floor in three different plants (dairy, pastry, and meat) and found that, even with routine sanitation (chlorinated alkaline cleaner quaternary ammonium-aldehyde), there was a buildup in microbial numbers. In the meat plant, numbers reached 10 7 /cm 2 for one surface. In another study (25), they investigated the hygienic quality of seven flooring products contaminated with 1-day biofilms of Pseudomonas fluorescens and Bacillus stearothermophilus spores. Hygiene quality was linked to cleanability rather than disinfectability. The cleanability of the flooring products was determined by the overall surface texture, specifically the depth of the inwardly directed portion of the profile. Their results would explain why combination B cleaner, which was extremely effective on the floor material, was less effective on the brick, which was porous as well as rough textured. Surprisingly, cells, overall, were not more resistant to cleaning and sanitizing than s. This finding is contradictory to previous studies showing increased resistance with biofilm age (20, 21); however, those biofilms were not developed in the presence of meat residue. Stopforth et al. (36) tested the sanitizer resistance of L. monocytogenes and resident microflora on stainless steel grown at 15 C in fresh carcass decontamination washings for up to 14 days. They showed L. monocytogenes was more resistant at day 7 than day 2 and most sensitive by day 14; peroxyacetic acid was more effective than the other sanitizers tested. In our study, the bacteria could have attached to the meat residue, which built up on the surface over time or have become trapped in fat. While these conditions could protect bacteria from sanitizers, they would be more susceptible to cleaning agents, which are designed to remove fat and protein. We found L. monocytogenes bacteria that were present in biofilms could survive storage at 4 or 10 C for at least 5 days. Low temperatures and high humidity are conducive to moisture retention. Generally, surfaces, like the porous brick, that remained moist after storage resulted in higher percent survival compared with surfaces that had dried. The presence of frankfurter or fat residue enhanced survival of biofilm cells on storage and, when conditions were favorable, growth occurred. Helke and Wong (16) showed that presence of milk soil combined with high humidity were conditions that allowed for growth of L. monocytogenes on stainless steel. Palumbo and Williams (28) found long-term survival occurred when Listeria was suspended in beef extract and stored at 5 C. In addition to meat residue, the low temperature used to develop biofilms may be a factor. Listeria has been shown to alter branch-chain fatty acid concentrations to maintain membrane fluidity when grown at low temperatures (2). Smith (34) showed that L. monocytogenes adapted to osmotic and chill stress by accumulating carnitine from processed meats when grown at low temperatures. These factors may have a positive affect on survival and resistance of the organisms used in our study. In conclusion, this study has shown that, in a simulated RTE meat-processing environment, L. monocytogenes can develop biofilms on a variety of food contact and non food

11 2228 SOMERS A WONG J. Food Prot., Vol. 67, No. 10 contact surfaces and survive at reduced temperatures for up to 5 days. The importance of using a cleaner followed by a sanitizer when examining the effect of inactivating or removing L. monocytogenes biofilms is stressed. The types of surface, as well as the presence of soil, are major factors on cleanability. Either cleaner significantly (P 0.05) reduced bacterial biofilm number regardless of the surface or type of soil. Combination B was far more effective on problematic surfaces like conveyor belt and non food contact material, with or without soil, than combination A. For all samples and conditions, using the benchmark efficacy of 3-log reduction, combination cleaner and sanitizer A was only effective 50.0% of the time. Combination cleaner and sanitizer B, at 86.1%, was the more effective. 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