Efficacy of Cleaning and Sanitizing Agents Against Attached Listeria Mono- Cytogenes on Meat Slicer Components Philip G

Efficacy of Cleaning and Sanitizing Agents against Attached Listeria monocytogenes on Meat Slicer Components Philip G. Crandall,1* Corliss A. O’Bryan,1 Elizabeth M. Martin,2 H. Michael Kuefner,1 Sean Pendleton,1 Erin M. Shannon,1 John A. Marcy3 and Steven C. Ricke1,3 1Center for Food Safety-IFSE and Food Science Dept., 2650 Young Ave., University of Arkansas, Fayetteville, AR 72704, USA; 2Dept. of Biological & Agricultural Engineering, University of Arkansas, Fayetteville, AR 72704, USA; 3Dept. of Poultry Science, Division of Agriculture, University of Arkansas, Fayetteville, AR 72704, USA

INTRODUCTION ABSTRACT Listeria monocytogenes, an intra- cellular Gram-positive pathogen, The objective of this study was to evaluate the resistance caused 0.34 cases of invasive list- of surface-attached Listeria innocua and Listeria monocytogenes eriosis per 100,000 persons in 2009, to sanitizing agents under laboratory conditions. Six strains of compared with Salmonella spp., L. monocytogenes and one strain of L. innocua were attached to which caused 15 cases per 100,000 stainless steel or aluminum coupons that had been cut from a persons (2). However, 89% of list- used deli meat slicer. One cleaner/sanitizer and two sanitizers eriosis patients were hospitalized and were tested against the attached cells. No sanitizer caused more there was an overall 12.7% case fatal- 2 ity rate (CFR), in contrast to 27.5% than a 1.5 log CFU/cm reduction of Listeria when treated and hospitalizations and a CFR of 0.34% untreated coupons were compared. Many delicatessens are for salmonellosis (2). Consumption using sanitizing wipes during operating times. Therefore, the of food contaminated by L. monocy- best performing sanitizer, sanitizer C, from the first experiment togenes is the primary mode of trans- was applied with a variety of cleaning cloths and compared with mission of this pathogen to humans a commercial sanitation wipe. No cloth produced more than a (7). L. monocytogenes contaminates one log reduction compared to controls. food from a variety of environ- mental sources and food processing facilities. If present in meats or cheese, L. monocytogenes can contaminate slicers in delicatessens, and the result- ing contaminated food contact sur- A peer-reviewed article faces may allow bacterial survival and multiplication and thus become *Author for correspondence: Phone: +1 479.575.7686; Fax: +1 479.575.6936 sources of cross-contamination of Email: [email protected]

68 FOOD PROTECTION TRENDS | FEBRUARY 2012 TABLE 1. Strains of Listeria used in sanitizer evaluation

Strain Serotype Origin

LM 27a 4b Philadelphia outbreak, CDC LM 98a 1/2c Spinal fluid, male, Scotland LM 187a 4b Cheese outbreak, CDC LM 189a 1/2a Sliced turkey outbreak LM 190a 1/2a Human illness isolate LM 191a 1/2a Human illness isolate Li 169a Known as M1 Antibiotic resistance to 50 ppm rifampicin & 250 ppm streptomycin

aAll strains were obtained from the culture collection of the Center for Food Safety at the University of Arkansas-Fayetteville.

foods that are not cooked before con- The objective of this study was ment, Australia) was cut into 2 × 2.5 cm sumption. Avoiding cross-contamination to determine the effectiveness of com- coupons, using a Flow Waterjet Cutting between foods and food contact surfaces monly used commercial cleaning and System (Flow International Corporation, is thus critical to minimizing the risk of sanitizing agents against surface-attached Kent, WA). This Waterjet cutting system listeriosis. L. monocytogenes on aluminum and was used to prevent heat-induced stress According to the FDA Food Code stainless steel coupons cut from a used that could change the physical proper- (4), food equipment used with poten- deli meat slicer. ties of the stainless steel. From the blade tially hazardous foods must have food guard of the same slicer, cast aluminum contact surfaces cleaned throughout the MATERIALS AND METHODS coupons (2 × 2 × 0.5 cm) were cut, using a Milwaukee Heavy-Duty cold-cutting day, at least every 4 hours. The equip- Bacterial strains and growth metal saw (Brookfield, WI) and a Well- ment must be disassembled as necessary, conditions saw metal-cutting band saw (Wells Man- scraped to remove food particulates, and ufacturing Corporation, Three Rivers, then washed to remove soils, using what- One strain of Listeria innocua and MI). Coupons were washed thoroughly ever means are necessary, including ap- six strains of L. monocytogenes were com- bined into a cocktail for use in this study. in Micro 90 cleaning solution (Interna- plication of detergents containing wet- tional Products Corp., Burlington, NJ) ting agents and emulsifiers; acid, alkaline, See Table 1 for details on strains used. Stock cultures were maintained frozen prepared as per directions of the manu- or abrasive cleaners; hot water; brushes; (–80°C) in tryptic soy broth contain- facturer and then rinsed in sterile deion- scouring pads; high-pressure sprays, ing 0.6% yeast extract (TSB-YE; Bacto, ized water. Coupons were autoclaved for and ultrasonic devices. After washing, Becton Dickinson Co., Sparks, MD) 15 min at 121°C for sterilization prior to the equipment must be rinsed so that supplemented with 16% glycerol. Each inoculation. any abrasive compounds and cleaning culture was inoculated from frozen chemicals are removed or effectively di- stocks onto plates of Bacto tryptic soy Cell attachment luted. Equipment food contact surfaces agar containing 0.6% yeast extract(TSA- Sterile coupons were laid individu- must be sanitized after washing and rins- YE; Bacto, Becton Dickinson Co.) and ally in alternating sequence, in multiwall ing and again before use by means of hot incubated at 37°C for 24 h. Overnight flat bottom plates (Falcon, Becton Dick- water or chemicals. Sanitizers are meant cultures of each strain were prepared by inson Labware, Franklin Lakes, NJ), and to be used on clean surfaces and are not inoculating a colony into 10 ml of TSB- designed to remove organic material or YE and incubating the mixture at 37°C 40 μl (approximately 8 log CFU) of the biofilms. for 18 to 20 h. Cocktails for inoculation Listeria cocktail was pipetted into the Properly cleaning and sanitizing the were prepared by placing equal aliquots middle of each coupon and carefully meat slicer can reduce the potential for of each overnight culture in a single ster- spread over the area with a sterile inocu- cross-contamination at food service. It ile tube and mixing by use of a vortex lation loop. The inoculum was allowed is generally agreed that commonly used mixer. to adhere during air drying for 2 h before disinfectants or sanitizers are effective treatment with sanitizer. against L. monocytogenes in suspension Preparing deli slicer coupons (1, 3); however, cells attached to surfaces from components Sanitizer evaluation may be more resistant to sanitizers than The stainless steel blade of a Hobart Sanitizers for testing were recom- cells in suspension (5, 9, 12). heavy duty slicer (Hobart Food Equip- mended to us by the deli managers of

FEBRUARY 2012 | FOOD PROTECTION TRENDS 69 TABLE 2. Cleaners/sanitizers evaluated for inactivation of Listeria cocktail surface inoculated on coupons from deli meat slicer

Designation Ingredients Per cent by weight

A Ethyl alcohol 5 Sodium xlylene sulfonate 5 Fatty acid alkanolamide 5 Sodium lauryl ether sulfate 5 Sodium dodecylbenzene sulfonate 20 Water 60

B n-alkyl dimethyl benzyl ammonium chlorides 5 n-alkyl dimethyl ethylbenzyl ammonium chlorides 5 Water 90

C Quaternary ammonium chloride 10 Ethanol 1 Water 89

D n-alkyl dimethyl benzyl ammonium chloride 0.0175 Isopropyl alcohol 5.48

local retail deli establishments. Sanitiz- W. The other two cloths were Textronic tence of L. monocytogenes on aluminum ers were prepared at the concentrations Microfibre Cloth (T) and Softronic has been published. The primary food recommended by the manufacturer (see Microfibre Cloth (S), both manufactured contact surface for the deli slicer is the Table 2 for composition). Compound A by VERMOP Salmon GmbH, Gilching, stainless steel blade, but the cast alumi- is a cleaner/sanitizer, while compounds B Germany. Coupons cut from the slicer num guard and other components of the and C are simply sanitizers. Test coupons were cleaned with the sanitizer-soaked slicer housing could also serve as a niche with attached cells were sprayed with cloths by wiping the coupon 3 times for survival of Listeria and thus could sanitizer and allowed to sit for one min- in the vertical direction and 3 times in lead to cross contamination. L. mono- ute. One ml of DE Neutralizing broth the horizontal direction. Each coupon cytogenes cells attached to the deli slicer (Difco, Becton Dickinson Co., Sparks, was placed in a sterile centrifuge tube may detach and contaminate food prod- MD) was added to each test and con- containing 10 ml sterile PBS and mixed ucts, and there is some indication that these detached cells could survive stress- trol coupon and allowed to sit for one with a vortex mixer; serial dilutions were ful conditions, even if they are older or minute. Excess DE broth was poured off made, dilutions were plated on Modified and coupons swabbed with sterile swabs. have been injured in some fashion (10). Oxford Agar (MOX; Becton Dickinson We were able to recover a high num- Swabs were placed separately into tubes Co., Sparks, MD), and the plates were of sterile PBS and mixed with a vortex ber of attached bacteria, approximately 7 incubated at 37°C for 48 hours. 2 mixer; 10-fold serial dilutions were then log/cm , when inocula were spotted on prepared. Dilutions were plated on TSA- coupons and allowed to dry. This result Statistical analysis YE and plates were incubated at 37oC for is comparable to those of Kastbjerg and Gram and Kim and others , who 24 hours. Mean number of colonies per ml (6) (8) survivors was converted to log CFU/cm2 recovered similar numbers of bacteria. This arrangement simulates situations Evaluation of cleaning cloths and means were calculated. Differences were determined by student’s t-test, with where insufficient cleaning and disinfec- Cleaning cloths evaluated included significance assigned atP < 0.05. tion allows L. monocytogenes to survive a commercial cloth with sanitizer (D, see through protection by organic residues. Coupons were inoculated with an aver- Table 2) and three other cloths evaluated RESULTS AND DISCUSSION with sanitizer C. One cloth evaluated age of 8 log CFU of Listeria cocktail. was a 100% terrycloth towel, commonly To the best of our knowledge, no Recovery from non-treated coupons known as a “bar towel,” designated as research specifically on survival or persis- (controls) was approximately 90%.

70 FOOD PROTECTION TRENDS | FEBRUARY 2012 Figure 1. log c f u /cm2 survivors of Listeria cocktail on aluminum and stain- L. monocytogenes tested on stainless steel less steel coupons from a deli meat slicer after use of cleaners and sanitizers. coupons. The description of ingredients Survival data for controls are the mean of three samples; survival data in sanitizer A indicates that it is intended for tests are the mean of 12 samples. a-dStainless steel bars not hav- primarily for cleaning rather than for ing the same letter are significantly different by student’s t-test (P < 0.05). disinfection. Taormina and Beuchat (13) x-z Aluminum bars not having the same letter are significantly different by student’s found that L. monocytogenes could survive t-test (P < 0.05). suspended in cleaning solutions commonly used in food processing plants. However, they also found that cells that survived in the cleaning solutions were still susceptible to sanitizers and heat. Sanitizers B and C were significantly more effective (P < 0.05) in reducing Listeria recovery than was sanitizer A on the cast aluminum coupons (Fig. 1), al- though sanitizer A significantly reduced recovery compared to the control (P < 0.05). Comparison of sanitizer treatments on coupons made from the stainless steel blade and cast aluminum guard of a deli slicer revealed no significant differences (P > 0.05) in recovery of List- eria from control (untreated) coupons or for sanitizer A. However, sanitizers B and C performed significantly better (P < 0.05) on stainless steel than on cast aluminum. Figure 2. Comparison of a commercial sanitation wipe to various cloths Sanitizer C, because it was the most and a sanitizer for cleaning stainless steel with surface-attached Listeria. D = effective in the study just completed, was commercial sanitizing wipe, W = white bar towel, S = Softronic Microfibre selected for further study using different cleaning cloths on stainless steel coupons Cloth and T = Textronic Microfibre Cloth. a-cDifferent superscripts between from the blade. The commercial sanita- treatments denote significant difference of means (P < 0.05). tion wipe (D) produced an approximate 1 log reduction of Listeria, as did sanitizer C used with the two microfiber cloths (S and T). These results are shown in Fig. 2. The most successful combination was the white bar towel with sanitizer C. These results are significant, because retail foodservice workers commonly wipe the slicer blade with a sanitation wipe or towel that was stored in sanitizer between products. In this scenario, if there was heavy L. monocytogenes contamination from a previously sliced food or cross- contamination, the practice of just using a sanitation wipe would not be adequate to prevent cross-contamination.

CONCLUSIONS In most foodservice operations, the retail meat slicer is used randomly and sporadically throughout the day while On stainless steel coupons, all three significantly betterP ( < 0.05) than A being kept at room temperature, unlike sanitizers were effective against the at- and B, effectiveness significantly better other equipment such as food proces- tached Listeria, removing 2 to 3 log (P < 0.05) in the order C, then B, then sors or mixers that are used and cleaned CFU/cm2 of inoculated cocktail, a sta- A (Fig. 1). Kastbjerg and Gram (6) also after each use. This study revealed that tistically significant reduction compared found that quaternary ammonium sani- all three sanitizers were effective against with untreated controls. Sanitizer C was tizers were effective against 14 strains of attached Listeria on stainless steel and

FEBRUARY 2012 | FOOD PROTECTION TRENDS 71 cast aluminum coupons, with sanitizer C 3. Chavant, P., B. Gaillard-Martinie, 9. Mafu, A. A., D. Roy, J. Goulet, being the most effective in both cases. and M. Hebraud. 2004. Antimicro- L. Savoie, and P. Magny. 1990. Effi- Sanitizers B and C were found to be more bial effects of sanitizers against ciency of sanitizing agents for effective on stainless steel than on alumi- planktonic and sessile Listeria mono- destroying Listeria monocytogenes num coupons. Of the various cloths test- cytogenes cells according to the on contaminated surfaces. J. Dairy ed, the common white bar towel was the growth phase. FEMS Microbiol. Lett. Sci. 73:3428–3432. most effective. Often the operators prefer 236:241–248. 10. Poimenidou, S., C. A. Belessi, E. D. using wiping cloths or sanitary wipes in 4. FDA. 2009. Food Code 2009. http:// Giaouris, A. S. Gounadaki, G. J. E. place of disassembling and cleaning (11), www.fda.gov/Food/FoodSafety/ Nychas, and P. N. Skandamis. 2009. RetailFoodProtection/FoodCode/ which this research indicates may not be Listeria monocytogenes attachment FoodCode2009/default.htm. Acc- adequate to insure safety. to and detachment from stain- essed 17 August, 2011. less steel surfaces in a simulated 5. Frank, J. F., and R. A. Koffi. 1990. ACKNOWLEDGMENTS Surface-adherent growth of Listeria dairy processing environment. Appl. Environ. Microbiol. 75:7182–7188. This research was supported by an monocytogenes is associated with 11. Powitz, R. W. 2009. Sanitation in the American Meat Institute Foundation increased resistance to sanitizers deli: contamination prone equip- grant to author Crandall and a USDA and heat. J. Food Prot. 53:550–554. ment. Food Safety Oct/Nov: 20–21, Food Safety Consortium grant to author 6. Kastbjerg, V. G., and L. Gram. 2009. Model systems allowing quantifica- 59. Ricke. tion of sensitivity to disinfectants 12. Stopforth, J. D., J. Samelis, J. N. Sofos, REFERENCES and comparison of disinfectant P. A. Kendall, and G. C. Smith. 2002. susceptibility of persistent and Biofilm formation by acid-adapted 1. Aarnisalo, K., S. Salo, H. Miettinen, presumed nonpersistent Listeria and nonadapted Listeria monocyto- M. L. Suihko, G. Wirtanen, T. Autio, monocytogenes. J. Appl. Microbiol. genes in fresh beef decontamina- J. Lunden, H. Korkeala, and A. M. 106:1667–1681. tion washings and its subsequent Sjoberg. 2000. Bactericidal efficien- 7. Kathariou, S. 2002. Listeria monocy- inactivation with sanitizers. J. Food cies of commercial disinfectants togenes virulence and pathogenicity, Prot. 65:1717–1727. against Listeria monocytogenes on a food safety perspective. J. Food 13. Taormina, P. J., and L. R. Beuchat. surfaces. J. Food Safety 20:237–250. Prot. 65:1811–1829. 2. Center for Disease Control, CDC. 8. Kim, H., J. H. Ryu, and L. R. Beuchat. 2002. Survival of Listeria monocy- 2010. Preliminary FoodNet data 2007. Effectiveness of disinfectants togenes in commercial food-pro- on the incidence of infection with in killing Enterobacter sakazakii in cessing equipment cleaning solu- pathogens transmitted commonly suspension, dried on the surface of tions and subsequent sensitivity to through food — 10 States, 2009. stainless steel, and in biofilm. Appl. sanitizers and heat. J. Appl. Microbiol. MMWR 59:418–422. Environ. Microbiol. 73:1256–1265. 92:71–80. CALL FOR APPLICATIONS! Apply now for the opportunity to attend IAFP 2012 in Providence, Rhode Island! Applications are now being accepted for the IAFP 2012 Student Travel Scholarship. Deadline is Tuesday, February 28, 2012. For more details, including criteria and application forms, visit the IAFP Web site at www.foodprotection.org.

72 FOOD PROTECTION TRENDS | FEBRUARY 2012