1 The impact of surface conditions on sanitizer efficacy against Listeria monocytogenes biofilm

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2 on food-contact surface

3 Zi Hua1, Mei-Jun Zhu1,*

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4 1School of Food Science, Washington State University, Pullman, WA, 99164

6 *Corresponding author: Dr. Mei-Jun Zhu, School of Food Science, Washington State University,

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7 Pullman, WA 99163; Tel: 509-595-9068; Email: meijun.zhu@wsu.edu

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This preprint research paper has not been peer reviewed. Electronic copy available at: https://ssrn.com/abstract=4377892
 9 Abstract

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10 Unsanitary food-contact surfaces could be potential sources of contamination for Listeria

11 monocytogenes in fresh produce packing facilities. This study evaluated the impact of surface

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12 conditions on the effectiveness of sanitizers against L. monocytogenes biofilm on the selected

13 food-contact surfaces. The 7-day-old L. monocytogenes biofilms grown on different food-contact

14 surfaces were subjected to 200 ppm chlorine, 400 ppm quaternary ammonium compound (QAC),

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15 or 160 ppm peroxyacetic acid (PAA). Our results showed that the effectiveness of sanitizers was

16 lowered as surfaces were worn, a 5-min treatment with 200 ppm chlorine reduced L.

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17 monocytogenes by 1.84 - 3.39 log10 CFU/coupon on worn food-contact surfaces, compared to

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19
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2.79 - 3.93 on new surfaces. The 5-min use of 400 ppm QAC treatment caused 2.51 - 3.66 and

2.05 - 2.88 log10 CFU/coupon reductions of L. monocytogenes on new and worn surfaces,
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20 respectively. Among all sanitizer treatments tested, 160 ppm PAA was the most effective

21 treatment that decreased L. monocytogenes by 3.77 - 4.35 log10 CFU/coupon on all worn food-

22 contact surfaces after 1-min exposure. Apple juice soiling lowered the efficacies of QAC and
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23 PAA on all worn surfaces. Data highlighted the importance of effective surface disinfection and

24 proper equipment maintenance.

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25

26 Keywords: L. monocytogenes, biofilm, food-contact surface, worn, sanitizer

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This preprint research paper has not been peer reviewed. Electronic copy available at: https://ssrn.com/abstract=4377892
 27 1. Introduction

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28 Listeria monocytogenes is increasingly identified as a critical foodborne pathogen for the

29 fresh produce industry, causing multiple listeriosis outbreaks related to fresh fruits and vegetables

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30 (CDC, 2012, 2015, 2020). Listeria spp. was frequently detected in the fresh apple and other tree

31 fruit packing facilities (Ruiz-Llacsahuanga et al., 2021; Simonetti et al., 2021; Tan et al., 2019).

32 This pathogen can form biofilms on various food-contact surfaces (Beresford et al., 2001; Doijad

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33 et al., 2015; Hua et al., 2019; Papaioannou et al., 2018). If food-contact surfaces are not thoroughly

34 cleaned and sanitized, they can represent potential and continuous sources of contamination to

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35 fresh produce, causing serious food safety problems. The unsanitary packinghouse equipment and

36

37
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environment are recognized as important sources of contamination associated with the nationwide

listeriosis outbreaks in cantaloupe (CDC, 2012) and caramel apple (CDC, 2015). The detection of
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38 L. monocytogenes on equipment surfaces near the packing area also led to recalls related to

39 multiple fresh-cut products (FDA, 2020). Thus, effective surface disinfection and good equipment

40 maintenance are needed to mitigate L. monocytogenes contaminations risks.

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41 To reduce or eliminate microbial loads on food-contact surfaces and prevent cross-

42 contamination, chemical sanitizers are used daily to sanitize the packing lines of fresh apples and
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43 other produce. However, L. monocytogenes in biofilm is more resistant to chemical sanitizers

44 (Robbins et al., 2005) and other disinfection treatments (Frank & Koffi, 1990; Liu et al., 2017)
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45 compared to planktonic cells. Even though the effectiveness of different sanitizer treatments

46 against L. monocytogenes biofilms has been widely evaluated by previous studies, most studies
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47 were conducted to test the efficacies of sanitizer on new food-contact surfaces free of scratch, pit,

48 and cracks (Dhowlaghar et al., 2018; Hua et al., 2019; Park et al., 2012; Park & Kang, 2017).
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49 However, the food-contact surfaces are subjected to natural aging and abrasion with usage and

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 50 time. Damaged/worn equipment and food-contact surfaces are more prone to Listeria

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51 contamination. L. monocytogenes was found on worn rubber surfaces (Tompkin, 2002) and

52 damaged plastic cutting board (Berzins et al., 2010) in the ready-to-eat meat facilities. Ruiz-

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53 Llacsahuanga et al. (2021) reported that Listeria spp. had an overall 4.6% prevalence in fresh apple

54 packing lines in Washington state; most Listeria-positive food-contact surfaces had cracks and

55 worn edges. In the caramel apple outbreak investigation, L. monocytogenes was detected on worn

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56 food-contact surfaces, including vinyl surfaces with frayed edges and damaged conveyor belts in

57 the fresh apple facility (Angelo et al., 2017). Yet, limited information is available about the

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58 practical efficacies of sanitizers against L. monocytogenes biofilm formed on worn food-contact

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surfaces.
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This study aimed to 1) compare the population of L. monocytogenes in biofilm on common
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61 food-contact surfaces under new and worn conditions; 2) assess the efficacies of commonly used

62 sanitizers, including chlorine, quaternary ammonium compound (QAC), and peroxyacetic acid

63 (PAA), against L. monocytogenes biofilm on new and worn surfaces, in the presence or absence
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64 of organic matter.

65
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66 2. Materials and Methods

67 2.1 L. monocytogenes strains and cocktail preparation

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68 L. monocytogenes NRRL B-33069 (1/2a), NRRL B-57618 (1/2a), NRRL B-33006 (1/2b),

69 NRRL B-33466 (1/2b), NRRL B-33071 (4b), and NRRL B-33385 (4b) were obtained from
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70 USDA-ARS culture collection of National Center (NRRL) for Agricultural Utilization Research

71 (Peoria, IL, US) and stored at -80 °C in Trypticase Soy Broth supplied with 0.6% Yeast Extract
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72 (TSBYE, Fisher Scientific, Fair Lawn, NJ, US) and 20% (v/v) glycerol. Each frozen culture was

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 73 activated in TSBYE at 37 °C for 24 h statically and then transferred to TSBYE for another 24-h

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74 incubation at 37 °C. To prepare a six-strain L. monocytogenes cocktail, an equal volume from each

75 L. monocytogenes suspension was pooled, centrifuged (8,000 × g, 5 min, 22 °C), and the resulting

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76 pellet was re-suspended with sterile Modified Welshimer’s Broth (MWB, HiMedia, West Chester,

77 PA, US) to reach a final concentration of ~108 CFU/ml.

78

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79 2.2 Preparation of food-contact coupons

80 The SS (AISI 316) sheets with 2B finish and 4 finish were acquired from the Washington

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81 State University Instrument Shops (Pullman, WA, US). The low-density polyethylene (LDPE),

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83
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polyvinyl chloride (PVC), and polyester (PET) sheets were purchased from Interstate Plastics

(Sacramento, CA, US), and the silicone rubber sheet was from Rubber Sheet Warehouse (Los
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84 Angeles, CA, US). To simulate the worn food-contact surfaces, the SS-2B and SS-4 sheets were

85 bead blasted (moderately worn) and sanded with 80-grit sandpaper (severely worn) based on

86 previous reports (Uesugi et al., 2007; Woodling & Moraru, 2005). LDPE, PVC, PET, and rubber
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87 are soft materials that are easily damaged; they have shorter longevity and are replaced more

88 frequently than SS. Thus, the worn LDPE, PVC, PET, and rubber used in this study were sanded
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89 with 80-grit sandpaper for 5 minutes. All coupons (1.5 cm × 0.75 cm) of respective surface type

90 were prepared as previously described (Hua et al., 2019).

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91

92 2.3 L. monocytogenes biofilm formation

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93 The surface coupons conditioned with or without apple juice were transferred into 24 well

94 plates (one coupon per well); each coupon was inoculated with 2 ml of the above-prepared six-
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95 strain L. monocytogenes cocktail (~108 CFU/ml) suspending in MWB. Then the L. monocytogenes

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 96 biofilm was formed by incubating statically at room temperature (RT, ~22 °C) for seven days (Hua

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97 et al., 2019).

98

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99 2.4 Sanitizer treatment of L. monocytogenes biofilm

100 The coupons carrying 7-day-old biofilm were washed with 2 ml of sterile PBS three times,

101 then treated with 160 ppm PAA prepared with Bioside HS (15% PAA, EnviroTech, Modesto, CA,

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102 US) for 1 or 5 min, 400 ppm QAC prepared with STOP IT (Pace International, Wapato, WA, US)

103 for 1 or 5 min, and 200 ppm chlorine with Accu-Tab (Pace International, Wapato, WA, US) for 1

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104 or 5 min at RT, respectively. Each coupon was immediately rinsed with 2 ml of Dey-Engley

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106
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Neutralizing Broth (Oxoid, US) to neutralize the residual sanitizer, followed by 2 ml PBS rinsing

once. Coupons without sanitizer treatments were used as a control. There were three replicates for
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107 the selected surface type and sanitizer treatment, and each treatment combination was repeated

108 three times independently. The concentration of the PAA solution was verified with a PAA test kit

109 (AquaPhoenix, Hanover, PA, US), and the concentrations of QAC and chlorine were confirmed
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110 using test kits from LaMotte (Chestertown, MD, US).

111
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112 2.5 Biofilm dislodgement and enumeration

113 Each coupon-bearing biofilm was transferred into 2-ml tubes containing 1.0 ml sterile PBS
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114 and 3~4 glass beads. The tubes were vortexed with a benchtop mixer (VWR, CA, United States)

115 for 2 min at maximal speed to dislodge L. monocytogenes from surfaces. The resulting suspension
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116 was 10-fold serial diluted with sterile PBS, and 100 l of appropriate dilution was plated onto

117 TSAYE plates in duplicates. The plates were enumerated after a 48-h incubation at 37 °C.
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118

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119 2.6 Surface roughness measurement

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120 The roughness of each type of surface coupon without inoculation was measured using a

121 profilometer (Model: PHD-II, SPN Technologies, US). The Rz (ten-point height), Ra (arithmetic

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122 mean roughness), Rq (mean square roughness), and Rp (maximal height of peaks) of each coupon

123 were averaged from four different scanning sites on the same coupon, and four replicates were

124 tested for each type of surface material (Hua et al., 2021).

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126 2.7 Statically analysis

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127 Data were analyzed by Two-way Analysis of Variance (ANOVA) and Uncorrected Fisher’s

128

129
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Least Significant Difference (LSD) using SPSS (BM SPSS Statistics, version 19.0 software,

Chicago, IL, United States). Data were presented as means ± SEM (standard error mean).
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130 Microbiological data were repeated three times independently, with three replicates per treatment

131 combination in each independent study. Roughness parameters were averaged by 16

132 measurements of the selected surface.

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133

134 3. Result
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135 3.1 L. monocytogenes population on food-contact surfaces

136 The counts of L. monocytogenes in biofilm formed on SS surfaces were comparable

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137 regardless of finish type and surface conditions, and there was a 6.93 - 7.10 log10 CFU/coupon L.

138 monocytogenes on SS (Fig. 1 A). L. monocytogenes populations on worn LDPE, PVC, PET, and
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139 rubber were significantly (P < 0.05) higher compared to the corresponding new surfaces; L.

140 monocytogenes levels on worn LDPE, PVC, PET, and rubber surface coupons was 7.89 - 8.64
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141 log10 CFU/coupon, compared to 7.05 - 7.50 log10 CFU/coupon on new surface coupons (Fig. 1 B).

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142

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143 3.2 Sanitizers efficacies against L. monocytogenes biofilm on new and worn surfaces

144 The efficacy of the selected sanitizers against L. monocytogenes biofilms treated at 1 min and

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145 5 min was first evaluated using new rubber surfaces (Fig. 2). Chlorine at 200 ppm and QAC at 400

146 ppm exhibited significant (P < 0.05) higher efficacy when used for 5 min than 1 min, causing 3.07

147 and 2.88 vs. 2.41 and 2.38 log10 CFU/coupon reduction after 5 min and 1 min treatment of chlorine

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148 or QAC, respectively. The efficacy of PAA at 160 ppm at 1 min contact time was not different

149 from 5 min contact (3.52 vs 3.75 log10 CFU/coupon reduction). Therefore, the antimicrobial

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150 efficacies of chlorine and QAC were evaluated at 5 min, and the effectiveness of PAA was tested

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for 1 min in the following studies.
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L. monocytogenes in biofilm on SS-2B exhibited higher resistance than that on SS-4,
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153 regardless of surface conditions and sanitizer treatments, and the surfaces with defects or damage

154 compromised the efficacies of sanitizers in removing L. monocytogenes from SS surface coupons

155 (Table 1). The 5 min exposure of 400 ppm QAC caused 2.38 and 2.88 log reductions on worn SS-
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156 2B and SS-4 surfaces, respectively, which was significantly (P < 0.05) less effective than 2.83 and

157 3.65 log reductions obtained on new SS-2B and SS-4 coupons. Similarly, PAA at 160 ppm for 1-
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158 min contact is more effective against L. monocytogenes biofilm on new SS surfaces than those on

159 worn ones: 4.05 and 4.32 vs. 3.41 and 3.83 log reduction on SS-2B and SS-4 surfaces in new vs.
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160 worn conditions, respectively (Table 1). The effectiveness of sanitizer treatments on defective

161 (moderate wear) and worn (severe wear) SS are comparable (Table 1).
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162 Similarly, the bactericidal effect of chlorine was significantly (P < 0.05) reduced on worn

163 PVC, PET, and rubber surfaces compared to that on new surfaces, which removed 3.35 vs. 3.06,
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164 3.23 vs. 1.84, 3.93 vs. 3.31, and 2.97 vs. 2.43 log10 CFU/coupon of L. monocytogenes from LDPE,

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165 PVC, PET, and rubber in new vs. worn conditions, respectively (Table 2). QAC is more effective

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166 in removing L. monocytogenes biofilm from new LDPE, PVC, and PET surfaces than from worn

167 surfaces, but it caused a comparable reduction on new and worn rubber coupons (Table 2). PAA

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168 at 160 ppm for 1-min contact led to similar L. monocytogenes reductions on new and worn LDPE,

169 PVC, PET, and rubber surfaces (Table 2). Given that the population of L. monocytogenes in

170 biofilm on all tested worn surfaces is significantly (P < 0.05) higher than that on new surfaces (Fig.

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171 1 & Table 2), PAA was less effective in sanitizing the worn surfaces. For each selected surface,

172 PAA at 160 ppm for 1-min treatment was more effective in removing L. monocytogenes biofilm

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173 than 5-min treatments of 200 ppm chlorine and 400 ppm QAC, regardless of the degree of wear

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(Tables 1 and 2).
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176 3.3 Sanitizers efficacies against L. monocytogenes biofilm on soiled food-contact surfaces

177 Food matrix conditioning on surfaces did not impact the L. monocytogenes numbers in

178 biofilm on worn SS, PVC, and rubber surface coupons but significantly (P < 0.05) increased the
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179 L. monocytogenes counts on worn LDPE and PET surfaces compared to that on clean ones (Fig.

180 3). When apple juice was present, there were 7.02 - 7.11 log10 CFU/coupon on worn SS coupons
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181 and 8.48 - 8.62 log10 CFU/coupon on worn non-SS surfaces, respectively (Fig. 3).

182 The efficacies of QAC and PAA against L. monocytogenes on worn SS surfaces, regardless
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183 of surface finish and the degree of wear, are compromised by organic matter conditioning (Fig. 4

184 A-B). When the organic matter was present, a 5-min exposure of 400 ppm QAC removed 1.69 and
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185 1.38 log10 CFU/coupon of L. monocytogenes on defective and worn SS-2B surfaces, 1.91 and 1.64

186 log10 CFU/coupon on defective and worn SS-4 surface, respectively, compared to 1.88 and 2.21
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187 log10 CFU/coupon reduction on new SS-2B and SS-4. The PAA (160 ppm, 1 min) treatment

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188 reduced L. monocytogenes by 2.78/2.58 and 3.11/2.93 log10 CFU/coupon on apple juice coated

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189 defective/worn SS-2B and SS-4 surfaces, respectively, compared to 3.24 and 3.50 log10

190 CFU/coupon reductions on new SS-2B and SS-4 (Fig. 4 A-B).

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191 Similarly, the sanitizer’s efficacies against L. monocytogenes biofilm on worn non-SS

192 surfaces were decreased in the presence of organic matter. QAC (400 ppm, 5 min) decreased

193 2.12/1.37, 2.05/1.64, 2.47/1.00, and 2.55/1.52 log10 CFU/coupon L. monocytogenes on

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194 clean/soiled worn LDPE, PVC, PET, and rubber surfaces (Fig. 4 C-E). PAA (160 ppm, 1 min)

195 removed 3.77/3.44, 3.93/3.80, 4.35/4.07, and 3.95/3.05 log10 CFU/coupon L. monocytogenes on

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196 clean/soiled worn LDPE, PVC, PET, and rubber surfaces, respectively (Fig. 4 C-E). Notably, ≥

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7.0 or 4.5 log10 CFU/coupon of L. monocytogenes were detected on all non-SS worn surfaces after

QAC or PAA treatment, respectively. Those data highlighted that worn food-contact surfaces
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199 soiled with food debris could be persistent L. monocytogenes contamination sources.

200

201 3.4 Roughness of food-contact surfaces

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202 The prepared surface coupons’ roughness, a key surface property affecting biofilm formation

203 (Awad et al., 2018), was evaluated. The roughness of all worn surface coupons was increased
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204 significantly (P < 0.05), evidenced by greatly improved Ra, Rz, Rp, and Rq values (Table 3). Worn

205 SS-2B was the roughest surface, followed by SS-4. The worn PET was the smoothest surface, and
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206 worn PVC, LDPE, and rubber showed a similar roughness level (Table 3).

207
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208 4. Discussion

209 4.1 Surface conditions and types impacted L. monocytogenes biofilm formation
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210 Food-contact surfaces could naturally abrade as they are in use; the Ra of polyesterurethane

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211 belts in a poultry facility was 28.8-times increased after a 5-year processing (Chaturongkasumrit

212 et al., 2011). The Ra of the electropolished SS blade increased from 0.653 - 0.752 um to 0.836 -

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213 0.935 um after 1-year use, with substantial wear and pitting observed on blade surfaces (Vorst et

214 al., 2006). To mimic the worn food-contact surfaces, each tested surface was artificially abraded,

215 which significantly (P < 0.05) increased the roughness of all surfaces. SS-2B was rougher than

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216 SS-4 under all conditions, which was consistent with a previous report on electropolished SS

217 (Woodling & Moraru, 2005).

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218 Although SS-2B (Ra: 0.28 - 1.56 μm) is rougher than SS-4 (Ra: 0.24 - 1.30 μm), the L.

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monocytogenes population size in 7-day-old biofilms on SS was not impacted by their finish and

wear condition. In support of our finding, the population of L. monocytogenes in 2-day-old

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221 biofilms formed on mechanically finished SS (Ra: 0.26 - 0.69 μm) and electropolished SS (Ra: 0.16

222 - 0.67 μm) was similar (Rodriguez et al., 2008). However, L. monocytogenes counts in biofilm

223 formed on the worn PVC, PET, LDPE, and rubber surfaces were ~ 1.0 log10 CFU/coupon higher
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224 than the respective new surfaces. In support of our finding, compared to a new polyesterurethane

225 conveyor belt, the numbers of L. monocytogenes in the 7-day-old biofilm were 1.6 - 1.8 log10
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226 CFU/coupon higher on a 5-year-old polyesterurethane conveyor belt (Chaturongkasumrit et al.,

227 2011). Similarly, greater Staphylococcus aureus populations were observed in 5-day-old biofilms
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228 on scratched polypropylene and polyethylene than the smooth ones (Kim et al., 2017). This is

229 likely due to the increased surface contact area and attachment niches for bacteria to harborage on
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230 worn surfaces.

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232 4.2 Surface conditions impacted sanitizer efficacies against L. monocytogenes biofilm

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233 L. monocytogenes biofilm on SS-2B was more difficult to decontaminate than on SS-4

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234 regardless of sanitizer treatments and damage degree of new, defective (moderately damaged),

235 and worn (severely damaged). In alignment with our findings, a 15-min exposure of 20 ppm

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236 gaseous chlorine dioxide was more effective against attached L. monocytogenes on SS-4 than on

237 SS-2B (Park & Kang, 2017). The surface defect decreased the tested sanitizer efficacies in

238 removing L. monocytogenes biofilm on SS coupons. Despite the limited studies comparing

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239 sanitizer efficacies against L. monocytogenes on SS surfaces, our findings were supported by

240 studies conducted on other bacteria on SS surfaces. A 10-min treatment of 50 ppm chlorine

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241 reduced Salmonella by 2.8 and 2.1 logs on new and worn SS surfaces (finish type not specified),

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respectively (Schlisselberg & Yaron, 2013); a 10-min exposure of 200 ppm chlorine removed ~

4.4 and 4.2 logs of 5-day-old S. aureus biofilms on new or scratched SS (finish type not
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244 provided), respectively (Kim et al., 2017); the acidic electrolyzed water showed better efficacy

245 against milk spoilage bacteria on smooth SS-2B than that on a coarse SS-2B (Jimenez-Pichardo

246 et al., 2016). In addition, the effectiveness of chlorine (200 ppm, 45 sec) and QAC (200 ppm, 35
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247 sec) in inactivating S. aureus was lowered on abraded SS-4 compared to the new SS-4 surface

248 (Frank & Chmielewski, 1997).

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249 The effectiveness of all sanitizers tested in this study, regardless of sanitizer type, in

250 removing L. monocytogenes biofilm on LDPE, PVC, PET, and rubber surfaces was compromised
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251 by abrasions and defects on the surface, based on log reduction and or the culturable L.

252 monocytogenes remained on each surface coupon. In alignment with our findings, a 10-min
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253 exposure of a 1% mixture of amphoteric alkyl and aminoethyl glycine solution caused 0.8 log10

254 CFU/coupon higher reduction of a 7-day-old L. monocytogenes from new polyesterurethane

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255 conveyor belt than that from a worn belt (Chaturongkasumrit et al., 2011). Treatments of QAC-

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256 based sanitizers and chlorine exhibited higher anti-Listeria effects on smooth high-density

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257 polyethylene surfaces than on old ones (Yang et al., 2009). Similarly, the effectiveness of chlorine

258 (200 ppm, 45 sec) and QAC (200 ppm, 35 sec) were compromised on abraded polycarbonate

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259 surfaces (Frank & Chmielewski, 1997). Chlorine (200 ppm, 10 min) was more effective in

260 removing 5-day-old S. aureus biofilm from smooth versus scratched polypropylene (Kim et al.,

261 2017).

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262 Organic soiling with apple juice significantly decreased the sanitizers’ effectiveness in

263 removing L. monocytogenes on all tested surfaces, regardless of surface type and wear conditions.

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264 This is consistent with our findings on new surface coupons (Hua et al., 2019; Korany et al., 2018)

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266
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and other studies (Ayebah et al., 2006; Kuda et al., 2008; Moretro et al., 2019; Vandekinderen et

al., 2009). The defects and irregularities on surface materials, especially those in worn condition,
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267 could entrap organic matter and further influence the effectiveness of sanitizers. Food soils on

268 surfaces could chemically and physically protect L. monocytogenes from sanitizer inactivation

269 because the sanitizer loses antimicrobial activity as it reacts with organic matter (Ayebah et al.,
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270 2006). In addition, the biofilm matrix and organic soils could build a barrier limiting sanitizer from

271 penetrating (Fernandes et al., 2015).

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272 This study once again indicated that surface conditions are important for effective surface

273 disinfection. The organic matter soiling on the worn surface causes additional challenges in
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274 eradicating L. monocytogenes biofilms. More than 4.5 log L. monocytogenes remained on PAA-

275 sanitized worn/soiled PVC, LDPE, PET, and rubber surfaces, suggesting that a more effective
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276 surface decontamination method is needed.

277
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278 Conclusion

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279 The population of L. monocytogenes in biofilms on all surface coupons except SS surfaces

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280 was significantly higher on the worn surface than on new ones. Worn food-contact surfaces

281 reduced the effectiveness of all sanitizer treatments, especially when organic matter was present.

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282 A 5-min use of 200 ppm chlorine reduced L. monocytogenes by 2.79 - 3.93 and 1.84 - 3.39 log10

283 CFU/coupon on new and worn surfaces, respectively. There were 2.51 - 3.66 vs. 2.05 - 2.88 log10

284 CFU/coupon reductions of L. monocytogenes on new vs. worn surfaces, after treatment with 400

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285 ppm QAC for 5 min. PAA (160 ppm, 1 min) was the most effective treatment, reducing 3.77 - 4.35

286 log10 CFU/coupon L. monocytogenes on worn surfaces. Food debris soiling on worn surfaces

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287 lowered the efficacies of QAC and PAA on all worn surfaces, and more than 4.5 log10 CFU/coupon

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289
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L. monocytogenes survived on non-SS coupons treated with PAA. Data highlights the importance

of surface maintenance and effective cleaning and sanitization.

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290

291 Acknowledgment

292 This study was supported by Washington Tree Fruit Research Commission. We
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293 acknowledged Pace International Inc. for their generous donations of Accu-Tab, Bioside HS, and

294 STOP-IT. We thanked Mrs. Tonia Green and Ms. Ritu Sharma for their assistance in preparing the
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295 experiment materials.

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296 References

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297 Angelo, K. M., Conrad, A. R., Saupe, A., Dragoo, H., West, N., Sorenson, A., Barnes, A., Doyle,
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299 Wise, M., & Jackson, B. R. (2017). Multistate outbreak of Listeria monocytogenes
300 infections linked to whole apples used in commercially produced, prepackaged caramel

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304 Ayebah, B., Hung, Y. C., Kim, C., & Frank, J. F. (2006). Efficacy of electrolyzed water in the
305 inactivation of planktonic and biofilm Listeria monocytogenes in the presence of organic
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307 028x-69.9.2143
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317 CDC. (2012). Multistate outbreak of listeriosis linked to whole cantaloupes from Jensen Farms,
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325
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341 Doijad, S. P., Barbuddhe, S. B., Garg, S., Poharkar, K. V., Kalorey, D. R., Kurkure, N. V.,

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369 Hua, Z., Korany, A. M., EI-Shinawy, S. H., & Zhu, M. (2019). Comparative evaluation of
370 different sanitizers against Listeria monocytogenes biofilms on major food-contact
371 surfaces. Frontiers in microbiology, 10, 1-12.
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372 https://doi.org/https://doi.org/10.3389/fmicb.2019.02462
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374 Hua, Z., Younce, F., Tang, J., Ryu, D., Rasco, B., Hanrahan, I., & Zhu, M.-J. (2021). Efficacy of
375 saturated steam against Listeria innocua biofilm on common food-contact surfaces. Food
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376 Control, 107988. https://doi.org/https://doi.org/10.1016/j.foodcont.2021.107988

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379 Garcia-Almendarez, B. E. (2016). Evaluation of electrolyzed water as cleaning and
380 disinfection agent on stainless steel as a model surface in the dairy industry. Food
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381 Control, 60, 320-328. https://doi.org/10.1016/j.foodcont.2015.08.011

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383 Kim, C. Y., Ryu, G. J., Park, H. Y., & Ryu, K. (2017). Resistance of Staphylococcus aureus on
384 food contact surfaces with different surface characteristics to chemical sanitizers. Journal
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385 of Food Safety, 37(4), 1-9. https://doi.org/https://doi.org/10.1111/jfs.12354

386

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387 Korany, A. M., Hua, Z., Green, T., Hanrahan, I., El-Shinawy, S. H., El-Kholy, A., Hassan, G., &

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388 Zhu, M. J. (2018). Efficacy of ozonated water, chlorine, chlorine dioxide, quaternary
389 ammonium compounds and peroxyacetic acid against Listeria monocytogenes biofilm on
390 polystyrene surfaces. Frontiers in microbiology, 9, 1-10.
391 https://doi.org/https://doi.org/10.3389/fmicb.2018.02296
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393 Kuda, T., Yano, T., & Kuda, M. T. (2008). Resistances to benzalkonium chloride of bacteria
394 dried with food elements on stainless steel surface. LWT-Food Science and Technology,
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397 Liu, F., Du, L. H., Zhao, T., Zhao, P., & Doyle, M. P. (2017). Effects of phenyllactic acid as
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399 72-78. https://doi.org/10.1016/j.foodcont.2017.02.050
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401 Moretro, T., Fanebust, H., Fagerlund, A., & Langsrud, S. (2019). Whole room disinfection with
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403 environments. International Journal of Food Microbiology, 292, 118-125.
404 https://doi.org/10.1016/j.ijfoodmicro.2018.12.015
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culture simulated fish processing conditions and chemical disinfection challenges.
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409 International Journal of Food Microbiology, 267, 9-19.
410 https://doi.org/10.1016/j.ijfoodmicro.2017.12.020
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412 Park, S. H., Cheon, H. L., Park, K. H., Chung, M. S., Choi, S. H., Ryu, S., & Kang, D. H. (2012).
413 Inactivation of biofilm cells of foodborne pathogen by aerosolized sanitizers.
414 International Journal of Food Microbiology, 154(3), 130-134.
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415 https://doi.org/10.1016/j.ijfoodmicro.2011.12.018
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417 Park, S. H., & Kang, D. H. (2017). Influence of surface properties of produce and food contact
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418 surfaces on the efficacy of chlorine dioxide gas for the inactivation of foodborne
419 pathogens. Food Control, 81, 88-95. https://doi.org/10.1016/j.foodcont.2017.05.015
420
421 Robbins, J. B., Fisher, C. W., Moltz, A. G., & Martin, S. E. (2005). Elimination of Listeria
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422 monocytogenes biofilms by ozone, chlorine, and hydrogen peroxide. Journal of Food
423 Protection, 68(3), 494-498. https://doi.org/10.4315/0362-028x-68.3.494
424
425 Rodriguez, A., Autio, W. R., & Mclandsborough, L. A. (2008). Effect of surface roughness and
426 stainless steel finish on Listeria monocytogenes attachment and biofilm formation.
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427 Journal of Food Protection, 71(1), 170-175. https://doi.org/https://doi.org/10.4315/0362-

428 028X-71.1.170
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430 Ruiz-Llacsahuanga, B., Hamilton, A., Zaches, R., Hanrahan, I., & Critzer, F. (2021). Prevalence
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431 of Listeria species on food contact surfaces in Washington State apple packinghouses.

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432 Applied and Environmental Microbiology.

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433 https://doi.org/https://doi.org/10.1128/AEM.02932-20
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435 Schlisselberg, D. B., & Yaron, S. (2013). The effects of stainless steel finish on Salmonella
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439 Simonetti, T., Peter, K., Chen, Y., Jin, Q., Zhang, G. D., LaBorde, L. F., & Macarisin, D. (2021).
440 Prevalence and distribution of Listeria monocytogenes in three commercial tree fruit
441 packinghouses. Frontiers in microbiology, 12. https://doi.org/ARTN 652708
442 10.3389/fmicb.2021.652708
443

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444 Tan, X. Q., Chung, T., Chen, Y., Macarisin, D., LaBorde, L., & Kovac, J. (2019). The
445 occurrence of Listeria monocytogenes is associated with built environment microbiota in
446 three tree fruit processing facilities. Microbiome, 7(1). https://doi.org/ARTN 115
447 10.1186/s40168-019-0726-2

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448
449 Tompkin, R. B. (2002). Control of Listeria monocytogenes in the food-processing environment.
450
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Uesugi, A. R., Woodling, S. E., & Moraru, C. I. (2007). Inactivation kinetics and factors of
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454 variability in the pulsed light treatment of Listeria innocua cells. Journal of Food
455 Protection, 70(11), 2518-2525. https://doi.org/https://doi.org/10.4315/0362-028X-
456 70.11.2518
457
458 Vandekinderen, I., Devlieghere, F., Van Camp, J., Kerkaert, B., Cucu, T., Ragaert, P., De
459 Bruyne, J., & De Meulenaer, B. (2009). Effects of food composition on the inactivation
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460 of foodborne microorganisms by chlorine dioxide. International Journal of Food

461 Microbiology, 131(2-3), 138-144. https://doi.org/10.1016/j.ijfoodmicro.2009.02.004
462
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463 Vorst, K. L., Todd, E. C., & Ryser, E. T. (2006). Transfer of Listeria monocytogenes during
464 slicing of turkey breast, bologna, and salami with simulated kitchen knives. Journal of
465 Food Protection, 69(12), 2939-2946. https://doi.org/https://doi.org/10.4315/0362-028X-
466 69.12.2939
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467
468 Wang, X., Kumagai, S., & Yoshimura, N. (1999). Relation between hydrophobicity and surface
469 roughness of silicone rubber insulator before and after aging in acid rain. IEEJ
470 Transactions on Fundamentals and Materials, 119(2), 216-223.
471 https://doi.org/https://doi.org/10.1541/ieejfms1990.119.2_216
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472
473 Woodling, S. E., & Moraru, C. I. (2005). Influence of surface topography on the effectiveness of
474 pulsed light treatment for the inactivation of Listeria innocua on stainless‐steel surfaces.
475 Journal of food science, 70(7), m345-m351.
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476 https://doi.org/https://doi.org/10.1111/j.1365-2621.2005.tb11478.x
477

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478 Yang, H., Kendall, P. A., Medeiros, L. C., & Sofos, J. N. (2009). Efficacy of sanitizing agents

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479 against Listeria monocytogenes biofilms on high-density polyethylene cutting board
480 surfaces. Journal of Food Protection, 72(5), 990-998. https://doi.org/Doi 10.4315/0362-
481 028x-72.5.990
482
483

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484

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485 Figure Legends

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486 Fig. 1 L. monocytogenes population in biofilms on food-contact surfaces with different

487 conditions. The L. monocytogenes biofilms were grown on each respective surface for 7 days

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488 statically at 22 °C. (A) Stainless steel (SS), SS-2B: stainless steel 2B finish, SS-4: stainless steel 4

489 finish. (B) Polyvinyl chloride (PVC), low-density polyethylene (LDPE), polyester (PET), and

490 rubber surfaces. New: new surfaces; Defective: SS was bead blasted; Worn: all surfaces were 80-

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491 grit sanded. a-b Bars topped with different letters differ significantly (P < 0.05). Mean  SEM was

492 averaged from three independent studies where three replicates were used within each independent

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493 study.

494

495
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Fig. 2 L. monocytogenes reduction on the new rubber surfaces after being treated with
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496 sanitizers at different times. The 7-day-old L. monocytogenes biofilms on the new rubber

497 surfaces were subjected to chlorine (200 ppm), quaternary ammonium compound (QAC, 400 ppm),

498 and peroxyacetic acid (PAA, 160 ppm) treatment for 1- and 5-min. a-b Bars topped with different
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499 letters differ significantly (P < 0.05). Mean  SEM was averaged from three independent studies

500 where three replicates were used within each independent study.
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501

502 Fig. 3 L. monocytogenes population in biofilms on food-contact surfaces soiled with organic
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503 matter. The L. monocytogenes biofilms were grown on each respective apple juice soiled

504 surface for 7 days statically at 22 °C. (A) Stainless steel (SS), SS-2B: stainless steel 2B finish,
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505 SS-4: stainless steel 4 finish. (B) Polyvinyl chloride (PVC), low-density polyethylene (LDPE),

506 polyester (PET), and rubber surfaces. New: new surfaces; Defective: SS was bead blasted; Worn:
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507 all surfaces were 80-grit sanded. a-b Bars topped with different letters differ significantly (P <

This preprint research paper has not been peer reviewed. Electronic copy available at: https://ssrn.com/abstract=4377892
508 0.05). Mean  SEM was averaged from three independent studies where three replicates were

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509 used for each selected surface coupon within each study.

510

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511 Fig. 4 The impact of organic matter on sanitizer’s efficacies against L. monocytogenes

512 biofilms on food-contact surfaces. The 7-day-old L. monocytogenes biofilm grown on apple

513 juice soiled (A) SS-2B, (B) SS-4, (C) LDPE, (D) PVC, (E) PET, and (F) rubber were treated

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514 with quaternary ammonium (QAC, 400 ppm, 5 min) and peroxyacetic acid (PAA, 160 ppm, 1

515 min), respectively. SS-2B: stainless steel 2B finish, SS-4: stainless steel 4 finish, LDPE: low-

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516 density polyethylene, PVC: polyvinyl chloride, PET: polyester. a-b Bars topped with different

517

518
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letters differ significantly (P < 0.05). Mean  SEM were averaged from three independent

studies where three replicates was used for each selected surface coupon within each study.
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519

520
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521 Figure 1

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522

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523

524 Figure 2

5.0 1 min
er 5 min
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Lm reduction on rubber

4.0 a
(Log10 CFU/coupon)

a
b
b
3.0
a a

2.0
ot

1.0

0.0
Chlorine QAC PAA
525
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527 Figure 3

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528

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529
530

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531 Figure 4

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532
A B
5.0 Defective,clean Defective, soiled 5.0
Worn,clean Worn,soiled
Lm reduction on SS-2B
a

iew
a

Lm reduction on SS-4
4.0 4.0

(Log10 CFU/coupon)
(Log10 CFU/coupon)
a a
a
b b
b a
3.0 a b 3.0
a

b
2.0 b 2.0 b
b

1.0 1.0

0.0 0.0

ev
QAC PAA QAC PAA

C D E F
5.0 Worn,clean 5.0 5.0 5.0
Worn,soiled a
a

Lm reduction on rubber
a a
Lm reduction on LDPE

a a
Lm reduction on PVC

4.0 4.0

Lm reduction on PET
4.0 4.0
(Log10 CFU/coupon)
(Log10 CFU/coupon)

(Log10 CFU/coupon)
(Log10 CFU/coupon)

r
b
b
3.0 3.0 3.0 a 3.0 a
a a

533
2.0

1.0

0.0
QAC
b

PAA
2.0

1.0

0.0
QAC
b

PAA
er 2.0

1.0

0.0
QAC
b

PAA
2.0

1.0

0.0
QAC
b

PAA
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534 Table 1 Sanitizer efficacies against L. monocytogenes biofilm on the stainless steel surface

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Reduction (Log10 CFU/coupon)
Treatment
Surface New Defective Worn

Chlorine SS-2B 2.79 ± 0.14aA 2.43 ± 0.16aA 2.44 ± 0.11aA

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SS-4 3.57 ± 0.09bA 3.31 ± 0.13bA 3.39 ± 0.16bA

QAC SS-2B 2.83 ± 0.21aA 2.55 ± 0.18aAB 2.38 ± 0.19aB

SS-4 3.65 ± 0.11bA 3.17 ± 0.16bB 2.88 ± 0.12bB
PAA SS-2B 4.05 ± 0.19aA 3.53 ± 0.10aB 3.41 ± 0.13aB

ev
SS-4 4.32 ± 0.15aA 3.85 ± 0.15aB 3.83 ± 0.13bB
535
536 The 7-day-old L. monocytogenes biofilms (6.93 - 7.10 log10 CFU/coupon) were used. SS-2B:
537 stainless steel 2B finish, SS-4: stainless steel 4 finish. New: new surfaces. Defective: SS was

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538 bead blasted. Worn: SS was 80-grit sanded. QAC: quaternary ammonium compound. PAA:
539 peroxyacetic acid. A-B means within a row without the same letter differ significantly (P < 0.05).
540
541 er
a-b means within a column without the same letter differ significantly (P < 0.05) for the same

sanitizer treatment.
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542

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543 Table 2 Sanitizer efficacies against L. monocytogenes biofilm on food-contact surfaces
Surface Reduction (Log10 CFU/coupon)
Condition Initial levels Chlorine QAC PAA
LDPE New 7.05 ± 0.11a 3.35 ± 0.11aA 2.97 ± 0.15aB 3.95 ± 0.15aC

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Worn 7.89 ± 0.11b 3.06 ± 0.14aA 2.12 ± 0.12bB 3.77 ± 0.19aC
PVC New 7.50 ± 0.12a 3.23 ± 0.13aA 3.37 ± 0.16aA 3.80 ± 0.11aB
Worn 8.74 ± 0.03b 1.84 ± 0.09bA 2.05 ± 0.16bA 3.93 ± 0.11aB

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PET New 7.34 ± 0.12a 3.93 ± 0.15aA 3.66 ± 0.15aA 4.64 ± 0.11aB
Worn 8.29 ± 0.09b 3.31 ± 0.07bA 2.47 ± 0.20bB 4.35 ± 0.09aC
Rubber New 7.45 ± 0.07a 2.97 ± 0.13aA 2.51 ± 0.08aB 3.68 ± 0.08aC

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Worn 8.32 ± 0.19b 2.43 ± 0.12bA 2.55 ± 0.17aA 3.95 ± 0.09aB
544 7-day-old L. monocytogenes biofilms formed on different surfaces were treated with chlorine,
545
546
547
548
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QAC, and PAA, respectively, and surviving L. monocytogenes counts on each surface were
detached and enumerated. LDPE: low-density polyethylene, PVC: polyvinyl chloride, PET:
polyester. New: new surfaces. Worn: surfaces were 80-grit sanded. QAC: quaternary
ammonium compound. PAA: peroxyacetic acid. A-C means within a row without the same letter
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549 differ significantly (P < 0.05). a-b means within a column without the same letter differ
550 significantly (P < 0.05) for the same surface type.
551
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553 Table 3 Roughness of food-contact surfaces under different conditions

ed
Roughness Surface Conditions (µm)
parameter
New Defective Worn

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SS-2B 0.28  0.02bcA 1.04  0.09bB 1.56  0.15bC
SS-4 0.24  0.01bA 0.81  0.10aB 1.30  0.08abC
LDPE 0.25  0.01bA 1.27  0.11abB
Ra
PVC 0.34  0.02cA 1.17  0.15aB

ev
PET 0.12  0.01aA 1.03  0.15aB
Rubber 0.51  0.06dA 1.14  0.10aB
SS-2B 1.71  0.15dA 3.97  0.16aB 5.81  0.25dC

r
SS-4 1.24  0.07cA 3.78  0.12aB 5.27  0.25cdC
LDPE 0.79  0.03bA 3.29  0.24aB
Rz
PVC
PET
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2.14  0.05eA
0.41  0.03aA
4.69  0.22bcB
3.29  0.12aB
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Rubber 2.20  0.18eA 4.16  0.23bB
SS-2B 0.96  0.17cA 2.67  0.24bB 3.21  0.22cC
SS-4 0.67  0.10bcA 1.62  0.10aB 2.39  0.18bC
LDPE 0.36  0.01aA 2.16  0.24bB
ot

Rp
PVC 1.33  0.14dA 2.10  0.13bB
PET 0.59  0.01abA 1.51  0.15aB
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Rubber 0.65  0.12abA 2.24  0.20bB

SS-2B 0.68  0.06dA 1.42  0.12bB 1.93  0.16cC
SS-4 0.44  0.06bcA 1.02  0.12aB 1.36  0.10abC
rin

LDPE 0.35  0.05abA 1.51  0.13bB

Rq
PVC 1.02  0.06eA 1.54  0.17bcB
PET 0.20  0.08aA 1.01  0.14aB
ep

Rubber 0.55  0.07cdA 1.39  0.15abB

554
555 SS-2B: stainless steel 2B finish, SS-4: stainless steel 4 finish, PVC: polyvinyl chloride, PET:
556 polyester, LDPE: low-density polyethylene. New: new surfaces. Defective: SS was bead blasted.
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557 Worn: all surfaces were 80-grit sanded. The roughness of each surface was averaged from 16
558 measurements and presented in µm; mean ± SEM. A-C Numbers topped with different letters

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559 within each row differ significantly (P < 0.05) for different surface conditions. a-e Numbers

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560 topped with different letters within each column differ significantly (P < 0.05) for the same
561 roughness parameter.
562
563

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